Compositions and methods for inhibiting creatine transport as a treatment for cancer - Patents.com

Abstract

The present disclosure features compositions and methods useful for treating cancer (e.g., gastrointestinal cancer, colorectal cancer) featuring a creatine transporter inhibitor (e.g., β-guanidinopropionic acid) in combination with a KRAS inhibitor or another chemotherapeutic agent (e.g., leucovorin (folinic acid - FOL), 5-fluorouracil (F), and irinotecan hydrochloride (IRI), and / or bevacizumab).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 223,926, filed July 20, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Colorectal cancer is the second leading cause of cancer deaths among men and women in the United States and the leading cause of death worldwide. In 2020, approximately 148,000 people were diagnosed with colorectal cancer, and 53,200 died from the disease. Early-stage colorectal cancer is primarily treated by surgical resection. For larger tumors and tumors that have spread to lymph nodes, 5-fluorouracil-based chemotherapy regimens are administered in the "adjuvant" setting after surgery to reduce the risk of metastatic recurrence. For cancer that has spread to distant organs such as the liver, i.e., the primary site of metastatic recurrence, systemic 5-fluorouracil-containing chemotherapy regimens are given. Targeted therapies, including antibodies that engage the epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF), can provide a modest survival benefit when combined with chemotherapy. However, most metastatic patients succumb to the disease, with a 5-year survival rate of only 14%. Recently, innovative small molecules that covalently bind and inhibit the Kirsten rat sarcoma (KRAS) G12C oncogenic driver variant, present in approximately 4% of colorectal tumors, have elicited clinical responses in patients with this mutant allele. Other approaches include the development of inhibitors that target downstream KRAS signaling in RAS-mutant tumors. Limitations of such therapies include the emergence of resistance, which can reduce the durability of objective responses, a lack of efficacy against other KRAS mutant alleles present in approximately 40% of CRC patients, and a predicted lack of efficacy in KRAS wild-type tumors.

[0003] Cancer has evolved multiple mechanisms to maintain proliferation and survival, making it a leading cause of death worldwide. One such adaptive mechanism employed by cancer cells is metabolic alteration, commonly referred to as metabolic rewiring. By altering various metabolic pathways, cancer cells promote the biosynthesis of cellular building blocks necessary for growth, such as nucleotides, amino acids, and lipids. However, certain metabolites may become limited during cancer progression, necessitating their extracellular transport via metabolic transporters. Gastrointestinal tumors, such as colorectal cancer (CRC) and pancreatic cancer, are highly hypoxic, as are the metastases formed by these cancers. Metabolic rewiring supports tumor growth in a hypoxic environment, providing a mechanism that allows tumors to become resistant to chemotherapy. Therefore, additional approaches and combination therapies are needed to address the wide range of cancer types and metabolic rewiring. Summary of the Invention

[0004] In one aspect, the invention features a method of inhibiting cancer cell proliferation or survival, the method including contacting cancer cells with a creatine transporter inhibitor and a KRAS inhibitor, thereby inhibiting cancer cell proliferation or survival.

[0005] In another aspect, the invention features a method of inhibiting cancer cell proliferation or survival, the method including contacting cancer cells with β-GPA, 5-fluorouracil, and leflunomide, thereby inhibiting cancer cell proliferation or survival.

[0006] In yet another aspect, the invention features a method of inhibiting cancer cell proliferation or survival, the method including contacting cancer cells with a creatine transporter inhibitor, FOLFOX, leucovorin (folinic acid - FOL), 5-fluorouracil (F), and FOLFIRI, including irinotecan hydrochloride (IRI), and / or bevacizumab, thereby inhibiting cancer cell proliferation or survival.

[0007] In another aspect, the invention features a method of treating cancer in a subject, the method including administering to the subject a creatine transporter inhibitor and a KRAS inhibitor.

[0008] In yet another aspect, the invention features a method of treating a subject having cancer, the method including administering to the subject β-GPA or a pharmaceutically acceptable salt, 5-fluorouracil, and leflunomide.

[0009] In yet another aspect, the invention features a method of treating cancer in a subject, the method including administering to the subject a creatine transporter inhibitor, FOLFOX, leucovorin (folinic acid - FOL), 5-fluorouracil (5-FU), and FOLFIRI, including irinotecan hydrochloride (IRI), and / or bevacizumab.

[0010] In yet another aspect, the invention features a method of treating a selected subject, the method including administering a creatine transporter inhibitor and a KRAS inhibitor to the subject, wherein the subject is selected as having a cancer characterized by increased levels of creatine and / or creatine kinase B expression compared to a control cancer.

[0011] In yet another aspect, the invention features a method of treating a selected subject, the method including administering to the subject a creatine transporter inhibitor and a KRAS inhibitor, wherein the subject is selected as having a cancer comprising a KRAS, HRAS, or NRAS mutation.

[0012] In yet another aspect, the invention features a method for treating a subject having cancer, the method including administering to the subject β-GPA or a pharmaceutically acceptable salt thereof and folinic acid-fluorouracil-irinotecan regimen (FOLFIRI), wherein the β-GPA or a salt form thereof is administered at a dosage of about 1000 mg to 3600 mg twice daily.

[0013] In another aspect, the invention features a method of inhibiting cancer cell proliferation or survival, the method comprising contacting the cancer cell with β-GPA and adagrasib, thereby inhibiting cancer cell proliferation or survival.

[0014] In various embodiments of any of the above aspects or any other aspects of the invention described herein, the method further comprises contacting the cancer cells with an EGFR inhibitor.

[0015] In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells are contacted with adagrasib and β-GPA.

[0016] In various embodiments of any of the above aspects or any other aspects of the invention described herein, the creatine transporter is SLC6A8. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the creatine transporter inhibitor is selected from those listed in Table 2 (e.g., β-guanidinopropionic acid, N-methylamidino-N-methylglycine, 1-carboxymethyl-2-imino-hexahydropyrimidine (cyclocreatine), DL-alpha-guanidinopropionic acid, N-methyl-N-amidino-beta-alanine, N-ethyl-N-amidinoglycine, DL-alpha-guanidinobutyric acid, DL-beta-guanidinobutyric acid, gamma-guanidinobutyric acid, and guanidinoacetic acid, or a pharmaceutically acceptable salt thereof). In one embodiment, the creatine transporter inhibitor is β-guanidinopropionic acid (β-GPA) or a pharmaceutically acceptable salt thereof. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the KRAS inhibitor is any one or more of the following: sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells are contacted with β-GPA and a KRAS inhibitor that is any one or more of the following: sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells are contacted with β-GPA, FOLFIRI, and bevacizumab. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells are breast cancer cells, gastrointestinal cancer cells, colorectal cancer cells, lung cancer, melanoma cells, or pancreatic cancer cells. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells are non-small cell lung cancer cells.In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer is breast cancer, gastrointestinal cancer, colorectal cancer, lung cancer, melanoma, or pancreatic cancer. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer is non-small cell lung cancer. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer is colorectal cancer. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cell is a colorectal cancer cell. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cell comprises a KRAS, HRAS, or NRAS mutation (e.g., the mutation is at amino acid position 12, 13, or 61). In various embodiments of any of the above aspects or any other aspects of the invention described herein, the mutation is one or more of G12C, G12D, G12V, G12S, G13C, G13D, Q61H, and Q61L. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer comprises a KRAS, HRAS, or NRAS mutation. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the KRAS, HRAS, or NRAS mutation is Q61H. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the KRAS, HRAS, or NRAS mutation is a G12C or G12D mutation. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the method further comprises contacting the cells with 5-fluorouracil (5-FU) and leflunomide. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells comprise one or more markers that are any one or more of carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 (CA19-9), and carbohydrate antigen 15-3 (CA15-3).In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells comprise an increased level of creatine or phospho-creatine compared to the level of creatine or phospho-creatine present in control cells. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the method further comprises contacting the cells with one or more additional chemotherapeutic agents, which are any one or more of atovaquone, brequinar sodium, leflunomide, teriflunomide, BAY-2402234, AG-636, leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the proliferation or survival of the cells is reduced by at least about 10% compared to untreated cancer cells. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the method further comprises contacting the cells with 5-fluorouracil (5-FU) and leflunomide. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells comprise a marker that is any one or more of carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 (CA19-9), and carbohydrate antigen 15-3 (CA15-3). In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer cells are characterized as having an increased level of creatine or phospho-creatine compared to the level of creatine or phospho-creatine present in a control cancer. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the subject is administered β-GPA or a pharmaceutically acceptable salt thereof and a KRAS inhibitor that is any one or more of sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the method further comprises administering an EGFR inhibitor to the subject.In various embodiments of any of the above aspects or any other aspects of the invention described herein, the subject is administered adagrasib and β-GPA. In various embodiments of any of the above aspects or any other aspects of the invention described herein, treatment is monitored by detecting urinary creatine excretion, and an increase in such excretion indicates SLC6A8 inhibition. In various embodiments of any of the above aspects or any other aspects of the invention described herein, β-GPA or a salt form thereof is administered twice daily at a dosage of about 2400 mg or about 3000 mg BID. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer is gastrointestinal (GI) adenocarcinoma or colorectal cancer. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer comprises a KRAS, HRAS, or NRAS mutation associated with constitutive GTPase activity. In various embodiments of any of the above aspects or any other aspects of the invention described herein, prior to treatment, the cancer has developed resistance to one or more previous therapies. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer has been resistant to treatment with oxaliplatin and / or capecitabine. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the subject receives irinotecan, folinic acid, and 5-FU on days 1 and 15 after initiation of treatment. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the irinotecan is at about 180 mg / m. 2 In various embodiments of any of the above aspects or any other aspects of the invention described herein, the folinic acid is administered intravenously at about 400 mg / m 2 In various embodiments of any of the above aspects or any other aspects of the invention described herein, 5-FU is administered intravenously at about 2400 mg / m 2In various embodiments of any of the above aspects or any other aspects of the invention described herein, the intravenous infusion is administered over a 46-hour period. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the method further comprises administering bevacizumab. In various embodiments of any of the above aspects or any other aspects of the invention described herein, bevacizumab is administered at the start of treatment and on day 15 after initiation of treatment. In various embodiments of any of the above aspects or any other aspects of the invention described herein, bevacizumab is administered at about 5 mg / kg. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the treatment is administered in a 28-day cycle. In various embodiments of any of the above aspects or any other aspects of the invention described herein, RGX-202-01 is administered at about 3000 mg twice daily on days 1-28 of a 28-day cycle. In various embodiments of any of the above aspects or any other aspects of the invention described herein, bevacizumab is administered at a dose of about 5 mg / kg on days 1 and 15 of each 28-day cycle. In various embodiments of any of the above aspects or any other aspects of the invention described herein, β-GPA is administered twice daily at a total daily dose of about 6000 mg / day on days 1-28 of a 28-day cycle. In various embodiments of any of the above aspects or any other aspects of the invention described herein, irinotecan is administered intravenously at about 180 mg / m over 90 minutes, concurrently with folinic acid (leucovorin) administered intravenously at 400 mg / m over 2 hours, followed by 5-FU administered intravenously at about 2400 mg / m over 46 hours, on days 1 and 15 of each 28-day cycle. In various embodiments of any of the above or any other aspects of the invention described herein, bevacizumab is administered at about 5 mg / kg on days 1 and 15 of each 28-day cycle. In various embodiments of any of the above or any other aspects of the invention described herein, treatment results in a reduction of target lesions.In various embodiments of any of the above aspects or any other aspects of the invention described herein, the target lesion is an abdominal wall metastasis. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the reduction in the target lesion is a reduction of about 20% or more. In various embodiments of any of the above aspects or any other aspects of the invention described herein, the cancer comprises an increased level of creatine or phospho-creatine compared to the level of creatine or phospho-creatine present in a control cancer.

[0017] Any composition or method provided herein may be combined with one or more of any of the other compositions and methods provided herein.

[0018] The compositions and articles defined by the present invention have been isolated or otherwise prepared in connection with the examples provided below. Other features and advantages of the present invention will become apparent from the detailed description and claims.

[0019] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (3 rd edition.2006), The Cambridge Dictionary of Science and Technology (Walker ed., 1990), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991), The Biology of Cancer (2 ndedition, Weinberg et al., 2013) and Cancer:Principles and Practice of Oncology Primer of Molecular Biology in Cancer(3 rd edition, LWW, 2020). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified:

[0020] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0021] "Alteration" or "modulation" refers to a change (increase or decrease) in the expression level, structure, or activity of a gene or polypeptide as detected by standard art-known methods, such as those described herein. As used herein, alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

[0022] By "ameliorate" is meant to decrease, suppress, attenuate, reduce, arrest, or stabilize the onset or progression of a disease.

[0023] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, an analog of a polypeptide retains the biological activity of the corresponding naturally occurring polypeptide while possessing certain biochemical modifications that enhance the analog's function relative to the naturally occurring polypeptide. Such biochemical modifications can increase the analog's protease resistance, membrane permeability, or half-life, for example, without altering ligand binding. Analogs can also include unnatural amino acids.

[0024] "Chemotherapeutic agent" refers to any agent that inhibits cancer cell proliferation, inhibits cancer cell survival, inhibits or stabilizes tumor growth, or is otherwise useful in the treatment of cancer. Exemplary anti-cancer agents provided herein may be used in combination with creatine transport inhibitors (e.g., RGX-202, RGX-202-01) and / or creatine kinase inhibitors provided herein to reduce cancer cell proliferation or reduce tumor size in a subject. Exemplary chemotherapeutic agents include, but are not limited to, irinotecan, oxaliplatin, cetuximab, bevacizumab (AVASTIN®), leucovorin, and 5-fluorouracil (5-FU). As used herein, chemotherapeutic agents encompass both chemical and biological agents. These agents function to inhibit cellular activities upon which cancer cells depend for continued survival. Categories of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones or hormone analogs, and other antitumor agents. Most, if not all, of these agents are directly toxic to cancer cells and do not require immune stimulation. In one embodiment, the chemotherapeutic agent is an agent used in the treatment of a neoplasm, such as a solid tumor, hi one embodiment, the chemotherapeutic agent is a radioactive molecule.Those skilled in the art can easily identify the chemotherapeutic agent to be used (see, for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., COPYRGT. 2000 Churchill Livingstone, Inc.; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). In embodiments, the agents and combinations described herein reduce cancer cell proliferation or survival by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, including inducing cell death (apoptosis) in one or more cells within a cell mass.

[0025] As used herein, the term "β-guanidinopropionic acid" or "β-GPA" refers to the following structure: TIFF2024528705000001.tif14128 refers to small molecules and their salts.

[0026] β-GPA is a creatine analog identified by CAS number 353-09-3. β-Guanidinopropionic acid (β-GPA), also known as guanidinopropionic acid, beta-guanidinopropionic acid, or N-(aminoiminomethyl)-beta-alanine, inhibits the creatine transporter by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. β-GPA, RGX-202, and RGX-202-01 are exemplary creatine transporter inhibitors.

[0027] As used herein, a "biological sample" refers to a sample obtained from a biological subject, including samples from biological tissues or fluids obtained, arrived at, or collected in vivo or in situ that contain or are suspected of containing nucleic acids or polypeptides associated with cancer, e.g., creatine kinase B (CKB) or KRAS mutations. Biological samples also include samples from areas of a biological subject that contain precancerous or cancerous cells or tissues. Such samples can be organs, tissues, fractions, and cells isolated from mammals, including, but not limited to, patients, mice, and rats, including humans. Biological samples can also include tissues, e.g., sections of biological samples, including frozen sections taken for histological purposes. Biological samples are typically of eukaryotic origin, e.g., mammalian (e.g., rat, mouse, cow, dog, guinea pig, rabbit, primate, e.g., chimpanzee, or human).

[0028] "Colorectal cancer" or "CRC" refers to cancer that affects the colon or rectum, located at the end of the digestive tract.

[0029] As used herein, "combination" therapy, unless otherwise clear from the context, is meant to encompass the administration of two or more therapeutic agents in a coordinated manner, including, but not limited to, sequential or substantially simultaneous administration. Specifically, combination therapy encompasses both coadministration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent is in some way conditioned on the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a predetermined period of time. See, e.g., Kohrt et al. (2011) Blood 117:2423.

[0030] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meaning ascribed to them in U.S. patent law, and can mean "includes," "including," and the like, and "consisting essentially of" or "consists essentially" likewise have the meaning ascribed to them in U.S. patent law, and the terms are open-ended, permitting the presence of more than what is recited so long as the basic or novel characteristics of the recited items are not changed by the presence of more than what is recited, but excluding prior art embodiments.

[0031] A "control sample" refers to a sample of biological material representing a healthy, cancer-free animal or an animal with cancer. The sample can be collected from an animal for use in the methods described herein or can be any biological material representing a normal, cancer-free animal suitable for use in the methods of the present invention. A control sample can also be obtained from normal tissue from an animal with or suspected of having cancer. A control sample can also refer to a given level of a marker representing a cancer-free population that has been previously established based on measurements from normal, cancer-free animals. Alternatively, a biological control sample can refer to a sample obtained from a different individual or can be a normalized value based on baseline data obtained from a population. Furthermore, a control sample can be defined by a specific age, sex, ethnicity, or other demographic parameter. In some situations, a control is implicit in a particular measurement. A typical control level for a gene is two copies per cell. An example of an implicit control is when a detection method can only detect a marker, such as creatine kinase type B (CKB), or the corresponding gene copy number, when a higher level than that typical of normal, cancer-free animals is present. Another example is in the context of an immunohistochemistry assay, where the control level for the assay is known. Other examples of such controls are within the knowledge of those skilled in the art.

[0032] As used herein, the term "creatine" refers to a molecule having the following chemical structure: TIFF2024528705000002.tif22128

[0033] Creatine can rapidly resynthesize ATP from ADP through the anaerobic conversion of phosphorylated creatine (phosphocreatine) to creatine in a reversible reaction by the enzyme creatine kinase.

[0034] "Creatine transporter polypeptide" refers to a polypeptide or fragment thereof having creatine transport activity. Creatine transporter polypeptides can be expressed on the outer membrane of cells (e.g., cancer cells). Exemplary creatine transporter polypeptides include solute carrier family 6 member 8 polypeptide (SLC6A8) and solute carrier family 16 member 12 polypeptide (SLC16A12).

[0035] "Creatine transport inhibitor" refers to an agent provided herein that reduces or inhibits the activity, or reduces the level or expression of a creatine transporter polypeptide. Exemplary creatine transport inhibitors are listed in Table 2. In one embodiment, the creatine transport inhibitor is RGX-202, RGX-202-01, and analogs and derivatives thereof.

[0036] "CKB" or "creatine kinase B polypeptide" refers to a cytoplasmic polypeptide involved in energy homeostasis, or a fragment thereof, having kinase activity, e.g., an enzyme capable of reversibly catalyzing the transfer of phosphate between ATP and various phosphogens, such as creatine phosphate, and having at least about 85% or greater amino acid sequence identity with NCBI Gene Specific Gene ID: 1152; NCBI Reference Sequence: NP_001814.2. An exemplary human CKB amino acid sequence is provided below. >NP_001814.2 Creatine kinase type B isoform 1 [Homo sapiens] MPFSNSHNALKLRFPAEDEFPDLSAHNNHMAKVLTPELYAELRAKSTPSGFTLDDVIQTGVDNPGHPYIM TVGCVAGDEESYEVFKDLFDPIIEDRHGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSRVRTGRSIRGF CLPPHCSRGERRAIEKLAVEALSSLDGDLAGRYYALKSMTEAEQQQLIDDHFLFDKPVSPLLLASGMARD WPDARGIWHNDNKTFLVWVNEEDHLRVISMQKGGNMKEVFTRFCTGLTQIETLFKSKDYEFMWNPHLGYI LTCPSNLGTGLRAGVHIKLPNLGKHEKFSEVLKRLRLQKRGTGGVDTAAVGGVFDVSNADRLGFSEVELV QMVVDGVKLLIEMEQRLEQGQAIDDLMPAQK

[0037] The terms "reduce," "reducing," "reducing," "decreasing," or "inhibiting" are all used herein to generally mean a decrease by a statistically significant amount. However, for the avoidance of doubt, "reduce," "reducing," or "reducing," or "inhibiting" means a decrease of at least 10% compared to a reference level, for example, 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 a 100% decrease compared to a reference level, including a 100% decrease (e.g., a deficient level compared to a reference sample), or any decrease between 10 and 100%.

[0038] "Detecting" refers to determining the presence, absence, or amount of the analyte being detected.

[0039] "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the molecule of interest detectable via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, or haptens.

[0040] The phrases "detecting cancer" or "diagnosing cancer" refer to determining the presence or absence of cancer or a precancerous condition in a subject. "Detecting cancer" can also refer to obtaining indirect evidence of the possible presence of precancerous or cancerous cells in an animal, or assessing a patient's predisposition to developing cancer.

[0041] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include, but are not limited to, breast cancer, colorectal cancer (CRC), lung cancer (e.g., non-small cell lung cancer (NSCLC)), melanoma, pancreatic cancer, or gastrointestinal cancer. In some examples, the cancer is associated with a mutation in a KRAS, HRAS, or NRAS polypeptide.

[0042] By "drug-resistant" cancer is meant a cancer that does not respond or shows a reduced response to one or more chemotherapeutic agents.

[0043] "Effective amount" refers to the amount of drug required to improve the symptoms of a disease compared to an untreated subject (e.g., a subject without cancer). The effective amount of the active compound(s) used to practice the present invention for the therapeutic treatment of a disease will vary depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0044] The present invention provides several targets useful for the development of very specific drugs to treat or characterize disorders characterized by the methods described herein. Furthermore, the methods of the present invention provide a facile means for identifying therapies that are safe for use in subjects. Furthermore, the methods of the present invention provide a route to analyze virtually any number of compounds for their effect on the diseases described herein with high volume throughput, high sensitivity, and low complexity.

[0045] The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.

[0046] As used herein, the terms "failed to respond to previous therapy" or "refractory to previous therapy" refer to cancer that has progressed despite treatment with therapy.

[0047] As used herein, the term "FOLFIRI" refers to a chemotherapy regimen that includes leucovorin (folinic acid - FOL), 5-fluorouracil (F), and irinotecan hydrochloride (IRI). FOLFIRI is the standard of care for gastrointestinal cancers (e.g., colorectal cancer).

[0048] As used herein, the term "FOLFOX" refers to a chemotherapy regimen that includes leucovorin (folinic acid - FOL), 5-fluorouracil (F), and oxaliplatin (OX). FOLFOX is the standard treatment for gastrointestinal cancers (e.g., colorectal cancer).

[0049] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0050] "HRAS" or "H-Ras" or "Harvey rat sarcoma polypeptide" or "HRAS polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or greater amino acid sequence identity to GenBank Accession No. CAG38816.1, reproduced below, and having GTPase activity. GTPase activity involves the conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). An exemplary human HRAS amino acid sequence is provided below. >CAG38816.1 HRAS [Homo sapiens] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQYREQIKRVKDSDDVPMVLVGNKCDLAARTVESRQAQDLARSYGIPYIETSAKTRQGVEDAFYTLVREIRQHKLRKLNPPDESGPGCMSCKCVLS

[0051] By "HRAS polynucleotide" is meant a polynucleotide that encodes an HRAS polypeptide.

[0052] As used herein, "HRAS mutant cancer" refers to a cancer that contains an alteration in the sequence of an HRAS polypeptide (i.e., an HRAS mutation).

[0053] "Hybridization" means hydrogen bonding between complementary nucleobases, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0054] The terms "increased," "increase," or "enhance," or "activate" are all used herein to generally mean a statically significant increase. For the avoidance of doubt, the terms "increased," "increase," or "enhance," or "activate" mean an increase of at least 10% 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%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0055] As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but within an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0056] As used herein, the term "in vivo" refers to events that take place within a multicellular organism, such as a non-human animal.

[0057] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components that normally accompany it as found in its native state. "Isolated" refers to a degree of separation from the original source or surroundings. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other materials so that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the invention is purified when it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein yields essentially one band in an electrophoretic gel. For proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can yield different isolated proteins that can be purified separately.

[0058] An "isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes that flank it in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, a recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule independent of other sequences (e.g., cDNA, or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0059] By "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, of the polypeptide of the invention. Isolated polypeptides of the invention can be obtained, for example, by extraction from a natural source, by expressing a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0060] "KRAS" or "K-Ras" or "Kirsten rat sarcoma polypeptide" or "KRAS polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid sequence identity to NCBI Gene Identification 3845, NCBI Reference Sequence: NP_001356715.1, and having GTPase activity. GTPase activity involves the conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). This definition is broad enough to encompass other RAS family members (e.g., HRAS and NRAS). An exemplary human KRAS amino acid sequence is provided below. NCBI sequence: >NP_001356715.1 GTPase KRas isoform a [Homo sapiens] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQ YMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIP FIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM >NP_001356716.1 GTPase KRas isoform b [Homo sapiens] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQ YMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIP FIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIM UNIPROTKB- G3V5T7: >tr|G3V5T7|G3V5T7_Human GTPase KRas OS=Homo sapiens OX=9606 GN=KRAS PE=4 SV=1 MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEGVDDAFYTLVREIRKHKEKMSKD GKKKKKKSKTKCVIM

[0061] In some embodiments, the KRAS polypeptide comprises an amino acid substitution of cysteine ​​for glycine at amino acid position 12 (KRAS G12C), an amino acid substitution of aspartic acid for glycine at amino acid position 12 (KRAS G12D). The amino acid codon and residue position assignments for human KRas are based on the amino acid sequence identified by UniProtKB / Swiss-Prot P01116:variant p.Gly12Cys.

[0062] KRAS, HRAS, and NRAS are canonical ras gene family members that encode four highly related protein isoforms. These Ras family members share over 85% amino acid sequence identity and all share GTPase activity. Oncogenic mutations at positions 12, 13, or 61 of the H-ras, N-ras, and K-ras genes are among the most common genetic lesions in mammalian tumors. See, for example, Castellano and Santos, Genes Cancer. 2011 Mar;2(3):216-231, incorporated herein by reference.

[0063] By "KRAS polynucleotide" is meant a polynucleotide that encodes a KRAS polypeptide.

[0064] As used herein, "KRAS mutant cancer" refers to a cancer that contains an alteration in the sequence of a KRAS polypeptide (i.e., a KRAS mutation) or an alteration in the sequence of another RAS family member (e.g., HRAS, NRAS) that falls within the definition of a KRAS polypeptide.

[0065] "KRAS inhibitor" refers to an agent that reduces KRAS expression or activity. Such reduction may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Exemplary KRAS inhibitors include, but are not limited to, those listed in Table 1 provided herein.

[0066] By "marker" is meant any protein or polynucleotide having an altered expression level or activity associated with a disease or disorder. In some embodiments, the marker is a cancer or tumor marker. Exemplary cancer markers include tumor antigens, such as carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 or CA19-9, and carbohydrate antigen 15-3 (CA15-3).

[0067] "Metabolic rewiring" refers to the process by which a cancer (e.g., a tumor) alters the flux of metabolites in one or more metabolic pathways to increase tumor growth. Metabolic pathways can include, for example, the biosynthesis of anabolic building blocks required for growth, such as nucleotides, amino acids, and lipids.

[0068] As used herein, the terms "malignant tumor," "malignant condition," "cancer," or "tumor" refer to the uncontrolled growth of cells that interferes with the normal function of bodily organs and systems. A subject with a malignant tumor (i.e., cancer or tumor) is one who has objectively measurable malignant or cancerous cells present in the subject's body. This definition includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers that migrate from their original location and seed vital organs can ultimately lead to the death of the subject through the deterioration of the affected organ's function.

[0069] As used herein, a "metastatic tumor" refers to a tumor or cancer in which the cancer cells forming the tumor are likely to or have begun to metastasize, or spread, from one location to another location or locations within a subject. In some embodiments, metastasis occurs via the lymphatic system or via hematogenous spread, e.g., giving rise to a secondary tumor within the subject. Such metastatic behavior may be indicative of malignancy. In some cases, metastatic behavior may be associated with increased cell migration and / or invasive behavior of tumor cells.

[0070] Examples of cancers that may be defined as metastatic include, but are not limited to, non-small cell lung cancer (NSCLC) (e.g., non-squamous non-small cell lung cancer), breast cancer, ovarian cancer, colorectal cancer, cholangiocarcinoma, biliary tract cancer, bladder cancer, brain cancer including glioblastoma and medulloblastoma, cervical cancer, choriocarcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal cancer, hematological malignancies, multiple myeloma, leukemia, intraepithelial neoplasia, liver cancer, lymphoma, neuroblastoma, oral cancer, pancreatic cancer, prostate cancer, sarcoma, skin cancer including melanoma, basal cell carcinoma, squamous cell carcinoma, testicular cancer, stromal tumors, germ cell tumors, thyroid cancer, and renal cancer.

[0071] As used herein, the term "modulate" is meant to refer to any change in a biological state, ie, an increase, a decrease, etc.

[0072] "NRAS" or "N-Ras" or "Kirsten rat sarcoma" or "NRAS polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or greater amino acid sequence identity to NCBI Gene Identification 3845, NCBI Reference Sequence Accession No. NP_002515.1, reproduced below, and having GTPase activity. GTPase activity involves the conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP).

[0073] >NP_002515.1 GTPase NRas [Homo sapiens] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNSKSFADINLYREQIKRVKDSDDVPMVLVGNKCDLPTRTVDTKQAHELAKSYGIPFIETSAKTRQGVEDAFYTLVREIRQYRMKKLNSSDDGTQGCMGLPCVVM

[0074] By "NRAS polynucleotide" is meant a polynucleotide that encodes an NRAS polypeptide.

[0075] As used herein, "NRAS mutant cancer" refers to a cancer that contains an alteration in the sequence of the NRAS polypeptide (i.e., an NRAS mutation).

[0076] As used herein, "obtaining," as in "obtaining a drug," includes synthesizing, purchasing, or otherwise acquiring a drug.

[0077] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound useful within the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickeners, and / or excipients. Pharmaceutical compositions facilitate administration of a compound to an organism.

[0078] Multiple techniques exist in the art for administering compounds, including but not limited to intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

[0079] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the composition and is relatively non-toxic, i.e., that may be administered to an individual without causing undesired biological effects or interacting in a deleterious way with any of the components of the composition in which it is contained.

[0080] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in the delivery or transport of a compound(s) of the invention in or to a subject so that the subject can perform its intended function. Typically, such compounds are delivered or transported from one organ or part of the body to another organ or part of the body. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials which can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients such as cocoa butter and suppository waxes, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil, glycols such as propylene glycol, polyols such as glycerin, sol Examples of suitable non-toxic and compatible substances for use in pharmaceutical formulations include sorbitol, mannitol, and polyethylene glycol, esters such as ethyl oleate and ethyl laurate, agar, buffers such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer solution, diluents, granulating agents, lubricants, disintegrants, wetting agents, emulsifiers, colorants, release agents, coating agents, sweeteners, flavorings, fragrances, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, solidifying agents, suspending agents, surfactants, humectants, carriers, stabilizers, and other non-toxic and compatible substances for use in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents that are compatible with the activity of the compound and physiologically acceptable to the subject. Supplementary active ingredients can also be incorporated into the composition.

[0081] As used herein, the term "pharmaceutically acceptable salts" refers to salts of the administered compound prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.

[0082] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the likelihood of developing a disorder or condition in a subject who does not have the disorder or condition but is at risk of developing or is susceptible to it.

[0083] "RAS polypeptide" refers to a RAS family member protein. Exemplary RAS proteins include, but are not limited to, HRAS, KRAS, or NRAS polypeptides. RAS polypeptides are described in E. Castellano and E. Santos, "Functional Specificity of Ras Isoforms," ​​Genes Cancer, 2:216-231 (2011), doi:10.1177 / 1947601911408081, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0084] "RAS polynucleotide" means a polynucleotide that encodes a RAS family member protein. Exemplary RAS polynucleotides include, but are not limited to, an HRAS, KRAS, or NRAS polynucleotide.

[0085] By "RAS mutated cancer" is meant a cancer that contains an alteration in the sequence of a RAS polypeptide or polynucleotide.

[0086] By "decreasing" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0087] "Reference" refers to a standard or control condition, e.g., a cancer cell, population thereof, or subject that has not been administered an agent provided herein.

[0088] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a particular sequence, for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is generally at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer therebetween or any integer therebetween.

[0089] " siRNA " refers to double-stranded RNA. Optimally, siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a two-base overhang at its 3' end. These dsRNAs can be introduced into individual cells or whole animals, for example, they can be introduced systemically through the bloodstream. Such siRNAs are used to down-regulate mRNA levels or promoter activity.

[0090] "SHP2" or "protein tyrosine phosphatase 2 polypeptide" or "Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 polypeptide" or "protein tyrosine phosphatase non-receptor type 11 polypeptide" refers to a polypeptide or fragment thereof that has phosphatase activity and has at least about 85% or more amino acid sequence identity with NCBI Gene Identification 5781, NCBI Reference Sequence: NP_002825.3. An exemplary human SHP2 amino acid sequence is provided below. >NP_002825.3 Tyrosine-protein phosphatase non-receptor type 11 isoform 1 [Homo sapiens] MTSRRWFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLYGGEK FATLAELVQYYMEHHGQLKEKNGDVIELKYPLNCADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRES QSHPGDFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVL QLKQPLNTTRINAAEIESRVRELSKLAETTDKVKQGFWEEFETLQQQECKLLYSRKEGQRQENKNKNRYK NILPFDHTRVVLHDGDPNEPVSDYINANIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQENS RVIVMTTKEVERGKSKCVKYWPDEYALKEYGVMRVRNVKESAAHDYTLRELKLSKVGQGNTERTVWQYHF RTWPDHGVPSDPGGVLDFLEEVHHKQESIMDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDV PKTIQMVRSQRSGMVQTEAQYRFIYMAVQHYIETLQRRIEEEQKSKRKGHEYTNIKYSLADQTSGDQSPL PPCTPTPPCAEMREDSARVYENVGLMQQQKSFR

[0091] By "SHP2 inhibitor" is meant an agent provided herein that reduces or inhibits the activity of SHP2 or reduces the level or expression of SHP2.

[0092] "SLC6A8" or "SLC6a8" or "solute carrier family 6 member 8 polypeptide" refers to a polypeptide or fragment thereof having creatine transport activity and having at least about 85% or more amino acid sequence identity with NCBI Gene Identification 6535, NCBI Reference Sequence: NP_005620.1. An exemplary human SLC6A8 amino acid sequence is provided below. >NP_005620.1 Sodium- and chloride-dependent creatine transporter 1 isoform 1 [Homo sapiens] MAKKSAENGIYSVSGDEKKGPLIAPGPDGAPAKGDGPVGLGTPGGRLAVPRETWTRQMDFIMSCVGFAV GLGNVWRFPYLCYKNGGGVFLIPYVLIALVGGIPIFFLEISLGQFMKAGSINVWNICPLFKGLGYASMVI VFYCNTYYIMVLAWGFYYLVKSFTTTLPWATCGHTWNTPDCVEIFRHEDCANASLANLTCDQLADRRSPV IEFWENKVLRLSGGLEVPGALNWEVTLCLLACWVLVYFCVWKGVKSTGKIVYFTATFPYVVLVVLLVRGV LLPGALDGIIYYLKPDWSKLGSPQVWIDAGTQIFFSYAIGLGALTALGSYNRFNNNCYKDAIILALINSG TSFFAGFVVFSILGFMAAEQGVHISKVAESGPGLAFIAYPRAVTLMPVAPLWAALFFFMLLLLGLDSQFV GVEGFITGLLDLLPASYYFRFQREISVALCCALCFVIDLSMVTDGGMYVFQLFDYYSASGTTLLWQAFWE CVVVAWVYGADRFMDDIACMIGYRPCPWMKWCWSFFTPLVCMGIFIFINVVYYEPLVYNNTYVYPWWGEAM GWAFALSSMLCVPLHLLGCLLRAKGTMAERWQHLTQPIWGLHLEYRAQDADVRGLTTLTPVSESSKVVV VESVM >NP_001136277.1 Sodium- and chloride-dependent creatine transporter 1 isoform 2 [Homo sapiens] MAKKSAENGIYSVSGDEKKGPLIAPGPDGAPAKGDGPVGLGTPGGRLAVPRETWTRQMDFIMSCVGFAV GLGNVWRFPYLCYKNGGGVFLIPYVLIALVGGIPIFFLEISLGQFMKAGSINVWNICPLFKGLGYASMVI VFYCNTYYIMVLAWGFYYLVKSFTTTLPWATCGHTWNTPDCVEIFRHEDCANASLANLTCDQLADRRSPV IEFWENKVLRLSGGLEVPGALNWEVTLCLLACWVLVYFCVWKGVKSTGKIVYFTATFPYVVLVVLLVRGV LLPGALDGIIYYLKPDWSKLGSPQVWIDAGTQIFFSYAIGLGALTALGSYNRFNNNCYNGTSFFAGFVVF SILGFMAAEQGVHISKVAESGPGLAFIAYPRAVTLMPVAPLWAALFFFMLLLLGLDSQFVGVEGFITGLL DLLPASYYFRFQREISVALCCALCFVIDLSMVTDGGMYVFQLFDYYSASGTTLLWQAFWECVVVAWVYGA DRFMDDIACMIGYRPCPWMKWCWSFFTPLVCMGIFIFNVVYYEPLVYNNTYVYPWWGEAMGWAFALSSML CVPLHLLGCLLRAKGTMAERWQHLTQPIWGLHLEYRAQDADVRGLTTLTPVSESSKVVVVESVM >NP_001136278.1 Sodium- and chloride-dependent creatine transporter 1 isoform 3 [Homo sapiens] MKAGSINVWNICPLFKGLGYASMVIVFYCNTYYIMVLAWGFYYLVKSFTTTLPWATCGHTWNTPDCVEIF RHEDCANASLANLTCDQLADRRSPVIEFWENKVLRLSGGLEVPGALNWEVTLCLLACWVLVYFCVWKGVK STGKIVYFTATFPYVVLVVLLVRGVLLPGALDGIIYYLKPDWSKLGSPQVWIDAGTQIFFSYAIGLGALT ALGSYNRFNNNCYKDAIILALINSGTSFFAGFVVFSILGFMAAEQGVHISKVAESGPGLAFIAYPRAVTL MPVAPLWAALFFFMLLLLGLDSQFVGVEGFITGLLDLLPASYYFRFQREISVALCCALCFVIDLSMVTDG GMYVFQLFDYYSASGTTLLWQAFWECVVVAWVYGADRFMDDIACMIGYRPCPWMKWCWSFFTPLVCMGIF IFNVVYYEPLVYNNTYVYPWWGEAMGWAFALSSMLCVPLHLLGCLLRAKGTMAERWQHLTQPIWGLHHLE YRAQDADVRGLTTLTPVSESSKVVVVESVM

[0093] By "specifically binds" is meant an agent that recognizes and binds to a polypeptide of the invention, but does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample that naturally contains a polypeptide of the invention.

[0094] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to pairing to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein), or portions thereof, under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0095] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, detergent concentration, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is performed at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization is performed at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / mL denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization is performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / mL ssDNA. Useful variations of these conditions will be readily apparent to those of skill in the art.

[0096] For most applications, wash steps following hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As noted above, wash stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for wash steps are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for wash steps typically include temperatures of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, wash steps are performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps are performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash steps are performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977), Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975), Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001), Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York), and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0097] By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0098] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program may be used, e.g., -3 ~e -100 A probability score of 0.05 indicates closely related sequences.

[0099] By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a cow, dog, horse, cat, sheep, or rodent.

[0100] Ranges provided herein are understood to be shorthand for all values ​​within that range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0101] As used herein, the terms "treat," "treating," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms. It should be understood that treating a disorder or condition does not require, although not precluded, that the disorder, condition, or its associated symptoms be completely eliminated.

[0102] The term "tumor" refers to a mass of tissue formed by or containing cells undergoing uncontrolled growth. Tumors can be benign or malignant. Benign tumors are characterized by the lack of metastasis. Malignant cells are cancer cells, which have the potential to metastasize. Tumors that may be treated with the therapies described herein (e.g., creatine inhibitors alone or in combination with KRAS inhibitors) include, but are not limited to, adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hamartoma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma. Tumors include acral lentiginous melanoma, optic fasciculation tumor, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and teratoma. Linear keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary, carcinoid, carcinoma, carcinosarcoma, cavernous, bile duct carcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal, epithelioid, Ewing's sarcoma, fibrolamellar type, focal nodular hyperplasia, gastrinocarcinoma melanoma, germ cell tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatocellular adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, interepithelial squamous cell tumor, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, lentigo maligna melanoma, malignant melanoma, malignant mesothelioma, medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial, metastatic carcinoma, mucoepidermoid carcinoma, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, oat cell The cancer may be selected from carcinoma, oligodendroglial, osteosarcoma, pancreatic, papillary serous adenocarcinoma, pineal cell, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, submesothelial, superficial spreading melanoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, well-differentiated carcinoma, and Wilms' tumor.

[0103] The term "tumor progression" refers to all stages of tumor development, including tumor initiation, tumor growth and proliferation, invasion, and metastasis.

[0104] The term "inhibition of tumor progression" means inhibiting the initiation, growth, proliferation, or spread of a tumor, and includes, but is not limited to, the following effects: inhibiting cell growth in a tumor, (2) inhibiting tumor growth to some extent, including slowing or completely halting growth, (3) reducing tumor cell count, (4) reducing tumor size, (5) inhibiting (i.e., reducing, slowing, or completely halting) tumor cell invasion into adjacent peripheral organs and / or tissues, (6) inhibiting (i.e., reducing, slowing, or completely halting) metastasis, (7) increasing the survival time of a patient or patient population after treatment of the tumor, and / or (8) reducing the mortality rate of a patient or patient population at a given time point after treatment of the tumor.

[0105] A tumor "responds" to a particular agent provided herein if tumor progression is inhibited as defined above.

[0106] As used herein, the term "or" is understood to be inclusive unless specifically stated otherwise or clear from context. As used herein, the terms "a," "an," and "the" are understood to be singular or plural unless specifically stated otherwise or clear from context.

[0107] As used herein, the term "about" is understood to mean within normal tolerances in the art, e.g., within two standard deviations of the mean, unless specifically stated otherwise or clear from the context. About may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0108] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. [Brief explanation of the drawings]

[0109] [Figure 1] Figures 1A-D show that RGX-202 and RGX-202-01 reduce cellular and tumor creatine and phosphocreatine levels. Figures 1A-1D are graphs. Figure 1A shows that C57BL / 6 wild-type or SLC6A8 knockout mice were injected (ip) with RGX-202 or vehicle control before receiving d3-creatine (ip). For d3-creatine quantification by LC-MS / MS, mice were euthanized, hearts were removed, and metabolites were extracted. n = 3-8 independent experiments; p = 0.0016 (100 mg / kg), p < 0.0001 (250 mg / kg), p = 0.0014 (500 mg / kg); mean + / - SEM. Figure 1B shows that UN-KPC-961 pancreatic tumor-bearing B6129SF1 / J mice were fed a control or RGX-202-01-supplemented diet (800 mg / kg) for 35 days as soon as tumors reached approximately 80 mm3. Mice were injected with 1 mg / kg d3-creatine (ip), tumors were extracted 1.5 hours later, and d3-creatine levels were quantified by LC-MS / MS. n = 4 per group, p < 0.0001. Figures 1C-1D show correlation analysis of plasma creatine and RGX-202-01 exposure (AUC0-t) (G) and mean urinary creatine and RGX-202-01 concentrations (H) measured over a 24-hour period in mice receiving either a control or RGX-202-01-supplemented diet at 100, 400, or 1200 mg / kg for 10 days. n = 6 mice per dose group. Grey lines indicate 95% confidence intervals. [Figure 2-1]Figures 2A-2M show that SLC6A8 inhibition exhibits antitumor activity against primary and metastatic CRC in different backgrounds. Graphs and micrographs are shown. Figures 2A-2M show graphs and micrographs. Figures 2A and 2B show subcutaneous tumor growth (2A) and Kaplan-Meier survival curves (2B) of 1x10 Lvm3b cells in athymic nude mice. Daily oral gavage administration of RGX-202-01 (200 mg / kg) was initiated at tumor sizes of approximately 50 mm3, with n=8-9 per group. Photographs show control and RGX-202-01-treated mice. Figure 2C shows subcutaneous tumor growth in athymic nude mice injected with 0.5x10 HCT116 cells. Treatment with a control diet or an RGX-202-supplemented diet (800 mg / kg) was initiated when tumors became palpable. n=5 per group. Figures 2D and 2E show subcutaneous tumor growth (D) and Kaplan-Meier survival curves (E) of 2 × 10 HT29 cells in athymic nude mice receiving a control diet or an RGX-202-01-supplemented diet (800 mg / kg) when tumors reached approximately 55 mm. Inserts represent individual tumor growth curves. n = 10 per group. Figure 2F shows subcutaneous tumor growth of 0.5 × 10 CT26 cells in BALB / c mice receiving a control diet or an RGX-202-01-supplemented diet (500 mg / kg) starting from tumors of approximately 100 mm. n = 7–8 per group. Figure 2G shows subcutaneous tumor growth of 0.5 × 10 MC38 cells in C57BL / 6 mice. Mice received a control diet or an RGX-202-01-supplemented diet (500 mg / kg) starting from tumors of approximately 150 mm. n=7 per group. Figures 2H and 2I show that MC38 tumors treated with RGX-202-01 for 9 days by oral gavage (QD 250 mg / kg p=0.0011; 500 mg / kg p=0.0032) or by diet (diet, p=0.0092) were extracted and immunostained for cleaved caspase-3 (CC3) (H). Quantification of the percentage of tumor area stained with cleaved caspase-3. n=4 per group + / - SEM (I). Figures 2J and 2M show representative images and quantification of control and RGX-202-treated Lvm3b xenografts immunostained for cleaved caspase-3 (J, K) and Ki67 (L, M) and counterstained with DAPI.n=3 tumors per group. Figures 2L and 2M show quantification of CC3 (K) and Ki67 (M) positive cells in control and treated Lvm3b xenograft tumors. n=3 tumors per group. Mice were imaged 14 days post-injection. n=4 per group. Mean + / - SEM (A, C, D, F, G, I, K, M) is shown. P values ​​are based on two-tailed t-tests (A, C, D, F-I), one-tailed t-tests (K and M), and log-rank Mantel-Cox tests (B and E). Scale bars (H, J, and L) 200 μm. [Figure 2-2] See description of Figure 2-1. [Figure 2-3] See description of Figure 2-1. [Figure 2-4] See description of Figure 2-1. [Figure 2-5] See description of Figure 2-1. [Figure 3-1]Figures 3A-3K show that RGX-202 inhibits the growth of KRAS wild-type and mutant CRC PDXs and synergizes with 5-FU and leflunomide. Figures 3A-3K show graphs. Figures 3A-3D show subcutaneous tumor growth from approximately 25 mm PDX fragments implanted into athymic nude mice receiving a control or RGX-202-supplemented diet (800 mg / kg), starting with tumor sizes of approximately 100 mm (CLR4), 250 mm (CLR7), 200 mm (CLR24), and 150 mm (CLR30). n=10 per group. Figure 3E shows a waterfall plot of the response to RGX-202-01 across 43 colorectal PDX models. Each bar represents an individual PDX, and shading indicates KRAS mutation. Mice were treated with approximately 400 mg / kg of control or RGX-202-01-supplemented diet for 21 days. Figures 3F–3G show subcutaneous tumor growth (F) and Kaplan-Meier survival curves (G) of 1.5 × 10 CT26 cells in BALB / c mice treated with control, RGX-202-formulated diet (800 mg / kg, p = 0.0006), 5-FU (50 mg / kg / week), or a combination of RGX-202 and 5-FU. n = 7–8 per group. Figure 3H shows subcutaneous tumor growth of 2.5 × 10 UN-KPC-961 cells in B6129SF1 / J mice receiving a control diet, an RGX-202-formulated diet (800 mg / kg, p = 0.021), gemcitabine (ip 100 mg / kg, p < 0.0001), or the combination of RGX-202 and gemcitabine (p < 0.0001). n = 10 per group, *p (combination) = 0.04, one-tailed t-test. Figure 3I shows subcutaneous tumor growth of 1 × 10 MC38 cells in C57BL / 6 mice receiving a control diet, an RGX-202-supplemented diet (200 mg / kg), leflunomide (2.5 mg / kg), or the combination of RGX-202 and leflunomide. n = 10 per group. Figure 3J shows subcutaneous tumor growth from CLR1 PDX fragments implanted in athymic nude mice. Treatment with a control diet, a diet supplemented with 800 mg / kg of RGX-202, leflunomide (7.5 mg / kg), or a combination of RGX-202 and leflunomide was initiated when tumors reached approximately 100 mm. n=6 per group.Figure 3K shows subcutaneous tumor growth of CLR28 PDX fragments in athymic nude mice. Treatment with control diet, RGX-202-supplemented diet (800 mg / kg), leflunomide (7.5 mg / kg), a combination of RGX-202 and leflunomide or uridine (1 g / kg), and leflunomide began at approximately 100 mm3. n = 6 per group. Mean + / - SEM is shown, and p values ​​are based on two-tailed t-tests (A-D, F, H-K) or log-rank Mantel-Cox test (G). [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 3-4] See description of Figure 3-1. [Figure 3-5] See description of Figure 3-1. [Figure 4A]Figures 4A-4I show tumor CKB expression and creatine levels as predictive and pharmacodynamic biomarkers of SLC6A8 inhibition. Figures 4A-4I show schematics, graphs, and photomicrographs. Figure 4A shows a schematic illustrating the experimental design. Figures 4B-4D show tumor growth by 5 x 10 HCT-8 (p = 0.0004) (B), 5 x 10 SW480 (p = 0.01) (C), and 2 x 10 Hs746T (D) cells injected subcutaneously into athymic nude mice receiving a control diet or an RGX-202-01-supplemented diet (approximately 800 mg / kg). Photographs show IHC of CKB expression (brown) counterstained with hematoxylin (blue) in control tumors. n = 8-10 per group. Mean + / - SEM is shown, and p values ​​are based on a two-tailed t-test. Figures 4E–4F show linear regression analysis of tumor growth inhibition (TGI) as a function of CKB mRNA (E) and TGI as a function of CKB tumor proportion score (TPS) (F). Each dot represents the mean value of data points from one xenograft model. n = 5–7 tumors / model. Figure 4G shows a nonparametric analysis of tumor growth inhibition compared to controls at day 21 from 43 PDX models, stratified into models with low (0–5%) or >5% CKB TPS. Figures 4H–4I show a correlation analysis of serum (H) or urinary (I) creatine concentrations with RGX-202 blood exposure (AUC0-t). Dashed lines indicate 95% confidence intervals. Patients were sampled on day 15 of cycle 1, with a total of n = 13 patients (H–I). Scale bars (B, C, and D) 100 μm. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 4I] See legend to Figure 4A. [Figure 5] Figures 5A-5F show that RGX-202 inhibits tumor cell proliferation and liver colonization of metastatic CRC and pancreatic cell lines. Graphs and micrographs are shown. Figures 5A-5F show that HCT-15 (A-C) or NCI-H508 (D-F) tumors treated with control or RGX-202-01 (800 mg / kg) (A, D, n = 7-11 per group) were dissected and immunostained for cleaved caspase 3 (CC3) or Ki67 and counterstained with DAPI (B and E). Graphs show quantification of CC3- and Ki67-positive cells in control and RGX-202-treated HCT-15 (C) or NCI-H508 (F) xenografts. N = 3 tumors per group + / - SEM. Scale bars (B and E) 200 μm. [Figure 6] Figures 6A-6B show the antitumor efficacy of RGX-202 and RGX-202-01 in combination with 5-FU and irinotecan. Figures 6A-6B show graphs. Figure 6A shows tumor growth of 1.5 x 10 CT26 cells injected subcutaneously into BALB / c mice. As soon as tumors reached an average of 50 mm, mice were randomly distributed and received either a control diet, an RGX-202-01-supplemented diet (approximately 500 mg / kg, p=0.092), or a combination of 5-FU (ip 30 mg / kg / week) and irinotecan (ip 15 mg / kg / week) (p=0.064), or a combination of RGX-202-01, 5-FU, and irinotecan (p=0.0006). n=7-8 per group. Means + / - SEM are shown. All p-values ​​are based on two-tailed t-tests. *p=0.02. Figure 6B shows the weight curves of BALB / c mice bearing CT26 tumors. Mice were treated with RGX-202 administered by oral gavage (500 mg / kg) or formulated in the diet (500 mg / kg), and body weights were measured twice weekly. n = 7–11 per cohort. [Figure 7]Graphs showing xenograft tumor studies are shown. Tumor growth of 4 x 106 NCI-N87 (p = 0.027), 2 x 106 HepG2, 5 x 106 Colo205 (p = 0.042), and 1 x 106 Lvm3b (p < 0.0001) cells injected subcutaneously into athymic nude mice. Treatment with control or RGX-202-01-supplemented diets (approximately 800 mg / kg / day) began as soon as tumors reached 10-60 mm3. n = 8-10 per group. All p values ​​are based on two-tailed t-tests. ns = not significant. Mean ± SEM is shown. [Figure 8A]Figures 8A-8K show linear regression analysis demonstrating the antitumor efficacy of RGX-202-01 in CKB-high-expressing cell lines. Graphs and micrographs are shown. Figures 8A-8K show linear regression analysis of the percentage of tumor growth inhibition (TGI) as a function of SLC6A8 mRNA (A), TGI as a function of CKB H-score (B), and CKB mRNA levels as a function of CKB H-score (C) in tumors from the xenograft models tested. mRNA levels were measured by qPCR. Each dot represents the mean value of data points from one xenograft model. n = 4-6 tumors / xenograft model. Figure 8D shows qPCR analysis of CKB, CKM, CKMT1, and CKMT2 in the xenograft models tested. Relative expression is shown as ΔCt values, n = 7-11 models, 4-5 tumors / model. Mean + / - SEM is shown. Figures 8E-8G show linear regression analysis of TGI as a function of CKM mRNA (E), CKMT1 mRNA (F), and CKMT2 (G) tumor mRNA levels. mRNA levels were measured by qPCR. Each dot represents the mean value of data points from one xenograft model, n = 4-5 tumors / xenograft model. Figures 8H-8I show linear regression analysis of TGI and fold change in cleaved caspase-3 (H) or Ki67 (I) positive tumor area assessed by immunohistochemistry (IHC) in RGX-202-01-treated tumors relative to control tumors, n = 3 tumors / model. Each dot represents the mean value of data points from one xenograft model. Figures 8J-8K show CKB expression assessed by IHC in 23 metastatic CRC specimens. Quantification of tumor proportion score (TPS) is shown in (J), and representative images are shown in (K). [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 8G]See legend to Figure 8A. [Figure 8H] See legend to Figure 8A. [Figure 8I] See legend to Figure 8A. [Figure 8J] See legend to Figure 8A. [Figure 8K] See legend to Figure 8A. [Figure 9-1] Provides a table showing the PDX models utilized in PDX studies. Summary of PDX tumor growth data, KRAS and BRAF status. TGI(21) = Tumor growth inhibition at day 21 = ((C21-C0) / C21)-((T21-T0) / T0). Change in tumor size relative to control. [Figure 9-2] See description of Figure 9-1. [Figure 10] A table showing information on cell lines is provided. Antitumor efficacy and mutational status of cell lines used in xenograft studies. TGI = tumor growth inhibition. [Figure 11] Figure 1 shows a timeline showing the treatment duration and clinical outcomes for individual patients, with the y-axis indicating KRAS status and the x-axis indicating treatment duration (weeks). [Figure 12] Timelines showing treatment duration and best response in all evaluable patients with RGX-202-01 monotherapy and combination dose escalation are shown. [Figure 13]

[0023] Figure 1 shows a graph demonstrating antitumor activity in RGX-202-01 treated PDX models at a dose of 300 mg / kg. Tumor response was assessed versus control on day 21. CKB tumor proportion score (TPS) was determined by immunohistochemistry. [Figure 14] 1 shows a graph depicting RGX-202-mediated changes in creatine metabolism in patients with progressive disease (PD) versus partial responders / stable disease (PR / SD). [Figure 15] 1 shows a graph depicting changes in subject levels in creatine metabolism. [Figure 16] 1 shows the chemical structures of exemplary creatine transporter inhibitors. [Figure 17A]Figures 17A and 17B provide plots showing the tumor growth inhibition achieved by administration of RGX-202 in combination with a low-dose KRAS inhibitor (i.e., MRTX849 (adagrasib)). Figure 17A provides a plot of tumor volume over time. Figure 17B provides a plot of final tumor volume after treatment. In Figure 17A, the lines represent, from top to bottom, "control," "RGX-202," "MRTX849," and "RGX-202+MRTX849." In Figures 17A and 17B, *=p<0.05. [Figure 17B] See legend to Figure 17A. [Figure 18] Figure 1 provides plots showing tumor growth inhibition by RGX-202 in NCI-H460 KRASQ61 xenografts in mice. Tumor growth was measured as change in tumor volume or change in mouse body weight. 4 x 106 NCI-H460 cells were injected into 6-8 week old female nude mice. Treatment with RGX-202 formulated diet was initiated when tumors reached approximately 200 mm3. [Figure 19] Figure 1 provides plots showing tumor growth inhibition by RGX-202 in NCI-H358 KRASG12C xenografts in mice. Tumor growth was measured as the change in tumor volume or the change in mouse body weight. 5 x 10 NCI-H358 cells were injected into 6-8 week old female nude mice. Treatment with RGX-202 formulated diet was initiated when tumors reached approximately 80 mm. DETAILED DESCRIPTION OF THE INVENTION

[0110] Detailed Description of the Invention As described below, the present invention features compositions and methods useful for treating cancer (e.g., gastrointestinal cancer, colorectal cancer) featuring a creatine transporter inhibitor (e.g., β-guanidinopropionic acid) in combination with a KRAS inhibitor or another chemotherapeutic agent (e.g., leucovorin (folinic acid - FOL), 5-fluorouracil (F), and irinotecan hydrochloride (IRI), oxaliplatin, and / or bevacizumab).

[0111] The present invention is based, at least in part, on the discovery that the exemplary creatine transporter inhibitor β-guanidinopropionic acid (β-GPA), a creatine mimetic that competitively inhibits the creatine transporter SLC6A8, is useful, alone or in combination with KRAS inhibitors (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236), vascular endothelial growth factor inhibitors (e.g., bevacizumab), and / or FOLFIRI, to reduce cancer cell proliferation and / or induce cancer cell death, particularly in cells that contain KRAS mutations or have increased levels of creatine or phospho-creatine.

[0112] Colorectal cancer (CRC) is a leading cause of cancer death. Creatine metabolism has previously been shown to critically regulate colon cancer progression. As reported herein, the oral small molecule SLC6A8 transporter inhibitor, β-GPA, robustly inhibits creatine transport in vitro and in vivo, reducing intracellular phosphocreatine and ATP levels and inducing tumor cell apoptosis in CRC. β-GPA suppressed tumor growth across KRAS wild-type and KRAS mutant xenografts, as well as syngeneic and patient-derived xenografts of colorectal cancer. Antitumor efficacy correlated with tumor expression of creatine kinase B. Combining RGX-202 with 5-fluorouracil or the DHODH inhibitor leflunomide caused regression of multiple colorectal xenograft and PDX tumors of different mutational backgrounds. β-GPA also disrupted creatine metabolism in patients with metastatic CRC enrolled in a phase 1 trial, mirroring the pharmacodynamic effects on creatine metabolism observed in mice. This is the first demonstration of preclinical and human pharmacodynamic activity for targeting creatine metabolism in oncology and identifies an important therapeutic target for CRC.

[0113] Thus, the compositions provided herein feature orally bioavailable small molecule creatine mimetics (β-GPA, RGX-202, RGX-202-01) that significantly inhibited the SLC6A8 transporter, suppressed colorectal cancer tumor growth, and induced tumor cell apoptosis in vivo in syngeneic, xenograft, and PDX models. β-GPA also strongly suppressed liver metastasis formation. Importantly, this therapy was effective against both KRAS wild-type tumors and tumors harboring various KRAS mutations, including the KRAS G12D allele, which is currently not druggable by clinical-stage KRAS inhibitors alone. Furthermore, the antitumor efficacy of β-GPA correlated with increased CKB expression in tumors.

[0114] Notably, the working examples provided herein demonstrate that β-GPA showed greater activity in combination with multiple standard treatment regimens, including 5-FU, gemcitabine, FOLFIRI, and bevacizumab, compared to single-agent efficacy. Thus, the anti-cancer therapies and agents provided herein are useful in combination with RGX-202 to reduce cancer cell proliferation, reduce tumor size, and improve clinical outcomes in patients with RAS-mutated tumors (e.g., KRAS, HRAS, NRAS).

[0115] colorectal cancer During the progression of colorectal cancer to liver metastasis, cancer cells upregulate creatine kinase brain type (CKB) and secrete it into the extracellular space (Loo et al., Cell 160, 393-406 (2015)). CKB uses ATP to phosphorylate the metabolite creatine, thereby generating phosphocreatine. The γ-phosphate of phosphocreatine has approximately 50% higher free energy than the γ-phosphate of ATP. Therefore, phosphocreatine functions as a rapidly mobilizable, high-energy phosphate store that can generate ATP in an oxygen-free reaction. Due to its high energy content, phosphocreatine is stored at high levels in metabolically active tissues such as muscle, brain, and kidney, allowing for the tight maintenance of ATP levels in the organism, which are important for numerous metabolic and homeostatic processes. Although extracellular ATP levels are normally low, the death of cancer cells and stromal cells substantially increases extracellular ATP concentrations in the tumor microenvironment, which have been observed to exceed 700 μM. Thus, excess ATP above circulating creatine levels in the 9-90 μM range favors phosphocreatine production in the tumor microenvironment, a reaction mediated by tumor-secreted CKB. Phosphocreatine transport by SLC6A8 enhanced tumor ATP levels and thus promoted cell survival under hypoxia (Loo et al., supra). Importantly, extracellular phosphocreatine supplementation rescued the metastatic defect and in vitro survival phenotype of CKB-depleted cells (Loo et al., supra). Gastrointestinal tumors, such as CRC and pancreatic cancer, are highly hypoxic, as are the metastases formed by these cancers. This metabolic axis provides a mechanism to support tumor growth in a hypoxic environment. Importantly, expression levels of CKB and SLC6A8 are associated with increased colorectal cancer metastasis in patients (Loo et al., supra).

[0116] The SLC6A8 transporter has been identified as a therapeutic target in colorectal cancer. An orally bioavailable small-molecule creatine mimetic (RGX-202, also known as β-GPA) significantly inhibited SLC6A8, suppressed colorectal cancer tumor growth in syngeneic, xenograft, and PDX models, and induced tumor cell apoptosis in vivo. RGX-202 also potently suppressed liver metastasis formation. Importantly, this therapy was shown to be effective in both KRAS wild-type tumors and tumors harboring various KRAS mutations, including the KRAS G12D allele, which is currently undruggable with clinical-stage KRAS inhibitors. RGX-202 antitumor efficacy correlated with increased CKB expression in tumors. RGX-202 demonstrated enhanced activity in combination with multiple standard-of-care regimens, including 5-FU and gemcitabine, compared with its efficacy as a single agent. RGX-202 was also shown to synergize with the dihydroorotate dehydrogenase (DHODH) enzyme inhibitor leflunomide, an oral compound previously shown to inhibit colorectal cancer cell growth under hypoxia by inhibiting nucleotide biosynthesis. Metabolic profiling studies revealed that RGX-202 suppressed intracellular phosphocreatine, creatine, and ATP levels. Finally, a drug-exposure-dependent increase in creatine was observed in the blood and urine of mice and in patients treated with oral RGX-202 therapy, confirming creatine transporter inhibition in patients and supporting further clinical testing of RGX-202 in late-stage clinical trials.

[0117] RGX-202 Using cell-based, ex vivo tissue-based, and in vivo assays, RGX-202 has been shown to inhibit the creatine transporter SLC6a8 with a reported K of 8.8-120 μM (Dai 1999; Wyss 2000; Pereal 2002). RGX-202-mediated inhibition of the creatine transporter SLC6a8 caused a decrease in intracellular creatine levels in colon cancer cells in vitro and in vivo within tumors. RGX-202 treatment reduced cell viability in vitro (15-60%), induced cell death in colon cancer cells in vitro (approximately 7-fold), and induced cell death in colon cancer cells in vivo within tumors (5-15-fold). In embodiments, the methods provided herein comprise administering RGX-202 to a subject to treat a RAS-mutated cancer (e.g., a KRAS, HRAS, or NRAS-mutated cancer).

[0118] In syngeneic (immune-competent) and human xenograft (immune-deficient) mouse tumor models of colon, gastric, and pancreatic cancer, RGX-202 inhibited primary tumor growth by 30–65%. The tumors tested included both KRAS wild-type and KRAS mutant colon cancer cells and KRAS mutant / p53 mutant pancreatic cancer cells. Long-term treatment of human xenograft and syngeneic tumors with RGX-202 demonstrated 38–90% tumor regression and a reduced risk of death (hazard ratio 0.1–0.3). In vivo tumor growth studies of 11 gastrointestinal xenografts demonstrated a positive correlation between the antitumor efficacy of RGX-202 and CKB protein levels in these tumors. Tumors with high levels of CKB responded significantly better to RGX-202 treatment than tumors with low or undetectable levels of CKB. This suggests that response may be related to CKB expression levels, consistent with the upregulation of the CKB / SLC6a8 pathway observed in metastatic colon cancer, which is necessary for tumor progression (Loo 2015). Additionally, additive effects have been observed when RGX-202 is administered with 5-FU in vitro and in vivo, and in combination with fluorouracil and irinotecan in vivo.

[0119] Furthermore, RGX-202 demonstrated additive tumor-suppressing efficacy when administered in combination with gemcitabine in vivo. In vitro, treatment of HCT-116 colon cancer cells with RGX202 reduced cell viability by approximately 15-35%, whereas treatment with 5-FU showed only a slight, non-significant decrease in cell viability. However, combined treatment with RGX202 and 5-FU further reduced cell viability by approximately 60-90% compared to control treatment, resulting in a significant synergistic effect in reducing cell viability compared to 5-FU alone. In vivo, combined treatment with RGX-202 and 5-FU inhibited CT-26 tumor growth by 99% (50% of mice exhibited complete tumor regression), significantly more effective than treatment with 5-FU or RGX-202 alone. Additive antitumor activity has also been observed in pancreatic tumors in combination with gemcitabine treatment (approximately 80% tumor growth inhibition).

[0120] The antitumor efficacy of RGX-202 has been noted with various dosing paradigms, including oral administration (by gavage once daily (QD) or supplemented in the diet) or subcutaneous administration (using an osmotic pump).

[0121] In tumor growth studies in rodents using RGX-202, effective doses ranged from 50 to 500 mg / kg / day administered orally. At the effective dose of 200 mg / kg, systemic exposure to RGX-202 (based on AUC 0-24 h estimates) was similar in the diet-administered group (144,810 ng-h / mL) compared with the gavage-administered group (149,415 ng-h / mL) based on pharmacokinetic (PK) sampling data.

[0122] In animal models, RGX-202 inhibits primary tumor growth in various gastrointestinal cancers, including colorectal, pancreatic, and gastric cancers, across different genetic backgrounds. As a single agent, RGX-202 inhibited the growth of LvM3b, HCT116 KRAS wild-type, Colo205, and KRAS mutant CT26 and KRAS wild-type MC38 mouse colon cancers, as well as human KRAS mutant cells in PANC1 human pancreatic cancer cells. In addition, RGX-202 in combination with gemcitabine inhibited tumor growth in KPC mouse pancreatic cancer, and RGX-202 in combination with 5-fluorouracil and both 5-fluorouracil and irinotecan inhibited tumor growth in CT26 mouse colon cancer. RGX-202's tumor growth inhibitory activity essentially depends on its ability to interfere with creatine metabolism. Importantly, other malignancies, including those of the prostate, lung, ovary, cervix, and head and neck, have been noted for SLC6a8 and CKB gene amplification, potentially rendering them sensitive to RGX-202 inhibition of this metabolic pathway. Target engagement and clinical relevance are demonstrated by the effect of SLC6a8 inhibition by RGX-202-01 administration on metabolite levels such as creatine, and the level of CKB expression by immunohistochemistry in tumor tissue. The relevance of serum creatinine and creatine levels as pharmacodynamic markers of creatine transport inhibition can be assessed by assessing the dose-response of changes in each of these markers following RGX-202-01 administration and combination therapy, such as RGX-202 plus a KRAS inhibitor from Table 1.

[0123] Pharmacokinetics of RGX-202 The permeability coefficient of RGX-202 measured in human colon adenocarcinoma (Caco-2) cell monolayers was low. RGX-202 is not a substrate for the efflux transporters P-gp or BCRP in MDCKII cells or vesicles expressing these transporters. However, there is evidence of active uptake of RGX-202 in the A to B direction in MDCKII cells, suggesting that RGX-202 is a substrate for an uptake transporter endogenously expressed in the MDCKII cell line. Although the endogenous transporter was not elucidated in that study, RGX-202 is a substrate for the amino acid transporter hPat1 (SLC36a1). This transporter is involved in the cellular transport of RGX-202 (i.e., endogenous β-GPA) in humans and is expressed primarily in intestinal epithelial cells, brain, colon, liver, and lung (Metzner 2009).

[0124] RGX-202 is moderately bound to plasma proteins in all species. The overall mean percentage of bound RGX-202 ranged from 31.5% to 39.6%. In pharmacology studies in mice, there was substantial distribution of RGX-202 to the brain after 17 days of dosing at an effective dose (800 mg / kg administered in the diet).

[0125] Metabolism does not appear to be the primary clearance mechanism for this compound. RGX-202 does not appear to interact with cytochrome P450 enzymes and is not metabolized by liver hepatocytes from humans, monkeys, dogs, rats, and mice. In addition, RGX-202 is not a substrate for any of the assayed human CYP enzymes (rCYP1A2, rCYP2A6, rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C19, rCYP2D6, rCYP2E1, rCYP2J2, rCYP3A4, and rCYP4F2).

[0126] The IC of RGX-202 for inhibition of CYP isoforms 2E1, 2A6, 1A2, 2B6, 2C19, 2C8, 2C9, 2D6, 3A4 / 5 (midazolam and testosterone) was 0.01, with no apparent induction for 1A2, 2B6, or 3A. 50The β-glucan-1-phosphate dehydrogenase (β-glucan-1) ...

[0127] Because RGX-202 does not appear to be metabolized, excretion of the unchanged parent is likely the primary clearance mechanism. In DRF studies in rats and dogs, significant amounts of RGX-202 were found in the urine, indicating that renal excretion is the likely clearance mechanism for this compound. The potential for biliary excretion of RGX-202 has not been evaluated.

[0128] FOLFIRI FOLFIRI is a chemotherapy regimen consisting of irinotecan, leucovorin, and 5-fluorouracil. Irinotecan is a topoisomerase inhibitor that prevents DNA from unwinding and replicating. 5-FU is a pyrimidine analog and antimetabolite that incorporates into DNA and stops its synthesis. Fluorouracil injection, a nucleoside metabolic inhibitor for intravenous administration.

[0129] Leucovorin (folinic acid) is a vitamin B derivative that increases the cytotoxicity of 5-FU. Leucovorin is one of several active, chemically reduced derivatives of folic acid.

[0130] Recommended dose modifications for FOLFIRI-related toxicities are listed below TIFF2024528705000003.tif38149

[0131] Bevacizumab Bevacizumab is a vascular endothelial growth factor inhibitor. It is a recombinant humanized monoclonal IgG1 antibody containing human framework regions and mouse complementarity-determining regions. It has a molecular weight of approximately 149 kDa. It is produced in a mammalian cell (Chinese hamster ovary) expression system. Bevacizumab binds to VEGF and prevents its interaction with its receptors (Flt-1 and KDR) on the surface of endothelial cells. The interaction of VEGF with its receptor leads to endothelial cell proliferation and new blood vessel formation in an in vitro model of angiogenesis. Administration of bevacizumab to a xenograft model of colon cancer in nude (athymic) mice resulted in a reduction in microvascular growth and inhibition of metastatic disease progression. Bevacizumab is intended for intravenous use. Avastin® contains bevacizumab at a concentration of 25 mg / mL in either 100 mg / 4 mL or 400 mg / 16 mL single-dose vials. Bevacizumab is administered intravenously at 5 mg / kg every 2 weeks on a 14-day schedule prior to FOLFIRI.

[0132] Cancer and Metabolic Rewiring Given the surprising results obtained with therapeutic combinations featuring an SLC6A8 transporter inhibitor (e.g., β-guanidinopropionic acid, RGX-202) and a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236), and / or another chemotherapeutic agent (e.g., FOLFIRI, FOLFOX, bevacizumab, 5-fluorouracil, leflunomide, irinotecan, and / or oxaliplatin), one skilled in the art would predict that other cancers with such properties (e.g., expressing the creatine transporter SLC6A8, elevated levels of creatine, and / or expressing KRAS mutations) could be treated using a similar approach.

[0133] Examples of cancer types include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, bile duct cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), lymphoma including Hodgkin's and non-Hodgkin's lymphoma, melanoma, myeloma, neuroblastoma, oral 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 cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, These include, but are not limited to, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulvar cancer, and other carcinomas and sarcomas, as well as B-cell lymphomas (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 blood vessel growth associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs syndrome.

[0134] Cancers evolve multiple mechanisms to maintain proliferative growth and survival. One such adaptive mechanism is metabolic change, commonly referred to as "metabolic rewiring." By altering the flow of metabolites in various metabolic pathways, cancer cells enhance the biosynthesis of anabolic building blocks necessary for growth, such as nucleotides, amino acids, and lipids. However, certain metabolites may become limited during cancer progression, requiring their extracellular transport via metabolic transporters. Metabolic signaling pathways upregulated in cancer are further discussed in, for example, N.S. Chandel, "Navigating metabolism," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2015, and T. Suzuki, et al., "Mutant KRAS drives metabolic reprogramming and autophagic flux in premalignant pancreatic cells," Cancer Gene Ther, (2021), the teachings of each of which are incorporated herein by reference in their entirety.

[0135] Several factors may increase a person's risk of developing cancer, including, but not limited to, inherited genetic mutations, acquired genetic mutations, family history of cancer, obesity, a diet high in red and processed meat, smoking, moderate to heavy alcohol use, a history of colorectal polyps (adenomas), a history of inflammatory bowel disease, a history of having Lynch syndrome (hereditary nonpolyposis colorectal cancer, or HNPCC), familial adenomatous polyposis (FAP), and / or type 2 diabetes. Additional risk factors for colorectal cancer are discussed, for example, in Johnson CM et al., "Meta-analyses of colorectal cancer risk factors." Cancer Causes Control. 2013 Jun;24(6):1207-22, the teachings of which are incorporated by reference in their entirety.

[0136] The diagnosis of cancer can be determined by the symptoms exhibited by the subject and a variety of different laboratory and medical tests. The diagnosis of cancer is generally confirmed by biopsy and histological analysis of the tumor to evaluate cell proliferation and the expression of tumor markers. A skilled physician can determine which tests are appropriate based on the symptoms exhibited by the subject and the affected organ.

[0137] The prognosis of colorectal cancer is related to the extent to which the tumor penetrates the bowel wall and the presence or absence of lymph node involvement. Therefore, early detection and treatment are particularly important. Currently, diagnosis is aided by the use of screening assays such as fecal occult blood, sigmoidoscopy, colonoscopy, and double-contrast barium enema. Treatment regimens are determined by the type and stage of cancer and include surgery, radiation therapy, and / or chemotherapy.

[0138] Early-stage cancer is primarily treated by surgical resection or removal. Colon cancer recurrence after surgery (the most common form of treatment) is a major problem and is often the ultimate cause of death. Larger tumors and tumors that have spread to the lymph nodes are treated with various chemotherapy agents administered in an "adjuvant" setting after surgery to reduce the risk of metastatic recurrence. However, most metastatic patients have a very poor prognosis. For example, patients previously diagnosed with colorectal cancer that becomes metastatic ultimately succumb to their disease, with a 5-year survival rate of only 14%.

[0139] In the most lethal cases, cancer cells can detach from the primary tumor, infiltrate lymphatic and blood vessels, circulate via the bloodstream, and grow at distant foci (metastases) in normal tissues elsewhere in the body. Metastasis can be local or distant. It is a continuous process that relies on tumor cells to detach from the primary tumor, travel through the bloodstream, and arrest at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within tumor cells regulate this behavior, and interactions between tumor cells and host cells at distant sites are also significant.

[0140] Metastases are most frequently detected by diagnostic methods known in the art, such as magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest x-rays, and bone scans, in addition to monitoring for specific symptoms.

[0141] The compositions and methods provided herein overcome the limitations of previous cancer therapies by targeting creatine transport signaling and improving metabolic rewiring induced by cancer cells. Additionally, the compositions provided herein are useful in treating subjects with KRAS-mutated cancers.

[0142] RAS mutations and RAS inhibition in cancer Three canonical members of the Ras gene family (H-ras, N-ras, and K-ras) were identified over a quarter century ago due to their frequent oncogenic activation in human tumors. They are the founding members of the broader Ras superfamily, which includes over 150 small GTPases, divided into at least five distinct subfamilies (Ras, Rho / Rac, Rab, Arf, and Ran) based on primary sequence relationships. In particular, the Ras subfamily encompasses the H-ras, N-ras, and K-ras genes, along with the closely related R-Ras / TC21, Ral, and Rap loci.

[0143] All Ras superfamily proteins share a highly similar molecular structure and a common ability to bind and hydrolyze guanine nucleotides. Ras proteins continuously cycle between active (GTP-bound) and inactive (GDP-bound) conformational states, primarily depending on structural changes occurring in two motile switch I and switch II regions, which are also involved in the functional interaction of these proteins with negative (GAP) and positive (GEF) cellular regulators. The binary behavior of these proteins allows them to function as molecular switches in a wide range of signaling processes related to the transmission of extracellular signals to the interior of the cell. Oncogenic mutations at positions 12, 13, or 61 of the H-ras, N-ras, and K-ras genes are among the most common genetic lesions in mammalian tumors. These mutations result in a significant impairment of the overall GTPase activity of carrier Ras proteins, even in the absence of extracellular stimuli, locking them in a constitutively activated state that signals downstream effectors.

[0144] Expression of the H-ras, N-ras, and K-ras genes is nearly ubiquitous and broadly conserved across species, although specific differences in expression levels exist depending on the tissue and developmental stage under study. In particular, these three loci are known to encode four distinct protein isoforms (H-Ras, N-Ras, K-Ras4A, and K-Ras4B), the latter two resulting from alternative splicing of exon 4 of the K-ras locus. These four Ras isoform proteins are highly homologous in terms of their primary amino acid sequence (approximately 80%), with the differences between them centered in the so-called hypervariable regions (HVRs) in their C-terminal domains. These mammalian ras genes are expressed in all cell lineages and organs, but there are differences in expression during prenatal and postnatal development, with specific adult tissues preferentially expressing one or more members of the family.

[0145] Mammalian Ras subfamily proteins (H-Ras, N-Ras, K-Ras4A, and K-Ras4B) are highly conserved across different species and play important functional roles in numerous cellular processes, including proliferation, differentiation, and cell death. The large number of Ras activators and effectors identified in mammalian cells places Ras proteins at the crossroads of a staggering number of cell signaling networks. Such a central role of Ras gene products in normal cell signaling is also consistent with the high frequency of oncogenic activation of Ras genes in human cancers. The importance of Ras signaling in tumor initiation and maintenance is underscored not only by the prevalence of Ras mutations but also by the deregulation of many of its activator or effector pathways, thus affecting Ras pathway activity. Indeed, studies of the contribution of Ras signaling to tumorigenesis have significantly improved our current understanding of the molecular basis for the pathogenesis of many human cancers.

[0146] The most frequent mechanism of oncogenic activation involves point mutations that affect the interaction of Ras with guanine nucleotides. Mutations detected in naturally occurring ras oncogenes affect codons 12, 13, 59, and 61. These mutations result in inhibition of GTP hydrolysis by reducing GTPase activity or (in the case of codon 59) by modulating the rate of guanine nucleotide exchange.

[0147] Oncogenic ras mutations are found in a wide variety of human cancers, although their incidence varies considerably depending on the tumor type. Qualitatively, H-ras mutations have been reported in melanoma, bladder, thyroid, and breast cancer, while K-ras mutations have been found in bladder, ovarian, thyroid, lung, colon, and rectum, and pancreatic cancer, neuroblastoma, rhabdomyosarcoma, and acute nonlymphocytic leukemia. Finally, N-ras mutations have also been shown in melanoma, thyroid cancer, teratocarcinoma, fibrosarcoma, neuroblastoma, rhabdomyosarcoma, Burkitt's lymphoma, acute promyelocytic leukemia, T-cell leukemia, and chronic myeloid leukemia. Quantitative analysis demonstrates that some ras oncogenes are preferentially associated with specific forms of human tumors. Thus, K-ras activating missense mutations are frequently detected in non-small cell lung cancer (15%-20%), colon adenoma (40%), and pancreatic adenocarcinoma (95%), making it the single most common mutationally activated human oncoprotein. Similarly, N-ras mutations are frequently present in hematologic malignancies such as acute myeloblastic leukemia (20%-30%). In contrast, other tumor types do not show any significant preference for specific ras oncogene isoforms. For example, more than half of malignant thyroid tumors (poorly differentiated or anaplastic) harbor mutations in K-ras, H-ras, or N-ras. Furthermore, in some thyroid adenomas and carcinomas, mutations in all three ras isoforms can occur within the same tumor, suggesting that each isoform may contribute to different aspects of tumor growth. Simultaneous mutations in K-ras and N-ras have also been detected in multiple myeloma. Finally, although ras mutations are rare in breast cancer, point mutations in H-ras or K-ras have been detected in primary tumors and in some breast cancer-derived cell lines.

[0148] Kirsten rat sarcoma, also known as KRAS, is a proto-oncogene that encodes a small 21-kD guanosine triphosphate (GTP) / guanosine diphosphate (GDP) protein that binds to proteins involved in regulating cellular responses to many extracellular stimuli. The KRAS gene is located on 12p12.1 and spans approximately 38 kb. KRAS normally functions in signaling cascades initiated by the binding of epidermal growth factor receptor (EGFR), hepatocyte growth factor, and insulin-like growth factor receptors to their receptors. When activated wild-type KRAS binds to GTP, this results in a conformational change that allows the protein to bind and activate over 20 known downstream effectors, including Raf, Braf, mTOR, MEK1 and 2, ERK, AKT, and PIK3CA. These downstream effectors exert many different effects, including apoptosis inhibition, cell growth promotion, cell transformation, angiogenesis, migration, and differentiation. KRAS functions as a molecular binary switch that alternates between a GTP-bound "active" state and a GDP-bound "off" state, each state having a specific molecular conformation.

[0149] KRAS has intrinsic GTPase activity, but the hydrolysis rate constant is too low to be physiologically relevant. However, specific "GTPase-activating proteins" can increase hydrolysis by approximately 100,000-fold. Guanine nucleotide exchange factor proteins then promote GTP binding by reducing the affinity of KRAS for bound GDP and catalyzing its replacement by GTP. Activating KRAS mutations are primarily point mutations affecting KRAS amino acid residues 12, 13, and 61, all of which reduce intrinsic KRAS- and GTP-activating protein-promoted GTP hydrolysis, resulting in constitutive KRAS activation.

[0150] KRAS mutations in cancer are associated with decreased survival and increased tumor aggressiveness. The prognostic significance of KRAS mutations is further discussed, for example, in Dinu D, et al. Prognostic significance of KRAS gene mutations in colorectal cancer--preliminary study. J Med Life. 2014;7(4):581-587, the teachings of which are incorporated herein by reference in their entirety.

[0151] Colorectal cancer frequently arises from preneoplastic lesions through the activation of oncogenes (KRAS and BRAF) and inactivation of tumor suppressor genes (APC, p16, p53, and DCC) and mismatch repair genes such as MLH1 and MSH2, and to a lesser extent PMS2 and hMSH6. Approximately 30% to 40% of colon cancers harbor KRAS mutations.

[0152] KRAS gene mutations are also common in pancreatic cancer, lung adenocarcinoma, colorectal cancer, gallbladder cancer, thyroid cancer, and bile duct cancer, and are associated with drug resistance to standard treatment chemotherapy regimens. Non-limiting examples of KRAS mutations that have been identified in human subjects include G12C, G12D, G12V, G12S, G13C, G13D, Q61H, and Q61L.Additional examples of KRAS mutation status in cancer include, for example, Aza A. Lyanova et al., “The KRAS mutation status and resistance to cetuximab in patients with squamous cell carcinoma of oral cavity.” Journal of Clinical Oncology (2020) 38:15; Erminia Massarelli et al., “KRAS Mutation Is an Important Predictor of Resistance to Therapy with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer.” Clin Cancer Res May 15 2007(13)(10)2890-2896; Westcott PM and To MD, “The genetics and biology of KRAS in lung cancer.” Chin J Cancer. 2013;32(2):63-70; Buscail, L., et al., “Role of oncogenic KRAS in the diagnosis, prognosis, and treatment of pancreatic cancer.” Nat Rev Gastroenterol Hepatol 17, 153-168 (2020), and Polom K, et al., “KRAS Mutation in Gastric Cancer and Prognostication Associated with Microsatellite Instability Status.” Pathol Oncol Res. 2019 Jan;25(1):333-340, the teachings of each of which are incorporated herein by reference in their entirety.

[0153] The methods provided herein for identifying KRAS mutant cancers are known in the art. For example, histological analysis, sequencing, high-resolution melting analysis (HRM), single-strand conformation polymorphism analysis (SSCP), denaturing gradient gel electrophoresis (DGGE), denaturing high-performance liquid chromatography (DHPLC), array / strip analysis, and allele-specific PCR can be used. Sanger sequencing and pyrosequencing of PCR-amplified DNA are commonly used in KRAS mutation analysis. Sanger sequencing of PCR-amplified genomic DNA is considered the gold standard for identifying KRAS mutant cancers. PCR primers are used to amplify genomic DNA immediately adjacent to known KRAS point mutations, such as those listed above.

[0154] Due to the evasive nature of KRAS-mutated cancers and the low survival rates of subjects with KRAS-mutated tumors, several chemotherapeutic agents have been developed to specifically target the most common KRAS-mutated tumors. Small molecules that covalently bind to and inhibit the KRAS G12C oncogenic driver variant, present in approximately 4% of colorectal tumors, have induced clinical responses in patients with this mutant allele. Additionally, inhibitors targeting downstream KRAS signaling in RAS-mutated tumors have also been developed. Non-limiting examples of KRAS inhibitors are listed in Table 1 below.

[0155] Table 1. Exemplary KRAS inhibitors TIFF2024528705000004.tif73170TIFF2024528705000005.tif254170

[0156] Additional KRAS inhibitors, their analogs, and derivatives are described, for example, in U.S. Pat. Nos. 8,546,421 B1, 10,640,504 B2, 10,125,134 B2, 10,125,134 B2, U.S. Patent Application Publication No. 2020 / 0360374 A1, and WO2021 / 041671 A1, the teachings of each of which are incorporated by reference herein in their entirety.

[0157] The KRAS inhibitors provided herein are administered to a subject in combination with an agent that inhibits creatine transport or creatine signaling in cancer cells (e.g., RGX-202, RGX-202-01). Certain combinations of KRAS inhibitors and creatine transport inhibitors can provide additive therapeutic effects in subjects with malignant cancer.

[0158] Creatine signaling in cancer Under normal physiological conditions, creatine is synthesized in the liver and kidneys and transported throughout the body via active transport systems to tissues with high energy demands. Creatine is used by the body to rapidly resynthesize ATP from ADP through the anaerobic conversion of phosphorylated creatine (phosphocreatine) to creatine in a reversible reaction by the enzyme creatine kinase.

[0159] Creatine has the following chemical structure: TIFF2024528705000006.tif26128

[0160] During times of low energy demand, excess ATP can be utilized to convert creatine to phosphocreatine. Due to its high energy content, phosphocreatine is stored at high levels in metabolically active tissues such as muscle, brain, and kidney, allowing the organism to closely maintain ATP levels, which are important for a vast number of metabolic and homeostatic processes.

[0161] Cancers such as colorectal and pancreatic cancers are highly hypoxic, as are the metastases they form. During cancer growth and progression to liver metastasis, cancer cells upregulate creatine kinase brain type (CKB) and secrete it into the extracellular space. CKB uses ATP to phosphorylate the metabolite creatine, thereby producing phosphocreatine. Increased expression of creatine kinase leads to the production of excess phosphocreatine, which can be used as an energy reserve to generate the ATP needed to withstand hepatic hypoxia. Specifically, the gamma phosphate of phosphocreatine has approximately 50% more free energy than the gamma phosphate of ATP. Therefore, phosphocreatine functions as a rapidly mobilizable, high-energy phosphate store that can generate ATP in an oxygen-free reaction.

[0162] Although extracellular ATP levels are normally low, the death of cancer cells and stromal cells substantially increases extracellular ATP levels in the tumor microenvironment. An excess of ATP above circulating creatine levels, which range from approximately 9 to 90 μM, favors phosphocreatine production in the tumor microenvironment, a reaction mediated by tumor-secreted CKB.

[0163] Phosphocreatine transport by creatine transporters (e.g., SLC6A8) on the cell membrane of cancer cells further enhances tumor ATP levels under hypoxic conditions, promoting cancer cell survival. Importantly, the working examples provided herein demonstrate that increased expression levels of CKB and SLC6A8 are associated with increased colorectal cancer metastasis and progressive disease in human patients. The working examples also demonstrate that targeting the creatine transporter with β-GPA and administering one or more additional chemotherapeutic agents (e.g., atovaquone, brequinar sodium, leflunomide, teriflunomide, BAY-2402234, AG-636, leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin) can be highly beneficial in treating subjects with wild-type and mutant KRAS, HRAS, or NRAS tumors or previously drug-resistant tumors. Thus, the compositions provided herein can be used to improve metabolic rewiring induced by cancer cells and reduce cell proliferation.

[0164] SLC6A8 As discussed above, inhibition of the phosphocreatine system by inhibiting creatine uptake and / or creatine kinase in cancer cells is a useful target for the treatment of cancer and / or metastasis. One example of a creatine transporter that can be targeted to reduce cancer cell proliferation and / or increase cancer cell apoptosis is solute carrier family 6 member 8 polypeptide, or SLC6A8.

[0165] SLC6A8 is a plasma membrane protein that functions to transport creatine in and out of cells. Defects in this gene can cause X-linked creatine deficiency syndrome. Under normal physiological conditions, SLC6A8 is highly expressed in the small intestine, heart, kidney, brain, and colon compared to other tissue types. Interestingly, SLC6A8 is one of the polypeptides that is upregulated in cancer cells as part of the metabolic rewiring process.

[0166] Specifically, in CKB-expressing cancer cells, phosphocreatine is transported into the cell via SLC6A8, which drives cancer cell survival in the hypoxic tumor niche. Knockdown of SLC6a8 in colon cancer cells has been shown to reduce intracellular phosphocreatine and ATP levels. Furthermore, depletion of SLC6A8 in colon cancer cells substantially reduces metastatic activity. Depletion of SLC6a8 in CKB-knockdown cells abolished the protective effect of phosphocreatine during hypoxic stress. See, for example, Loo JM, Scherl A, Nguyen A, et al. "Extracellular metabolic energetics can promote cancer progression." Cell. 2015;160(3):393-406, the teachings of which are incorporated herein by reference in their entirety.

[0167] therapeutic composition Provided herein are compositions comprising a therapeutic combination featuring an SLC6A8 transporter inhibitor (e.g., β-guanidinopropionic acid, RGX-202) and a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236), and / or another chemotherapeutic agent (e.g., FOLFIRI, FOLFOX, bevacizumab, 5-fluorouracil, leflunomide, irinotecan, and / or oxaliplatin). In some embodiments, the composition comprises RGX-202 in combination with a KRAS inhibitor. Such compositions reduce cancer cell proliferation and / or increase cancer cell apoptosis and can be administered as a therapeutic treatment for cancer (e.g., colorectal cancer). Specifically, the agents provided herein inhibit creatine transport in cells (e.g., cancer cells, tumor cells, colorectal cells, liver cells, kidney cells, etc.), and, as shown in the working examples provided herein, additional agents can be used to synergistically improve clinical outcomes.

[0168] In one embodiment, there is provided a method of inhibiting cancer cell growth or proliferation, comprising: a. administering one or more creatine transporter inhibitors to cancer cells; b. administering one or more anti-proliferative agents to the cancer cells.

[0169] In another aspect, provided herein is a pharmaceutical composition comprising: (a) an effective amount of a creatine transporter inhibitor, or a pharmaceutically acceptable salt thereof; and (b) an effective amount of one or more additional antiproliferative agents.

[0170] In some embodiments of any of the aspects, the creatine transporter inhibitor is a compound comprising the structure: TIFF2024528705000007.tif27128 Formula I (In the formula, Q 1 is optionally substituted amidino or optionally substituted 2-pyridyl; m is 1 or 2; R 7 is hydrogen, optionally substituted C1-C6 alkyl, or optionally substituted C6-C10 arylC1-C6 alkyl, R 8 and R 9 are independently hydrogen, deuterium, halo, hydroxyl, NH, optionally substituted C1-C3 alkyl, or R 8 and R 9 are combined with the atoms to which they are attached to form an optionally substituted C3-C6 cycloalkyl ring, or R 8 or R 9 But R 10 or R 11 and R, taken together with the atom to which they are attached, form an optionally substituted C3-C4 cycloalkyl ring, or 8 or R 9 But R 12 and, in combination with the atoms to which they are attached, form an optionally substituted C3-C5 heterocycle, R 10 and R 11are independently hydrogen, deuterium, optionally substituted C1-C4 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 alkynyl, or R 10 and R 11 are combined with the atoms to which they are attached to form an optionally substituted C3-C6 cycloalkyl ring, or R 10 or R 11 combines with R or R and the atom to which they are attached to form an optionally substituted C3-C4 cycloalkyl ring, or R 10 or R 11 combine with R12 and the atom to which they are attached to form an optionally substituted C3-C4 heterocycle, R 12 is hydrogen, optionally substituted C1-C6 alkyl, or R 12 But R 8 or R 9 and R, in combination with the atom to which they are attached, form an optionally substituted C3-C5 heterocycle, or 12 But R 10 or R 11 and, in combination with the atoms to which they are attached, form an optionally substituted C3-C4 heterocycle, m is 1 and R 8 is hydrogen, halo, hydroxyl, or methyl, R, R 10 , and R 11 At least one of the is not hydrogen, m is 1 and R 10 is methyl, R8, R9, and R 11 At least one of the is not hydrogen, m is 1 and R 8 is NH2 and R 10 is hydrogen, methyl, or -CH2CH2OH, R9 or R 11 At least one of the is not hydrogen, When m is 1, R8 is halo, and R10 is optionally substituted C1-C4 alkyl, R9 and R 10at least one of which is not hydrogen) or a pharmaceutically acceptable salt thereof.

[0171] In some embodiments of any of the aspects, the creatine transporter inhibitor is selected from one of the agents in Table 2 provided below.

[0172] Table 2. Creatine transporters and creatine kinase inhibitors TIFF2024528705000008.tif254170TIFF2024528705000009.tif230170TIFF2024528705000010.tif247170TIFF2024528705000011.tif229170TIFF2024528705000012.tif228170TIFF2024528705000013.tif146170TIFF2024528705000014.tif245170TIFF2024528705000015.tif242170TIFF2024528705000016.tif237170TIFF2024528705000017.tif247170TIFF2024528705000018.tif226170TIFF2024528705000019.tif216170TIFF2024528705000020.tif229170TIFF2024528705000021.tif246170TIFF2024528705000022.tif240170TIFF2024528705000023.tif232170TIFF2024528705000024.tif234170TIFF2024528705000025.tif231170TIFF2024528705000026.tif237170TIFF2024528705000027.tif228170TIFF2024528705000028.tif230170TIFF2024528705000029.tif239170TIFF2024528705000030.tif232170TIFF2024528705000031.tif246170TIFF2024528705000032.tif237170TIFF2024528705000033.tif220170TIFF2024528705000034.tif236170TIFF2024528705000035.tif56170TIFF2024528705000036.tif153170TIFF2024528705000037.tif223170TIFF2024528705000038.tif228170TIFF2024528705000039.tif237170TIFF2024528705000040.tif232170TIFF2024528705000041.tif235170TIFF2024528705000042.tif234170TIFF2024528705000043.tif219170TIFF2024528705000044.tif208170TIFF2024528705000045.tif235170TIFF2024528705000046.tif226170TIFF2024528705000047.tif233170TIFF2024528705000048.tif228170TIFF2024528705000049.tif210170TIFF2024528705000050.tif231170TIFF2024528705000051.tif229170TIFF2024528705000052.tif220170TIFF2024528705000053.tif228170TIFF2024528705000054.tif219170TIFF2024528705000055.tif239170TIFF2024528705000056.tif214170TIFF2024528705000057.tif223170TIFF2024528705000058.tif234170TIFF2024528705000059.tif227170TIFF2024528705000060.tif216170TIFF2024528705000061.tif206170TIFF2024528705000062.tif223170TIFF2024528705000063.tif239170TIFF2024528705000064.tif234170TIFF2024528705000065.tif55170TIFF2024528705000066.tif186170TIFF2024528705000067.tif41170TIFF2024528705000068.tif195170TIFF2024528705000069.tif237170TIFF2024528705000070.tif206170TIFF2024528705000071.tif204170.

[0173] Additional non-limiting examples of agents that can be used in the methods and compositions provided herein are described, for example, in U.S. Pat. Nos. 10,308,597 B2 and 10,717,704 B2, the teachings of each of which are incorporated herein by reference in their entirety.

[0174] β-guanidinopropionic acid In some embodiments of any of the aspects, the creatine transporter inhibitor is β-guanidinopropionic acid (also referred to herein as β-GPA or "RGX-202"), or a pharmaceutically acceptable salt thereof (e.g., RGX-202-01). β-GPA is a creatine analog identified by CAS number 353-09-3. β-GPA (or RGX-202) provided herein is a small molecule having the following structure: TIFF2024528705000072.tif27128

[0175] β-GPA (Formula II) is a zwitterion and is highly soluble in water (greater than about 50 mg / mL), but poorly soluble in organic solvents.

[0176] Pharmaceutically acceptable salt forms may include, but are not limited to, those described in, for example, U.S. Pat. Nos. 9,884,813, 9,827,217, and 10,512,623, the teachings of which are incorporated herein by reference in their entireties.

[0177] RGX-202-01 In some embodiments of any of the aspects, the creatine transporter inhibitor is RGX-202-01. RGX-202-01 is a small molecule provided herein that is a pharmaceutically optimized form of RGX-202 (the endogenous compound, also known as β-guanidinopropionic acid or β-GPA) suitable for clinical administration. RGX-202-01 is also a small molecule inhibitor of the creatine transporter SLC6A8.

[0178] In some embodiments of any of the aspects, the one or more creatine transporter inhibitors for use in the methods and compositions provided herein are selected from the group consisting of β-guanidinopropionic acid, N-methylamidino-N-methylglycine, 1-carboxymethyl-2-imino-hexahydropyrimidine (cyclocreatine), DL-alpha-guanidinopropionic acid, N-methyl-N-amidino-beta-alanine, N-ethyl-N-amidinoglycine, DL-alpha-guanidinobutyric acid, DL-beta-guanidinobutyric acid, gamma-guanidinobutyric acid, guanidinoacetic acid, RGX-202-01, combinations thereof, and pharmaceutically acceptable salts thereof.

[0179] Additional examples of chemical structures of the above creatine transporter inhibitors can be found, for example, in FIG.

[0180] Any one of the creatine transporter inhibitors described above or provided in Table 2 can be used in combination with any one or more of the additional agents or KRAS inhibitors provided herein to reduce cancer cell proliferation or tumor growth and / or as a treatment for cancer in a subject. Combination therapies including such agents are further described below.

[0181] Combination therapy The present disclosure provides therapeutic combinations featuring an SLC6A8 transporter inhibitor (e.g., β-guanidinopropionic acid, RGX-202, RGX-202-01) and a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236), and / or another chemotherapeutic agent (e.g., FOLFIRI, FOLFOX, bevacizumab, 5-fluorouracil, leflunomide, irinotecan, and / or oxaliplatin). In embodiments, the therapeutic combinations provided herein can be used in combination with one or more additional anti-cancer therapies (e.g., surgery, radiation therapy), and / or one or more additional therapeutic agents.

[0182] Specifically, combination therapy encompasses both coadministration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent is in some way conditioned on the administration of another therapeutic agent. For example, one therapeutic agent (e.g., β-GPA) may be administered only after a different therapeutic agent (e.g., a KRAS inhibitor in Table 1 and / or an agent provided in Table 3 below) has been administered and allowed to act for a predetermined period of time.

[0183] Non-limiting examples of agents that can be combined with the creatine transporter inhibitors provided herein are provided in Table 3 below.

[0184] Table 3: Exemplary Anti-Cancer and Anti-Proliferative Agents TIFF2024528705000073.tif224168TIFF2024528705000074.tif222169TIFF2024528705000075.tif24816 8TIFF2024528705000076.tif103168TIFF2024528705000077.tif214168TIFF2024528705000078.tif97168

[0185] In some embodiments of any of the aspects, the one or more additional agents are a KRAS inhibitor (e.g., those listed in Table 1), a SHP2 inhibitor, a HER2 inhibitor, an EGFR inhibitor, a SOS1 inhibitor, a Raf inhibitor, a MEK inhibitor, an ERK inhibitor, a PI3K inhibitor, a polo-like kinase 1 (PLK1) inhibitor, an adenosine inhibitor, a PTEN inhibitor, an AKT inhibitor, an mTORC1 inhibitor, a BRAF inhibitor, a PD-L1 inhibitor, a PD-1 inhibitor, a CDK4 / 6 inhibitor, a dihydroorotate dehydrogenase (DHODH) inhibitor, or any combination thereof.

[0186] Exemplary EGFR inhibitors include, but are not limited to, erlotinib (Tarceva), afatinib (Gilotrif), gefitinib (Iressa), osimertinib (Tagrisso), dacomitinib (Vizimpro), and panitumumab (Vectibix). Non-limiting examples of EGFR inhibitors that can be used in the methods provided herein include those described below (i.e., erlotinib, osimertinib, neratinib, gefitinib, cetuximab, panitumumab, dacomitinib, lapatinib, necitumumab, mobocertinib, and vandetanib), pharmaceutical salts, analogs, derivatives, and combinations thereof.

[0187] Erlotinib (Tarceva) - Erlotinib is a tyrosine kinase receptor inhibitor used in the treatment of advanced or metastatic pancreatic cancer or non-small cell lung cancer. Erlotinib is a quinazoline derivative with antitumor properties. In competition with adenosine triphosphate, erlotinib reversibly binds to the intracellular catalytic domain of epidermal growth factor receptor (EGFR) tyrosine kinase, thereby reversibly inhibiting EGFR phosphorylation and blocking signaling events and oncogenic effects associated with EGFR activation.

[0188] Osimertinib (Tagrisso) - Tagrisso (osimertinib) is a targeted cancer therapy, an EGFR-TKI, designed to inhibit both activating sensitizing mutations (EGFRm) and T790M, a genetic mutation associated with EGFR-TKI treatment resistance. Tagrisso (osimertinib) is a kinase inhibitor of the epidermal growth factor receptor (EGFR) that irreversibly binds to specific mutant forms of EGFR (T790M, L858R, and exon 19 deletion) at approximately nine-fold lower concentrations than the wild-type.

[0189] Neratinib (Nerlynx) - Neratinib is a potent, irreversible tyrosine kinase inhibitor (TKI) of HER1, HER2, and HER4. Neratinib irreversibly binds to the intracellular signaling domains of HER1, HER2, HER3, and epidermal growth factor receptors, inhibiting phosphorylation and several downstream HER signaling pathways, resulting in decreased proliferation and increased cell death.

[0190] Gefitinib (Iressa) - Gefitinib is an inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase that binds to the adenosine triphosphate (ATP) binding site of the enzyme.

[0191] Cetuximab (Erbitux) - Erbitux is a recombinant human / mouse chimeric monoclonal antibody. The antibody binds to the epidermal growth factor receptor (EGFR, HER1, c-ErbB-1) on both normal and tumor cells, competitively inhibiting the binding of other ligands such as epidermal growth factor (EGF) and transforming growth factor-alpha. Erbitux is composed of the Fv region of a murine anti-EGFR antibody with human IgG1 heavy chain and kappa light chain constant regions.

[0192] Panitumumab (Vectibix) - Vectibix specifically binds to EGFR on both normal and tumor cells and competitively inhibits ligand binding to EGFR. Preclinical studies show that panitumumab's binding to EGFR prevents ligand-induced receptor autophosphorylation and activation of receptor-associated kinases, resulting in inhibition of cell growth, induction of apoptosis, reduced production of proinflammatory cytokines and vascular growth factors, and internalization of EGFR.

[0193] Dacomitinib (Vizimpro) - Vizimpro (dacomitinib) is an irreversible inhibitor of the kinase activity of the human EGFR family (EGFR / HER1, HER2, and HER4) and certain EGFR activating mutations (exon 19 deletion or exon 21 L858R substitution mutation). In vitro, dacomitinib also inhibits the activity of DDR1, EPHA6, LCK, DDR2, and MNK1 at clinically relevant concentrations.

[0194] Lapatinib (Tykerb) - Tykerb is an inhibitor of the intracellular tyrosine kinase domains of both the epidermal growth factor receptor (EGFR [ErbB1]) and human epidermal growth factor receptor type 2 (HER-2 [ErbB2]) receptors. When the binding site is blocked, signaling molecules can no longer bind to it and activate tyrosine kinases, enzymes that function to stimulate cell division.

[0195] Necitumumab (Portrazza) - Portrazza (necitumumab) is a recombinant human IgG1 monoclonal antibody that binds to the human epidermal growth factor receptor (EGFR) and blocks EGFR binding to its ligands. EGFR expression and activation are correlated with malignant progression, induction of angiogenesis, and inhibition of apoptosis. Necitumumab binding induces EGFR internalization and degradation in vitro. In vitro, necitumumab binding also resulted in antibody-dependent cellular cytotoxicity (ADCC) in EGFR-expressing cells.

[0196] Mobocertinib (Exkivity) - Exkivity (mobocertinib) is a kinase inhibitor designed to selectively target epidermal growth factor receptor (EGFR) Exon 20 insertion mutations, specifically at lower concentrations than wild-type (WT) EGFR. Two pharmacologically active metabolites (AP32960 and AP32914) with inhibitory profiles similar to those of mobocertinib have been identified in plasma following oral administration of mobocertinib. In vitro, mobocertinib also inhibited the activity of other EGFR family members (HER2 and HER4) and one additional kinase (BLK) at clinically relevant concentrations (IC50 values ​​<2 nM).

[0197] Vandetanib (Caprelsa) - Vandetanib is a kinase inhibitor. It inhibits the activity of tyrosine kinases, including members of the epidermal growth factor receptor (EGFR) family, vascular endothelial growth factor (VEGF) receptors rearranged during transfection (RET), protein tyrosine kinase 6 (BRK), TIE2, members of the EPH receptor kinase family, and members of the Src family of tyrosine kinases. Vandetanib inhibits endothelial cell migration, proliferation, survival, and new blood vessel formation in in vitro models of angiogenesis. Vandetanib inhibits EGFR-dependent cell survival in vitro. In addition, vandetanib inhibits epidermal growth factor (EGF)-stimulated receptor tyrosine kinase phosphorylation in tumor cells and endothelial cells, as well as VEGF-stimulated tyrosine kinase phosphorylation in endothelial cells. In vivo, vandetanib administration reduced tumor cell-induced angiogenesis, tumor vascular permeability, and inhibited tumor growth and metastasis in mouse models of cancer.

[0198] In some embodiments of any of the aspects, the one or more additional agents are selected from the group consisting of sotorasib (LUMAKRAS™, AMG-510), adagrasib (MRTX849), MRTX1133, RMC-6291, RMC-6236, atovaquone, brequinar sodium, leflunomide, teriflunomide, BAY-2402234, AG-636, leucovorin (folinic acid), 5-fluorouracil (5-FU), irinotecan, oxaliplatin, FOLFIRI, FOLFOX, combinations thereof, and pharmaceutically acceptable salts thereof.

[0199] In some embodiments of any of the aspects, the subject has been administered at least one previous anti-cancer therapy or agent prior to being administered a creatine transporter inhibitor and / or one or more additional anti-proliferative agents provided herein.

[0200] It will also be understood that the compounds and pharmaceutical compositions of the present invention can be formulated and utilized in combination therapy, i.e., the compounds and pharmaceutical compositions can be formulated or administered simultaneously with, prior to, or after one or more other desired therapeutic agents or medical procedures. The particular combination of therapies (therapeutic agents or procedures) utilized in a combination regimen will take into account the compatibility of the desired therapeutic agents and / or procedures with the desired therapeutic effect to be achieved. It will also be understood that the therapies utilized can achieve a desired effect for the same disorder (e.g., reducing cancer cell growth or tumor size in a subject), or they can achieve different effects (e.g., controlling any adverse effects).

[0201] The term "therapeutic synergy" is used when, for example, the combination of two drugs at a given dose is more effective than the best of the two products alone, given the same dose. To study therapeutic synergy, each combination can be compared with a single drug using estimates obtained from a two-way analysis of variance with repeated measures (time factor) on in vivo or cell growth parameters tumor volume.

[0202] Pharmaceutical Composition Pharmaceutical compositions featuring an SLC6A8 transporter inhibitor (e.g., β-guanidinopropanoic acid, RGX-202) and a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236), and / or another chemotherapeutic agent (e.g., FOLFIRI, FOLFOX, bevacizumab, 5-fluorouracil, leflunomide, irinotecan, and / or oxaliplatin) are formulated separately or in combination for delivery to a subject in need thereof. In some embodiments, the therapeutic combination is delivered with a pharmaceutically acceptable carrier, appropriate for delivering the compound via a selected route of administration. Suitable pharmaceutically acceptable carriers are those conventionally used with small molecules, such as diluents, excipients, etc. See, for example, "Remington's Pharmaceutical Sciences," 17th Ed., Mack Publishing Company, Easton, Pa., 1995, for guidelines regarding drug formulation.

[0203] Within the scope of the present invention are compositions containing a suitable carrier and one or more of the above-mentioned therapeutic agents. The composition may be a pharmaceutical composition containing a pharmaceutically acceptable carrier, a dietary composition containing a suitable dietarily acceptable carrier, or a cosmetic composition containing a cosmetically acceptable carrier.

[0204] The term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use. A "pharmaceutically acceptable carrier" is one that does not cause undesirable physiological effects after or upon administration to a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense of being compatible with and capable of stabilizing the active ingredient. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of the active compound. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a usable composition in dosage form. Other examples of carriers include colloidal silicon oxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

[0205] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, or allergic response, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977), incorporated herein by reference. Salts can be prepared during the final isolation and purification of the compounds of the invention, or in situ by separately reacting a free base or free acid functional group with a suitable reagent, as generally described below. For example, a free base functional group can be reacted with a suitable acid. Furthermore, when a compound of the invention contains an acidic moiety, suitable pharmaceutically acceptable salts thereof can include metal salts such as alkali metal salts, e.g., sodium or potassium salts, as well as alkaline earth metal salts, e.g., calcium or magnesium salts. Pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acid, or using other methods used in the art, such as ion exchange.Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydrogen iodide, and 2-hydroxy-ethanesulfonate. , lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Additional pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0206] As mentioned above, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the present invention, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated within the scope of the present invention. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; natural and synthetic phospholipids, including, but not limited to, soybean and egg yolk phosphatides, lecithin, hydrogenated soybean lecithin, dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, dioleyl lecithin, hydroxylated lecithin, lysophosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, diasteroylphosphatidylethanolamine (DSPE) and its pegylated esters, e.g., DSPE-PEG750 and DSPE-PEG2000, phosphatidic acid, phosphatidylglycerol, and phosphatidylserine.Preferred commercially available grades of lecithins include those available under the tradename Phosal® or Phospholipon®, including Phosal 53 MCT, Phosal 50 PG, Phosal 75 SA, Phospholipon 90H, Phospholipon 90G, and Phospholipon 90 NG, with soybean-phosphatidylcholine (SoyPC) and DSPE-PEG2000 being particularly preferred. Buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solutions; and other non-toxic compatible 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 may also be present in the composition, according to the judgment of the formulator.

[0207] The pharmaceutical compositions of the present invention can be administered parenterally, orally, nasally, rectally, topically, or buccally. As used herein, the term "parenteral" refers to subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, and any suitable infusion technique.

[0208] Sterile injectable compositions can be solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents. Such solutions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as solvents or suspending media (e.g., synthetic mono- or diglycerides). Fatty acids, such as, but not limited to, oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as, but not limited to, olive oil or castor oil, and their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as, but not limited to, carboxymethylcellulose or similar dispersants. Other commonly used surfactants, such as, but not limited to, Tween or Span, or other similar emulsifiers or bioavailability enhancers commonly used in the production of pharmaceutically acceptable solid, liquid, or other dosage forms, can also be used for formulation purposes.

[0209] In some embodiments of any of the aspects, one or more agents provided herein are formulated for oral administration.The composition for oral administration can be any orally acceptable dosage form, including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions.For tablets, commonly used carriers include, but are not limited to, lactose and corn starch.Lubricants, such as, but not limited to, magnesium stearate, are also typically added.For oral administration in capsule form, useful diluents include, but are not limited to, lactose and dried corn starch.When aqueous suspensions or emulsions are orally administered, the active ingredient can be suspended or dissolved in an oily phase combined with an emulsifier or suspending agent.If desired, certain sweeteners, flavors, or colorants can also be added.

[0210] For oral administration, the pharmaceutical compositions may take the form of tablets, troches, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., ationed oils, oily esters, ethyl alcohol or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Preparations may also contain buffer salts, flavoring agents, coloring agents and sweetening agents, as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

[0211] Pharmaceutical compositions for topical administration according to the invention described can be formulated as solutions, ointments, creams, suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils. Alternatively, topical formulations can be in the form of patches or bandages impregnated with the active ingredient(s), which can optionally contain one or more excipients or diluents. In some preferred embodiments, topical formulations include materials that enhance absorption or penetration of the active agent(s) through the skin or other affected areas.

[0212] Topical compositions contain a safe and effective amount of a dermatologically acceptable carrier suitable for application to the skin. A "cosmetically acceptable" or "dermatologically acceptable" composition or ingredient refers to a composition or ingredient suitable for use in contact with human skin without undue toxicity, incompatibility, instability, or allergic response. The carrier enables the active agent and optional ingredients to be delivered to the skin at an appropriate concentration(s). Thus, the carrier can function as a diluent, dispersant, solvent, etc. to ensure that the active is applied to the selected target at the appropriate concentration and evenly distributed over the selected target. The carrier can be solid, semi-solid, or liquid. The carrier can be in the form of a lotion, cream, or gel, particularly one that has a sufficient thickness or yield point to prevent the active from settling. The carrier can be inert or have dermatological benefits. It should also be physically and chemically compatible with the active ingredients described herein and should not unduly impair the stability, efficacy, or other use benefits associated with the composition.

[0213] Pharmaceutical compositions that may oxidize and lose biological activity, particularly in liquid or semi-solid form, may be prepared in a nitrogen atmosphere or may be sealed in capsules and / or foil packages of the type that exclude oxygen (e.g., Capsugel™).

[0214] For administration by inhalation, the drug is conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example, of gelatin, for use in an inhaler or insufflator can be formulated to contain a powder mix of the drug and a suitable powder base, such as lactose or starch.

[0215] The pharmaceutical composition can be formulated for parenteral administration by injection, e.g., bolus injection or continuous infusion. Preparations for injection can be provided in unit dosage form, e.g., in ampoules or multi-dose containers, with added preservatives. The agent can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The agent can be formulated in rectal compositions such as suppositories or retention enemas, containing, for example, conventional suppository bases such as cocoa butter or other glycerides.

[0216] In addition to the aforementioned formulations, pharmaceutical compositions can also be formulated as depot preparations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the drug can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt. Controlled-release formulations also include patches, for example, transdermal patches. Patches can be used with an ultrasonic applicator that deploys ultrasound waves with a unique combination of waveforms to introduce drug molecules through the skin that normally cannot be effectively delivered transdermally.

[0217] The pharmaceutical composition may contain a non-dissolving, non-disintegrating sustained-release suppository base consisting essentially of a linear polymer such as methylcellulose, polyvinylpyrrolidone, and water.

[0218] Pharmaceutical compositions can be incorporated into gel formulations, which are generally semi-solid systems consisting of either small inorganic particles (two-phase systems) or suspensions composed of large organic molecules distributed substantially uniformly throughout a carrier liquid (single-phase gels). Single-phase gels can be made, for example, by combining the active agent, carrier liquid, and a suitable gelling agent, such as tragacanth (2-5%), sodium alginate (2-10%), gelatin (2-15%), methylcellulose (3-5%), sodium carboxymethylcellulose (2-5%), carbomer (0.3-5%), or polyvinyl alcohol (10-20%), and mixing until a characteristic semi-solid product is produced. Other suitable gelling agents include methylhydroxycellulose, polyoxyethylene-polyoxypropylene, hydroxyethylcellulose, and gelatin. Gels typically use an aqueous carrier liquid, although alcohols and oils can also be used as carrier liquids.

[0219] Pharmaceutical compositions may be incorporated into microemulsions, which are generally thermodynamically stable, isotropically transparent dispersions of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9). The preparation of a microemulsion requires a surfactant (emulsifier), a cosurfactant (co-emulsifier), an oil phase, and an aqueous phase. Suitable surfactants include any surfactant useful in the preparation of emulsions, such as emulsifiers typically used in the preparation of creams. The co-surfactant (or "co-emulsifier") is generally selected from the group consisting of polyglycerol derivatives, glycerol derivatives, and fatty alcohols. Preferred emulsifier / co-emulsifier combinations are generally, but not necessarily, selected from the group consisting of glyceryl monostearate and polyoxyethylene stearate, polyethylene glycol and ethylene glycol palmitostearate, caprylic and capric triglycerides, and oleoyl macrogolglycerides. The aqueous phase typically contains not only water but also buffers, glucose, propylene glycol, polyethylene glycol, preferably low molecular weight polyethylene glycols (e.g., PEG300 and PEG400), and / or glycerol, while the oil phase generally contains, for example, fatty acid esters, modified vegetable oils, silicone oils, mixtures of mono-, di-, and triglycerides, mono- and di-esters of PEG (e.g., oleoyl macrogolglycerides), and the like.

[0220] In some embodiments, pharmaceutical formulations are provided for oral or parenteral administration, in which case the formulation may comprise an active compound-containing microemulsion as described above, or may contain alternative pharmaceutically acceptable carriers, vehicles, excipients, etc. that are particularly suitable for oral or parenteral drug administration. Alternatively, the active compound-containing microemulsion may be administered orally or parenterally substantially as described above without modification.

[0221] In some embodiments, the formulation containing the compound / agent comprises one or more additional components, which are at least one of an osmolality component that provides an isotonic or near-isotonic solution compatible with human cells or blood, and a preservative.

[0222] In some embodiments, the osmolality component is a salt such as sodium chloride, or a sugar, or a combination of two or more of these components. In some embodiments, the sugar can be a monosaccharide such as dextrose, a disaccharide such as sucrose or lactose, a polysaccharide such as dextran 40, dextran 60, or starch, or a sugar alcohol such as mannitol. Osmolality components are readily selected by one of ordinary skill in the art.

[0223] In some embodiments, the preservative is at least one of a paraben, chlorobutanol, phenol, sorbic acid, and thimerosal.

[0224] In some embodiments, the formulation containing the compound / agent is in the form of a sustained release formulation and optionally further comprises one or more additional ingredients, the additional ingredients being at least one of an anti-inflammatory agent and a preservative.

[0225] Delivery method Therapeutic compositions comprising an SLC6A8 transporter inhibitor (e.g., β-guanidinopropionic acid, RGX-202) and a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236) provided herein, and / or another chemotherapeutic agent (e.g., FOLFIRI, FOLFOX, bevacizumab, 5-fluorouracil, leflunomide, irinotecan, and / or oxaliplatin) can be administered via a variety of methods. Such methods include, but are not limited to, intratumoral, intravesical, intralesional (e.g., colon or GI tract), oral, intravenous (iv), subcutaneous (sc or sq), intraperitoneal, intramuscular, intradermal, or rectal administration to a subject (e.g., mammal) in need thereof. In some embodiments, sustained-release formulations are administered as suppositories. In some embodiments, sustained-release formulations are administered in implants designed for subcutaneous implantation. Exemplary subcutaneous implants are known to those skilled in the art and may include ports, catheters, etc. In certain embodiments, the port or catheter is implanted in the digestive tract.

[0226] Treatment medications and regimens The combination, which features an SLC6A8 transporter inhibitor (e.g., β-guanidinopropionic acid, RGX-202) and a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236), and / or another chemotherapeutic agent (e.g., FOLFIRI, FOLFOX, bevacizumab, 5-fluorouracil, leflunomide, irinotecan, and / or oxaliplatin), is administered in an amount suitable to increase patient survival, reduce tumor size, or otherwise stabilize the disease. The most suitable therapeutic dosage and regimen for treating a subject (e.g., a human patient) will vary depending on the disorder or condition being treated, as well as the patient's weight and other parameters. The dose of at least one compound / agent described herein may be administered, for example, from about 1000 to a maximum of about 3600 mg twice daily over the course of cancer treatment.

[0227] In some embodiments of any of the aforementioned methods, the creatine transporter inhibitor, or a pharmaceutically acceptable salt thereof, is administered in an amount of 0.01 to 100 mg / kg per day.

[0228] Lower doses and shorter or longer treatment durations or treatment frequencies are also contemplated to produce a therapeutically useful result, i.e., a statistically significant reduction in cell proliferation and / or tumor size. Furthermore, it is contemplated that local administration to the gastrointestinal tract (e.g., colon) can be optimized based on the response of gastrointestinal cells (e.g., epithelial cells) therein.

[0229] Effective dosage and treatment protocol can be determined by conventional methods, starting with a low dose in experimental animals, then increasing the dose while monitoring the effect, and similarly systematically changing the dosage regimen.When determining the optimal dosage for a given subject, clinicians can consider many factors, including the size, age, and general condition of the patient, the specific disorder being treated, the severity of the disorder, and the presence of other drugs in the patient.Test dosage can be selected after considering the results of animal studies and clinical literature.

[0230] A typical human dose of the compounds / agents provided herein can be from about 10 μg / kg body weight / day to about 10 mg / kg / day, more specifically, from about 50 μg / kg / day to about 5 mg / kg / day, and even more specifically, from about 100 μg / kg / day to 1 mg / kg / day.

[0231] In some embodiments of any of the aspects, the creatine transporter inhibitor (e.g., β-GPA, RGX-202, RGX-202-01), or a pharmaceutically acceptable salt thereof, is orally administered in an amount of at least 1000 milligrams (mg), 1200 mg, 1400 mg, 1600 mg, 1800 mg, 2000 mg, 2200 mg, 2400 mg, 2600 mg, 2800 mg, 3000 mg, 3200 mg, 3400 mg, or up to about 3600 mg.

[0232] In some embodiments of any of the aspects, the creatine transporter inhibitor, or a pharmaceutically acceptable salt thereof, is orally administered at least about once daily, twice daily, three times daily, four times daily, or five times daily.

[0233] In some embodiments of any of the aspects, the creatine transporter inhibitor, or a pharmaceutically acceptable salt thereof, is administered orally twice daily in an amount of at least about 2400 mg to about 3600 mg.

[0234] In some embodiments of the aspects, the KRAS inhibitor adagrasib, or a pharmaceutically acceptable salt thereof, is orally administered in an amount of at least about 150 mg or more, at least about 300 mg or more, at least about 600 mg or more, 1400 mg or more, 1600 mg or more, 1800 mg or more, 2000 mg or more, 2200 mg or more, 2400 mg or more, 2600 mg or more, 2800 mg or more, 3000 mg or more, 3200 mg or more, 3400 mg or more, up to about 3600 mg.

[0235] In some embodiments of any of the aspects, the KRAS inhibitor sotorasib or a pharmaceutically acceptable salt thereof is orally administered in an amount of at least about 150 mg or more, at least about 300 mg or more, at least about 600 mg or more, 1400 mg or more, 1600 mg or more, 1800 mg or more, 2000 mg or more, up to about 2500 mg.

[0236] In some embodiments of any of the aspects, the KRAS inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof, and is administered orally or intravenously in an amount of at least about 0.01 mg / kg or more, at least about 1 mg / kg or more, at least about 10 mg / kg or more, at least about 100 mg / kg or more, at least about 300 mg / kg or more, up to about 500 mg / kg.

[0237] In some embodiments of any of the aspects, the KRAS inhibitor RMC-6291, or a pharmaceutically acceptable salt thereof, is administered orally or intravenously in an amount of at least about 0.01 mg / kg or more, at least about 1 mg / kg or more, at least about 10 mg / kg or more, at least about 100 mg / kg or more, at least about 300 mg / kg or more, up to about 500 mg / kg.

[0238] In some embodiments of any of the aspects, the KRAS inhibitor RMC-6236, or a pharmaceutically acceptable salt thereof, is administered orally or intravenously in an amount of at least about at least about 0.01 mg / kg or more, at least about 1 mg / kg or more, at least about 10 mg / kg or more, at least about 100 mg / kg or more, at least about 300 mg / kg or more, up to about 500 mg / kg.

[0239] In some embodiments of any of the aspects, the KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and / or RMC-6236), or a pharmaceutically acceptable salt thereof, is orally administered at least about once daily, twice daily, three times daily, four times daily, or five times daily.

[0240] In some embodiments of any of the aspects, leucovorin (folinic acid) or a pharmaceutically acceptable salt thereof is orally administered in an amount of at least about at least about 1 mg or more, at least about 5 mg or more, at least about 10 mg or more, at least about 15 mg or more, at least about 20 mg or more, at least about 25 mg or more, at least about 30 mg or more, at least about 35 mg or more, at least about 40 mg or more, at least about 45 mg or more, up to about 50 mg.

[0241] In some embodiments of any of the aspects, leucovorin (folinic acid) or a pharmaceutically acceptable salt thereof is administered at a dose of at least about 100 mg / m 2 or more, at least about 200 mg / m 2 or more, at least about 300 mg / m 2or more, at least about 400 mg / m 2 or more, up to approximately 500 mg / m 2 The above doses are administered intravenously.

[0242] In some embodiments of any of the aspects, irinotecan or a pharmaceutically acceptable salt thereof is administered at a dose of at least about 100 mg / m 2 or more, at least about 200 mg / m 2 or more, at least about 300 mg / m 2 or more, at least about 400 mg / m 2 or more, up to approximately 500 mg / m 2 It is administered intravenously in the amount of

[0243] In some embodiments of any of the aspects, 5-fluorouracil (F) or a pharmaceutically acceptable salt thereof is administered at a dose of at least about 300 mg / m 2 or more, at least about 600 mg / m 2 or more, at least about 900 mg / m 2 or more, at least about 1200 mg / m 2 or more, at least about 1500 mg / m 2 or more, at least about 1800 mg / m 2 or more, at least about 2100 mg / m 2 or more, at least about 2400 mg / m 2 or more, at least about 2800 mg / m 2 or more, at least about 3000 mg / m 2 or more, at least about 3300 mg / m 2 or more, up to approximately 3600 mg / m 2 It is administered intravenously in the amount of

[0244] In some embodiments of any of the aspects, FOLFIRI (e.g., leucovorin (folinic acid - FOL), 5-fluorouracil (F), and irinotecan hydrochloride (IRI)) is administered orally and / or intravenously at least about once daily, twice daily, three times daily, four times daily, or five times daily.

[0245] In some embodiments of any of the aspects, bevacizumab or a pharmaceutically acceptable salt thereof is administered intravenously in an amount of at least about 1 mg / kg or more, at least about 2 mg / kg or more, at least about 3 mg / kg or more, at least about 4 mg / kg or more, at least about 5 mg / kg or more, at least about 7.5 mg / kg or more, at least about 10 mg / kg or more, at least about 15 mg / kg or more, up to about 20 mg / kg or more.

[0246] In some embodiments of any of the aspects, bevacizumab or a pharmaceutically acceptable salt thereof is administered intravenously at least about once daily, twice daily, three times daily, four times daily, or five times daily. In some embodiments of any of the aspects, bevacizumab or a pharmaceutically acceptable salt thereof is administered intravenously at least about weekly, at least about every two weeks, or at least about every three weeks.

[0247] The therapeutic efficacy of a compound / agent and / or composition comprising the same can be determined by assessing and comparing a patient's symptoms and quality of life before and after administration. Such methods apply regardless of the mode of administration. In certain embodiments, pre-administration refers to assessing a patient's symptoms and quality of life before the initiation of therapy, and post-administration refers to assessing a patient's symptoms and quality of life at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 29, or 20 weeks after the initiation of therapy. In certain embodiments, post-administration assessments are performed about 2-8, 2-6, 4-6, or 4 weeks after the initiation of therapy. In certain embodiments, a patient's symptoms (e.g., gastrointestinal distress) and quality of life before and after administration are assessed clinically and by questionnaire assessment.

[0248] Treatment effectiveness Therapeutic combinations featuring an SLC6A8 transporter inhibitor (e.g., β-guanidinopropionic acid, RGX-202) and a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236), and / or another chemotherapeutic agent (e.g., FOLFIRI, FOLFOX, bevacizumab, 5-fluorouracil, leflunomide, irinotecan, and / or oxaliplatin) are useful for treating cancer (e.g., colorectal cancer, gastrointestinal cancer, pancreatic cancer), or any other disease or condition described herein. An effective amount refers to the amount of active compound / drug required to confer a therapeutic effect on the treated subject. As will be recognized by those skilled in the art, the effective dose will vary depending on the type of disease being treated, the route of administration, the use of excipients, and the possibility of co-administration with other therapeutic treatments.

[0249] The compositions and methods provided herein can be used to reduce the proliferation or survival of cancer cells in vivo or in vitro.

[0250] Methods for assessing tumor progression or cell proliferation are well known in the art. In some embodiments, overall response is assessed from time point response assessments (based on tumor burden) as follows: Complete response (CR): Disappearance of all target lesions. All pathological lymph nodes (target or non-target) must be reduced to less than 10 mm in short axis. • Partial response (PR): At least a 30% reduction in the sum of the diameters of the target lesions, based on the baseline sum diameter. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of the target lesions (this includes the baseline sum if it is the smallest on the study) taking as reference the smallest sum on the study. In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also considered progression.) • Stable disease (SD): Taking the smallest sum diameter on the trial as reference, there is neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD.

[0251] In another embodiment, an in vitro cancer cell proliferation assay is used to assess the effectiveness of a creatine transporter inhibitor and / or one or more additional agents provided herein.

[0252] The compositions and methods provided herein result in a reduction in cancer cell proliferation or survival. For example, after treatment with one or more of the agents provided herein, cancer cell proliferation or survival is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to cell proliferation or survival before treatment.

[0253] The compositions and methods provided herein can result in a reduction in tumor size or volume. For example, after treatment, tumor size is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to its size before treatment. Tumor size can be measured by any reproducible means of measurement. Tumor size can be measured as tumor diameter or by any reproducible means of measurement.

[0254] Treating cancer can also result in a reduction in tumor number. For example, after treatment, the tumor number is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to the number before treatment. The tumor number can be measured by any reproducible means of measurement. The tumor number can be measured by counting tumors visible to the naked eye or at a specified magnification (e.g., 2x, 3x, 4x, 5x, 10x, or 50x).

[0255] Treatment of cancer can result in a reduction in the number of metastatic nodules in other tissues or organs distant from the primary tumor site. For example, after treatment, the number of metastatic nodules is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to the number before treatment. The number of metastatic nodules can be measured by any reproducible means of measurement. The number of metastatic nodules can be measured by counting metastatic nodules visible to the naked eye or at a specified magnification (e.g., 2x, 10x, or 50x).

[0256] Treating cancer can result in an increase in the mean survival time of a population of subjects treated with the present invention compared to a population of untreated subjects. For example, the mean survival time is increased by more than 30 days (60, 90, 120, or more days). The increase in mean survival time of a population can be measured by any reproducible means. The increase in mean survival time of a population can be measured, for example, by calculating the mean survival time of a population after the start of treatment with a compound of the present invention. The increase in mean survival time of a population can also be measured, for example, by calculating the mean survival time of a population after the completion of a first round of treatment with a compound of the present invention.

[0257] Treating cancer can also result in a reduction in mortality in a treated population compared to an untreated population. For example, mortality is reduced by more than 2% (e.g., 5%, 10%, 25%, or more). The reduction in mortality in a treated population can be measured by any reproducible means, for example, by calculating the average number of disease-related deaths per unit time for a population after the start of treatment with a compound of the invention. The reduction in mortality in a population can also be measured, for example, by calculating the average number of disease-related deaths per unit time for a population after the completion of a first round of treatment with a compound of the invention.

[0258] Methods for characterizing cancer and selecting subjects for treatment with creatine transporter inhibitors - Patent Application 20070122999 The present invention features a method for administering a personalized cancer therapy to a subject having or at risk of developing cancer. The method includes characterizing KRAS, HRAS, NRAS, CKB, and / or SLC6A8 expression levels, activities, or sequences in a tumor and selecting the subject for treatment if the tumor contains one or more of: (1) a KRAS, HRAS, and / or NRAS mutation; (2) an increased level or activity of creatine, CKB; and / or (3) an increased level or activity of SLC6A8, where the increase is measured relative to the level, activity, or sequence of KRAS, CKB, and / or SLC6A8 in a corresponding control cell (e.g., a colorectal cell not affected by cancer).

[0259] Thus, the present disclosure provides for characterizing a biological sample from a subject having or suspected of having cancer (e.g., colorectal cancer). Such characterization includes characterizing KRAS, HRAS, and / or NRAS polynucleotide sequences in a biological sample obtained from the subject and detecting the presence or absence of an alteration in the KRAS, HRAS, and / or NRAS polynucleotide sequence relative to a reference sequence, wherein detection of an alteration in the KRAS, HRAS, and / or NRAS polynucleotide sequence selects the subject for treatment with a therapeutic agent described herein, e.g., a creatine transporter inhibitor, a KRAS inhibitor, and / or another chemotherapeutic agent.

[0260] Another method for characterizing a biological sample from a subject having or suspected of having cancer (e.g., colorectal cancer) includes detecting the level of a creatine kinase B (CKB) polypeptide or polynucleotide in the biological sample obtained from the subject relative to a reference level, wherein an increase in the level or activity of CKB relative to the reference level selects the subject for treatment with a creatine transporter inhibitor and a KRAS inhibitor. In some embodiments, a biological sample from a subject having or suspected of having cancer (e.g., colorectal cancer) is characterized for the level of CKB and the presence or absence of alterations in KRAS, HRAS, and / or NRAS polynucleotide sequences.

[0261] KRAS, HRAS, and / or NRAS mutations and CKB levels can be detected, for example, in tissue and liquid biopsy samples.Changes in KRAS, HRAS, and / or NRAS polynucleotide sequences can be characterized using any method known in the art (e.g., sequencing, Sanger sequencing, next-generation sequencing, primer / probe hybridization, immunohistochemistry, Western blot, ELISA).Changes in CKB levels can be characterized, for example, using immunoassays (e.g., sequencing, Sanger sequencing, next-generation sequencing, primer / probe hybridization, immunohistochemistry, Western blot, ELISA) or any other method known in the art.

[0262] In some embodiments of any of the aspects, the biological sample is further evaluated for the presence or absence of a tumor marker. Non-limiting examples of tumor markers include carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 or CA19-9, and carbohydrate antigen 15-3 (CA15-3). Carbohydrate antigen (CA) 19-9 is a type of antigen released by pancreatic cancer cells. An increased level of CA19-9 indicates that the subject has a growing tumor compared to a reference level (e.g., a subject without cancer). CA15-3 is a tumor antigen expressed in breast cancer. Elevated levels of CEA compared to reference levels can be indicative of certain types of cancer, including cancer of the colon and rectum, prostate, ovary, lung, thyroid, and / or liver.

[0263] In some embodiments of any of the aspects, a biological sample (eg, a tumor specimen) obtained from the subject is further evaluated for altered levels of CKB and / or SLC6A8.

[0264] Methods for characterizing, assessing, and quantifying creatine transporter or creatine kinase levels or activity are provided herein in the working examples of Example 12. For example, CKB and / or SLC6A8 levels can be assessed by immunohistochemistry of biological samples, e.g., tissue or liquid biopsies.

[0265] kit A therapeutic combination, e.g., a therapeutic composition featuring an SLC6A8 transporter inhibitor (e.g., β-guanidinopropionic acid, RGX-202) and a KRAS inhibitor (e.g., sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236), and / or another chemotherapeutic agent (e.g., FOLFIRI, FOLFOX, bevacizumab, 5-fluorouracil, leflunomide, irinotecan, and / or oxaliplatin), can be provided in a kit. In one embodiment, an SLC6A8 transporter inhibitor (e.g., RGX-202) and a KRAS inhibitor can be provided in a kit. In one embodiment, the kit includes (a) a container containing the therapeutic composition, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing, or other material related to the use of the agent for the methods described herein and / or therapeutic utility. In one embodiment, the kit also includes an additional therapeutic agent. For example, the kit includes a first container containing the composition and a second container for an additional therapeutic agent.

[0266] The informational material of the kit is not limited in its form. In one embodiment, the informational material can include information about the production, concentration, expiration date, batch or production site information, etc. In one embodiment, the informational material relates to how to administer the composition, e.g., at a suitable dose, dosage form, or method of administration (e.g., a dose, dosage form, or method of administration described herein), to treat a subject in need thereof. In one embodiment, the instructions provide a dosing regimen, administration schedule, and / or route of administration of the composition or additional therapeutic agent. The information can be provided in a variety of formats, including printed text, computer-readable material, video or audio recording, or information that includes a link or address to the substantive material.

[0267] In addition to the composition, the kit can contain other components such as a solvent or buffer, a stabilizer, or a preservative. The composition can be provided in any form, for example, a liquid, a dry, or a lyophilized form, preferably substantially pure and / or sterile. When the agent is provided in a liquid solution, the liquid solution is preferably an aqueous solution. When the agent is provided in a dry form, reconstitution is generally performed by adding a suitable solvent and an acidulant. The acidulant and a solvent, for example, an aprotic solvent, sterile water, or a buffer, can optionally be provided in the kit.

[0268] The kit can include one or more containers for a composition or compositions comprising a KRAS inhibitor and / or a creatine transporter inhibitor provided herein. In some embodiments, the kit includes separate containers, dividers, or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe having informational material attached thereto in the form of a label. In some embodiments, the kit includes multiple (e.g., packs) of individual containers, each containing one or more unit dosage forms of a drug (e.g., a dosage form described herein). The container can include a combination unit dosage, e.g., a unit containing both a KRAS inhibitor and a creatine transporter inhibitor in a desired ratio. For example, the kit includes multiple syringes, ampoules, foil packets, blister packs, or medical devices, each containing a single combination unit dose. The containers of the kits can be airtight, waterproof (eg, impermeable to moisture change or evaporation), and / or light-tight.

[0269] The kit optionally includes a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device may be provided pre-loaded with one or both agents, or may be empty but suitable for loading.

[0270] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of one in the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual," second edition (Sambrook, 1989), "Oligonucleotide Synthesis" (Gait, 1984), "Animal Cell Culture" (Freshney, 1987), "Methods in Enzymology," "Handbook of Experimental Immunology" (Weir, 1996), "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987), "Current Protocols in Molecular Biology" (Ausubel, 1987), "PCR: The Polymerase Chain Reaction" (Mullis, 1994), and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments are discussed in the following sections.

[0271] The following examples are put forward so as to provide those of ordinary skill in the art with a complete disclosure and description of how the assay, screening, and treatment methods of the present invention are made and used, and are not intended to limit the scope of what the inventors regard as their invention. [Example]

[0272] Example 1: Oral RGX-202 inhibits creatine transport and depletes cellular phosphocreatine and ATP levels in vivo RGX-202 (β-guanidinopropionic acid, β-GPA) is a creatine mimetic that competitively inhibits cellular creatine transport by inhibiting the creatine transporter SLC6A8. RGX-202 and its pharmaceutically optimized form, RGX-202-01, suitable for clinical administration, were used herein to determine the effect of SLC6A8 inhibition on CRC progression. To assess the extent to which RGX-202 inhibits SLC6A8, creatine transport was monitored in vivo. Increasing concentrations of RGX-202 were administered, followed by injection of deuterium-labeled creatine (d3-creatine) into wild-type and SLC6A8 knockout mice. Mouse tissues were extracted and analyzed for d3-creatine levels by LC-mass spectrometry (LC-MS / MS) (23). RGX-202 treatment inhibited d3-creatine tissue uptake by up to 75% at 500 mg / kg in a dose-dependent manner (Figure 1A). D3-creatine levels in SLC6A8 knockout animals were below the lower limit of quantification, confirming that creatine transport was exclusively mediated by SLC6A8 (Figure 1A). To determine whether RGX-202 could inhibit tumor SLC6A8, we performed a similar study in mice bearing syngeneic UN-KPC-961 pancreatic tumors. Dietary supplementation of 800 mg / kg of RGX-202 for 35 days indeed suppressed tumor d3-creatine transport by 50% (Figure 1B). These findings demonstrated that administration of the creatine mimetic RGX-202 significantly inhibited creatine transport into tissues in vivo.

[0273] If RGX-202-01 inhibits cellular transport of creatine, changes in circulating creatine concentrations should be observed in RGX-202-01-treated mice. Increasing doses of RGX-202-01 (100, 400, and 1200 mg / kg) were administered, and RGX-202-01 was measured in plasma by LC-MS / MS. An RGX-202-01 exposure-dependent increase in circulating plasma creatine levels was observed, manifested by the accumulation of plasma creatine upon blockade of SLC6A8 in tissues (Figure 1C). In the same animals, a substantial exposure-dependent increase in urinary creatine levels was also observed (Figure 1D), consistent with inhibition of creatine transport to tissues, resulting in increased circulating creatine and subsequent urinary excretion, thus reducing the levels of creatine available to the tumor.

[0274] Example 2: SLC6A8 inhibition exhibits broad anti-tumor activity against diverse primary and metastatic CRCs. Primary tumors exhibit hypoxia, especially as they progress to larger sizes, and must acquire metabolic adaptation to the hypoxic microenvironment in order to survive. Approximately 40% of colorectal cancers harbor mutations in the KRAS oncogene. KRAS mutations have been shown to result in increased metabolic demands on cancer cells, including increased reliance on energy-producing metabolic pathways. Without intending to be bound by theory, it was hypothesized that these tumors may exhibit sensitivity to the effects of SLC6A8 blockade with RGX-202. To determine whether SLC6A8 inhibition affects the growth of KRAS-mutant CRC tumors, highly aggressive metastatic Lvm3b (KRAS G12D) cells were implanted subcutaneously into athymic nude mice and treated after tumors became palpable (30 mm). 3 RGX-202-01 treatment was initiated when tumors reached a larger size (>500 mm). Oral administration of RGX-202-01 caused approximately 50% tumor growth inhibition (Figure 2A). Interestingly, the effect of RGX-202-01 was not observed when tumors reached a larger size (>500 mm). 3These findings are consistent with the previously demonstrated role of creatine metabolism and SLC6A8 in hypoxic survival (Loo et al., supra). RGX-202-01-treated mice demonstrated a significant improvement in survival, experiencing a doubling of median survival from 23 to 48 days. One of nine immunocompromised mice experienced a complete tumor regression response, with the tumor remaining undetectable for more than 38 days after the end of treatment (Figure 2B). Antitumor efficacy was also observed in HCT116 (KRAS G13D) and HT29 (KRAS wild-type) human CRC tumors upon oral RGX-202 (Figure 2C) or RGX-202-01 (Figures 2D-2E). Similar to Lvm3b, treatment of HT29 tumors significantly improved tumor survival, even after tumors reached a large size (900 mm). 3 Reaching a 500 mg / kg / day (>100 mg / kg / day) induced regression in 7 out of 10 mice, and treatment substantially extended overall survival (Figures 2D and 2E). To determine whether SLC6A8 inhibition could suppress the progression of murine CRC in an immunocompetent model, KRAS G12D mutant CT26 murine CRC cells were implanted into syngeneic mice. RGX-202-01 significantly inhibited CT26 tumor growth (Figure 2F). To determine whether this approach would be effective in treating larger tumors, murine KRAS wild-type MC38 CRC tumors were implanted into mice approximately 150 mm in volume. 3Treatment was performed after reaching a tumor size of 1000. In this immunocompetent model (Figure 2G), RGX-202-01 administration substantially inhibited tumor growth. Drug treatment significantly enhanced in vitro tumor apoptosis in a dose-dependent manner, as quantified by cleaved caspase-3 immunohistochemistry (Figures 2H and 2I). Increased apoptosis was also observed in RGX-202-01-treated Lvm3b and HCT-15 tumors (Figures 2J and 2K, and Figures 5A–5C). Next, we investigated whether RGX-202-01 modulated tumor cell proliferation by quantifying Ki67-positive cells. RGX-202 treatment inhibited tumor cell proliferation in both Lvm3b and HCT15 tumor models (Figures 2L and 2M, Figures 5B and 5C). In contrast, no effect on apoptosis or proliferation was observed in NCI-H508, a xenograft that did not respond to RGX-202-01 treatment (Figures 5D-5F). These results indicated that the antitumor response was associated with inhibition of tumor cell proliferation and induction of tumor apoptosis.

[0275] Example 3: SLC6A8 inhibition shows broad activity in CRC PDX models Patient-derived xenografts (PDX) are believed to better recapitulate human tumor pathology and clinical drug responses (27-30). The efficacy of SLC6A8 therapeutic inhibition was assessed using established (100-250 mm 3) We evaluated the growth of human KRAS wild-type and KRAS mutant CRC PDX tumors. Oral RGX-202 administration reduced the growth of the CLR4 KRAS wild-type PDX model by 65% ​​(Figure 3A). Furthermore, treatment also induced tumor regression in three PDX models harboring different KRAS mutations (Figures 3B–3D; CLR7 KRAS G12V, CLR24 KRAS G12C, and CLR30 KRAS G12R). To further characterize the range of efficacy of RGX-202-01 across various CRC tumor subtypes, including KRAS wild-type and KRAS mutant CRC PDX tumors, we performed a mouse PDX study based on a 1 × 1 study design in which two mice were inoculated with the same PDX model, one mouse receiving RGX-202-01 and the other mouse receiving a control treatment (31). Such a study design was developed to mimic patient clinical trials and demonstrates the reproducibility and translatability of treatment responses (31). We included 43 well-documented colorectal cancer PDXs of various mutational backgrounds, including both KRAS wild-type and KRAS mutant subtypes (Figure 9). Among these models, 49% of tumors with diverse KRAS mutation subtypes demonstrated greater than 30% single-agent antitumor efficacy when treated with RGX-202-01 compared with matched control animals (Figure 3E). RGX-202 treatment also demonstrated antitumor efficacy in 30% of BRAF mutant tumors (2 / 7) (Figure 9). These findings demonstrate that SLC6A8 inhibition by oral RGX-202 treatment exhibits single-agent antitumor efficacy against a broad range of CRC tumor subtypes, suggesting potential clinical benefit in both KRAS wild-type and KRAS mutant cancers.

[0276] Example 4: SLC6A8 inhibition synergizes with 5-FU and oral leflunomide therapy Next, we determined whether SLC6A8 inhibition could cooperate or synergize with other therapeutic agents. 5-FU is the backbone of many chemotherapy regimens used in CRC. CT26 subcutaneous tumors were treated in immunocompetent syngeneic mice with vehicle control, oral RGX-202, 5-FU, or a combination regimen containing RGX-202 and 5-FU. Single-agent RGX-202 or 5-FU significantly suppressed tumor growth (66% inhibition RGX-202, 85% inhibition 5-FU), whereas the RGX-202 / 5-FU combination resulted in a 99% tumor growth reduction (Figure 3F). Importantly, survival studies revealed that combination RGX-202 / 5-FU therapy dramatically improved mouse survival, with 40% of mice experiencing complete regression responses and long-term (>240 days) survival (Figure 3G), despite treatment being discontinued on day 85, 160 days before the end of the study. RGX-202 also induced enhanced antitumor efficacy in combination with 5-FU and irinotecan, a standard-of-care regimen for metastatic colorectal cancer (Figure 6A). G12D ;Trp53 R172HCombination treatment of pancreatic cancer tumors with RGX-202 and gemcitabine (a deoxycytidine analog and approved pancreatic cancer drug) induced greater tumor suppression compared with either gemcitabine or RGX-202 alone, but the enhanced effect was modest compared with the combination effect observed in CRC (Figure 3H). Under hypoxia, metastatic CRC tumor growth is highly dependent on the pyrimidine nucleotide biosynthesis pathway, and these cells have previously been shown to be sensitized to inhibition of the dihydroorotate dehydrogenase (DHODH) enzyme, which catalyzes a key step in pyrimidine nucleotide biosynthesis. Treatment of mice with leflunomide, an oral DHODH inhibitor used as a rheumatoid arthritis drug, inhibited CRC primary tumor growth and metastatic progression. Combination treatment of established MC38 KRAS wild-type syngeneic CRC tumors with RGX-202 / leflunomide induced synergistic activity compared with single-agent treatment with either drug (Figure 3I). Next, we tested CLR1 KRAS G12D mutant and CLR28 KRAS G13D mutant PDXs and found similar synergistic tumor regression upon RGX-202 / leflunomide combination treatment compared to tumor growth suppression responses with either agent alone (Figures 3J-3K). To determine whether the effect of leflunomide on tumor growth regression was secondary to pyrimidine nucleotide depletion, we included a cohort in the CLR28 model treated with leflunomide and the pyrimidine nucleotide uridine. Uridine supplementation rescued leflunomide-induced tumor growth inhibition, consistent with pyrimidine nucleotide levels limiting tumor growth (Figure 3K).

[0277] RGX-202-treated animals did not exhibit any adverse effects, except for a lack of weight gain in animals fed RGX-202-formulated diets, but not when mice received treatment by oral gavage (Figure 6B). Overall, our findings demonstrate that RGX-202 is well tolerated as an oral therapy and can synergize or cooperate with the standard-of-care drug 5-FU and the FDA-approved oral rheumatoid drug leflunomide.

[0278] Example 5: Tumor CKB expression as a predictive biomarker of SLC6A8 inhibition response To identify predictive biomarkers for therapeutic response to SLC6A8 inhibition with RGX-202, we performed the following experiments. Therapeutic efficacy was observed upon SLC6A8 inhibition in both KRAS mutant, KRAS wild-type, and mismatch repair-mutant CRC lines, suggesting that sensitivity to SLC6A8 inhibition is not solely due to oncogenic or genomic instability mutational backgrounds (Figures 9 and 10). It has previously been shown that the CKB enzyme acts upstream of the SLC6A8 transporter by generating the energy metabolite phosphocreatine, which is released from CRC cells and transported via the SLC6A8 transporter (Loo et al., supra). Extracellular phosphocreatine supplementation rescued hypoxic survival impairment caused by CKB depletion in an SLC6A8-dependent manner (Loo et al., supra). CKB expression was also shown to be suppressed by microRNA-mediated silencing, raising the possibility that variations in its expression may predict sensitivity to therapeutic targeting of this axis (Loo et al., supra). A collection of gastrointestinal cell lines as xenografts was tested for responsiveness to RGX-202-01 (cell line details are shown in Figure 10). Independent cohorts of animals were inoculated simultaneously with the same xenografts, and tumors were grown to approximately 500 mm 3At that time, tumor SLC6A8 and CKB gene expression was assessed by qPCR and CKB protein expression by immunohistochemistry (IHC) (Figure 4A). SLC6A8 was not tested by IHC due to the lack of availability of an SLC6A8-specific antibody. This assessment included tumors that responded to treatment and tumors that did not respond (Figures 4B-4D, 10, and 7). Therapeutic response to RGX-202-01 was positively correlated with increased CKB mRNA expression (Figure 4E), but not with SLC6A8 mRNA expression (Figure 8A). Consistent with this, the degree of CKB protein expression, as assessed by tumor proportionality score (TPS), also positively correlated with the degree of therapeutic response (Figure 4F). Tumors expressing elevated CKB protein levels (Figure 4B) showed a greater tumor growth inhibitory response compared with tumors expressing decreased CKB levels (Figure 4D and Figures 8B and 8C). To determine whether this observation could be replicated in an independent dataset, we evaluated the association between CKB protein expression and treatment response in the murine RGX-202-01 1x1 PDX study described above. A similar predictive association between elevated CKB protein expression by IHC and antitumor response was observed (Figure 4G). Creatine kinase is a dimeric enzyme consisting of two subunits: CKB (brain-type) or CKM (muscle-type). Thus, three distinct isoenzymes exist: CK-MM, CK-BB, and CK-MB. To determine whether any of the other isoforms may contribute to efficacy, we performed a study measuring CKM expression in tumors by qPCR. Analysis of the 11 xenografts described above revealed significantly lower expression of CKM compared to CKB (Figure 8D), and no correlation between CKM expression and antitumor efficacy was observed (Figure 8E). Expression of two mitochondrial creatine kinases, CKMT1 and CKMT2, was also assessed. Although both enzymes were detectable, CKMT2 expression was significantly lower than CKB expression, with 4 / 11 xenografts having undetectable CKMT2 expression. No correlation between CKMT expression and response to RGX-202 treatment was observed (Figures 8D, 8F, and 8G).These results demonstrate that elevated tumor CKB mRNA and protein expression predicts enhanced responsiveness to SLC6A8 inhibition by RGX-202-01, consistent with CKB being an upstream component of the CKB / SLC6A8 phosphocreatine metabolic axis in CRC and a gene that exhibits post-transcriptional regulation and mutation in cancer. Consistent with the proposed mechanism of action of RGX-202-01, a positive correlation of response to RGX-202-01 was observed with induction of cleaved caspase-3, a marker of apoptosis, and a negative correlation of response to RGX-202-01 was observed with induction of the proliferation marker Ki67 (Figures 8H-8I).

[0279] To assess the proportion of patients with metastatic CRC with tumors containing elevated CKB protein expression, 23 human metastatic CRC specimens were immunohistochemically stained for CKB. CKB positivity (TPS > 5%) was observed in the majority of tumor tissue samples (56%; 13 / 23). Overall, these findings identify CKB as a patient stratification biomarker for the clinical development of SLC6A8 inhibitors. Next, CKB expression was assessed in tissue microarrays containing specimens from 10 human tissues. The highest CKB expression, with a TPS of 100%, was observed in the brain, with heterogeneous expression patterns observed in several tissues, including the colon, appendix, bladder, kidney, myometrium, and pancreas.

[0280] Example 6: RGX-202-01 increases serum and urinary creatine excretion in patients with cancer Motivated by the robust preclinical efficacy of RGX-202-01, we initiated a multicenter Phase 1a / b clinical trial in patients with advanced gastrointestinal cancer who had progressed on standard treatment regimens (ClinicalTrials.gov, NCT03597581). During the Phase 1a dose-escalation phase of this study, patients received oral RGX-202-01 treatment twice daily at doses ranging from 600 mg to 3600 mg in a continuous regimen. Blood and urine samples were collected from 13 patients on day 15 during the first cycle (28 days), and bioanalysis was performed by an independent contract laboratory using a commercially available, proprietary assay. Creatine concentrations in both serum and urine positively correlated with systemic exposure to RGX-202-01 (Figures 4H and 4I). These findings provide proof-of-concept for therapeutic targeting of creatine metabolism in humans and mirror experimental observations in mice.

[0281] The creatine / phosphocreatine bioenergetic shuttle is a critical system that enables highly metabolic tissues to rapidly respond to energy stress by generating high-energy ATP in reactions that do not require oxygen. CRC and pancreatic cancer are particularly hypoxic malignancies. The ability of these cancers to overexpress and release CKB as a means of generating extracellular phosphocreatine for transport via the SLC6A8 transporter allows cancer cells to increase the availability of high-energy phosphate for ATP generation under conditions of hypoxia and metabolic stress. SLC6A8 inhibition with RGX-202-01 reduced tumor growth in multiple syngeneic, xenograft, and PDX mouse models. Antitumor efficacy was associated with enhanced tumor apoptosis and reduced tumor cell proliferation, consistent with previous findings supporting the critical role of creatine metabolism in CRC progression and hypoxic survival (Loo et al., supra). These results provide clear evidence that RGX-202-01 targets the SLC6a8 creatine / phosphocreatine axis. Without intending to be bound by theory, it is possible that RGX-202-01 may mediate additional anti-tumor effects through additional target(s).

[0282] This study revealed that mutational background was not a significant predictor of response to SLC6A8 inhibition, and RGX-202-01 impaired the growth of a broad set of CRC tumors with different KRAS and mismatch repair mutation backgrounds, suggesting that diverse CRC tumors utilize creatine / phosphocreatine metabolism to drive progression. Consistent with a critical role for CKB in this pathway, CKB expression levels were associated with response to RGX-202. Tumors with high CKB expression responded better than tumors with reduced levels, suggesting that CKB-overexpressing tumors are more dependent on phosphocreatine as an energy source and therefore more sensitive to its depletion. These findings support the use of patient tumor CKB expression as a molecular biomarker for patient stratification in clinical trials.

[0283] These data also demonstrate that inhibition of SLC6A8 and blockade of cellular creatine / phosphocreatine uptake leads to increased urinary creatine excretion in both mice and patients. Urinary creatine levels, which positively correlate with blood RGX-202-01 concentrations, represent a direct measure of SLC6A8 inhibition. The opportunity for noninvasive monitoring of the pharmacokinetics of creatine transport inhibition by urine analysis of creatine and potentially additional metabolites provides a simple and rapid method for assessing target engagement in patients.

[0284] These findings demonstrate both the single-agent activity and combination efficacy of RGX-202-01 in CRC. Because 5-FU is the primary chemotherapeutic agent used in CRC, we tested the activity of RGX-202 in combination with 5-FU and 5-FU / irinotecan. The data demonstrated synergistic effects of SLC6A8 inhibition and complete tumor regression with these standard of care regimens. Overall, these results provide a rationale for incorporating RGX-202-01 into combination regimens, including standard of care agents or those targeting nucleotide synthesis.

[0285] Overall, this study supports the therapeutic targeting of creatine metabolism in CRC through inhibition of the SLC6A8 transporter. This therapeutic approach is currently being tested in a multicenter, national Phase 1b / 2 clinical trial for CRC (ClinicalTrials.gov, NCT03597581).

[0286] Example 7: RGX202-01 + FOLFIRI dose escalation clinical trial RGX202-01 monotherapy was shown to be safe and effective in subjects with gastrointestinal (GI) adenocarcinoma. To determine whether RGX202-01 could be effective in combination with the folinic acid-fluorouracil-irinotecan regimen (FOLFIRI), a conventional therapy for colorectal cancer, a combined dose-escalation study of RGX-202-01 with FOLFIRI was conducted. Eleven patients were enrolled, including those with gastrointestinal (GI) adenocarcinoma (median prior treatment history). Dose levels of 1800 mg twice daily (BID), 2400 mg BID, and 3000 mg BID were eliminated without significant adverse events. 600 mg tablets were available in a highly compressed salt form of the API.

[0287] RGX-202-01 and FOLFIRI were administered to seven patients with colorectal cancer (CRC). Six of the seven evaluable patients (85.7%) had stable disease. The median duration of treatment (PFS surrogate) was approximately 14 weeks. Patients had a median progression-free survival (PFS) of only approximately 8 weeks with TAS-102 and regorafenib, both FDA-approved in patients with late-line CRC.

[0288] Example 8: Clinical Activity in Second-Line (2L) CRC with RGX202-01 + FOLFIRI / Bevacizumab Eight patients with colorectal cancer were enrolled in a second-line dose-escalation cohort and received 2400 mg or 3000 mg of RGX-202-01 twice daily (BID). Five patients remained on treatment (Figure 11). One patient was pending initial efficacy evaluation, and two patients were not evaluable (off-treatment). 40% of patients (2 of 5) had a partial response to treatment. 60% (3 of 5) had stable disease, and none of the patients had progressive disease. This result was particularly encouraging, considering that previous overall response rates with FOLFIRI plus bevacizumab in 2L CRC have been approximately 5-10% and median progression-free survival approximately 5-6 months (see, e.g., Bennouna et al., Lancet Oncol 2013;14:29 37).

[0289] Example 9: Confirmed partial response (PR) in second-line CRC patients treated with RGX202-01 2400 mg twice daily plus FOLFIRI / bevacizumab Patient 2408 was further evaluated in a second-line trial (Example 8) and confirmed to be a partial responder to the combination therapy of RGX202-01 and FOLFIRI / bevacizumab. Patient 2408 was a 52-year-old man with metastatic KRAS G12D-mutated colorectal cancer (CRC). Prior to treatment, the patient was refractory to oxaliplatin-capecitabine with progressive disease. Response Evaluation Criteria in Solid Tumors (RECIST), a published set of rules that defines when a cancer patient's tumor improves, was used to measure how well the cancer patient responded to treatment. This is based on whether the tumor shrinks, stays the same, or grows. To use RECIST, there must be at least one tumor that can be measured by X-ray, CT scan, or MRI scan. The types of responses a patient can have are complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD). RECIST 1.1 patients were evaluable for response if they had measurable disease and at least one follow-up scan with at least one cycle of treatment.

[0290] At the end of the cycle, patient 2408 had a 230% reduction in target lesions (multiple liver and lung metastases). A confirmatory scan was completed 4 weeks later and confirmed a partial response to treatment with a 31% reduction in tumor size. At the end of the C4 scan, stable disease was observed with 19% tumor growth from nadir (i.e., regional minimum relative tumor size change value).

[0291] Example 10: PR in 2L CRC patients treated with RGX202-01 3000 mg twice daily + FOLFIRI / bevacizumab Patient 105-2501 was another partial responder. Patient 105-2501 was a 57-year-old woman with metastatic KRAS G12C-mutated colorectal cancer (CRC). The patient had previously been treated with oxaliplatin-capecitabine, with stable disease as the best response. A partial response by RECIST 1.1 was observed in patient 105-2501 on the first scan at the end of cycle 2. A 31% reduction in target lesions (multiple abdominal wall metastases) was observed.

[0292] Example 11: Clinical Activity Observed in RGX-202-01 Monotherapy and Combination Dose-Escalating Cohorts Patients enrolled in the monotherapy and combination dose-escalation cohorts were highly refractory and not preselected based on CKB status (CKB positivity was not required for enrollment in dose escalation). Clinical responses were observed in CRC patients receiving RGX-202-01 at ≥2400 mg BID as monotherapy or in combination with chemotherapy. A total of 20 single-agent / combination patients were evaluable for response. Four evaluable patients received RGX202 at doses ≥2400 mg twice daily. Of the nine KRAS-mutated patients evaluated, two had partial responses (PR) (22%), six had stable disease (SD) (67%), and one had progressive disease (PD) (11%) when treated with RGX-202-01 monotherapy and combination therapy. Of the five KRAS WT / unknown patients, 0 were partial responders (0%), 1 had stable disease (20%), and 4 still showed progressive disease (PD) (80%) to RGX-202-01 monotherapy and combination therapy. Thus, the greatest clinical benefit was observed in KRAS mutant patients in both the monotherapy and combination cohorts (Figure 12).

[0293] Example 12: RGX-202-01 activity in mouse PDX models is associated with tumor CKB expression CKB expression (by tumor immunohistochemistry) was evaluated in human patient-derived xenograft (PDX) models. Tumor response data for the same models were available for 54 of 60 PDX animals with colorectal cancer (CRC) (n=43), gastric cancer (n=6), and pancreatic cancer (n=5). Significantly higher RGX-202 therapeutic efficacy activity was observed in CKB-positive PDX models (tumor proportion score (TPS) cutoff of 5% or greater) compared to CKB-negative models (TPS less than 5%) (Figure 13).

[0294] Example 13: RGX-202-01 has activity in CRC and gastric cancer PDX mouse models. The therapeutic activity of RGX-202-01 was observed in various genetic subtypes of colorectal cancer (CRC) and gastric cancer PDX models, including: 1) KRAS wild-type and mutant CRC (G12C, G12D, G12S, and G12V), 2) KRAS wild-type and mutant gastric cancer, 3) HER2+ (mutated and amplified) CRC and gastric cancer, and 4) Activity in PDX models previously treated with chemotherapy and / or EGFR inhibitors.

[0295] Example 14: Pharmacokinetic and Pharmacodynamic Data for RGX-202-01 and FOLFIRI Pharmacokinetic and pharmacodynamic data were obtained from 28 subjects treated with RGX-202-01 as monotherapy or in combination with FOLFIRI across multiple dose-escalation cohorts. Systemic exposure to RGX-202-01 was slightly greater than dose-proportional both as monotherapy and in combination with FOLFIRI. Steady state was achieved by Day 15 of Cycle 1.

[0296] The effect of RGX-202-01 on creatine metabolism was monitored by measuring creatine, creatinine, guanidinoacetic acid, and creatine phosphokinase levels in urine and serum / plasma obtained from treated subjects. By day 15 of cycle 1, a dose-dependent increase in serum and urinary creatine levels was observed. Systemic exposure to RGX-202-01 (area under the curve: AUC) positively correlated with urinary and serum creatine. Patients with partial response or stable disease demonstrated higher and more sustained increases in serum and urinary creatine concentrations than patients with progressive disease.

[0297] CKB protein expression in tumor samples from patients was assessed using a Clinical Laboratory Improvement Amendments (CLIA)-validated immunohistochemistry (IHC) assay. Total treatment duration tended to be higher in patients with CKB+ tumors. Tumor samples from patients with better treatment outcomes, i.e., SD / PR, were CKB+. Baseline levels of urinary creatine, creatinine, and guanidinoacetic acid (GAA) were higher in patients with CKB+ tumors.

[0298] RGX-202-01-mediated changes in creatine metabolism in patients with progressive disease (PD) versus patients with partial responders / stable disease (PR / SD). Patients with favorable treatment outcomes demonstrated more robust and sustained changes in pharmacodynamic markers of RGX-202-01 target engagement. After two cycles of treatment, absolute serum and urinary creatine levels in patients with PR / SD showed an increase over time compared to those observed in patients with PD. This was more pronounced in patients treated with RGX-202-01 + FOLFIRI. The pharmacodynamic effect was more pronounced in urine than in serum (Figure 14). Figure 15 shows the target level changes in creatine metabolism.

[0299] Example 15: Combination therapy with RGX-202-01 and a KRAS inhibitor for the treatment of KRAS-mutated cancer The target engagement and clinical relevance of RGX-202-01 as a single agent and in combination with injectable 5-fluorouracil (5-FU) and leucovorin plus irinotecan (FOLFIRI), as well as in combination with FOLFIRI plus bevacizumab, will be demonstrated by assessing the effect of SLC6a8 inhibition by RGX-202-01 administration on metabolite levels such as creatine, and by the level of CKB expression by immunohistochemistry in tumor tissue. The relevance of serum creatinine and creatine levels as pharmacodynamic markers of creatine transport inhibition will be assessed by assessing the dose-responsiveness of changes in each of these markers following RGX-202-01 administration and combination therapy, such as RGX-202-01 plus a Kras inhibitor from Table 1. The primary objective during the dose-escalation phase is to identify the maximum tolerated dose (MTD) of RGX-202-01, or the highest test dose at which no multiple dose-limiting toxicities (DLTs) are observed, as a single agent and separately, in combination with infusional 5-fluorouracil (5-FU) and leucovorin plus irinotecan (FOLFIRI), and in combination with FOLFIRI plus bevacizumab.

[0300] The main objectives during the expansion phase are: ●The primary efficacy objective is to estimate the antitumor activity of RGX-202-01 in combination with FOLFIRI + bevacizumab in patients with previously treated advanced or metastatic colorectal cancer and tumors expressing the CKB biomarker. The primary safety objective is to characterize the safety profile of RGX-202-01 in combination with FOLFIRI plus bevacizumab at the MTD or maximum tested dose. Secondary objectives are to evaluate the pharmacokinetic (PK) profile of RGX-202-01 and potential metabolites in plasma and urine.

[0301] Expression of CKB and other related markers involving creatine metabolism in tumor samples will be assessed prior to RGX-202-01 treatment and correlated with clinical parameters reflecting anti-cancer activity after treatment. Assessment of pharmacodynamic markers, including but not limited to creatine, creatinine, guanidinoacetic acid (GAA), and lipid levels (including cholesterol and triglycerides), will be measured before, during, and after RGX-202-01 treatment.

[0302] Multiple doses of orally administered RGX-202-01, with or without FOLFIRI ± bevacizumab (monotherapy or combination therapy), will be evaluated in patients with advanced gastrointestinal tumors (i.e., locally advanced and unresectable, or metastatic) who have had progressive disease (PD) on available standard systemic therapy or for whom there is no standard systemic therapy with relevant clinical impact.

[0303] The clinical starting dose for oral administration of RGX-202-01 as a single agent is 1200 mg / day.

[0304] The starting dose of RGX-202-01 in combination with FOLFIRI and bevacizumab was at least one dose level below the last dose of RGX-202-01 evaluated as monotherapy and was not considered the MTD. The MTD will be determined for RGX-202-01 as monotherapy and in combination with FOLFIRI. The MTD is defined as the highest dose level at which fewer than 33% of patients experience dose-limiting toxicity (DLT) during the DLT evaluation period. Toxicity will be assessed using the National Cancer Institute's (NCI) Common Terminology Criteria for Adverse Events (CTCAE), version 5.

[0305] For all subjects receiving RGX-202-01 combination therapy, the FOLFIRI dose and schedule was the conventional FOLFIRI dosing regimen most commonly used to treat a wide variety of gastrointestinal tumors: irinotecan 180 mg / m on days 1 and 15 of each 28-day cycle. 2 intravenously over 90 minutes, concomitantly with folinic acid 400 mg / m 2intravenously over 2 hours, followed by 5-FU at 2400 mg / m 2 The drug is infused intravenously over 46 hours.

[0306] For all subjects receiving RGX-202-01 in combination with bevacizumab, the bevacizumab dose and schedule will be 5 mg / kg on days 1 and 15 of each 28-day cycle, which is one of the approved bevacizumab dosing regimens for the treatment of colorectal cancer.

[0307] In one embodiment, the starting dose regimen for single-agent RGX-202-01 is 600 mg BID on days 1-28 of a 28-day cycle. The starting dose of RGX-202-01 in combination with FOLFIRI is at least one dose level below the last dose of RGX-202-01 completed as monotherapy that did not meet the MTD. The starting dose of RGX-202-01 in combination with FOLFIRI plus bevacizumab is at least one dose level below the last dose of RGX-202-01 evaluated in combination with FOLFIRI that did not meet the MTD.

[0308] Patients receive their first dose on day 1 of cycle 1. Treatment cycles are 28 days long. All patients receive RGX-202-01 orally (PO) on a continuous daily schedule. For all RGX-202-01 patients receiving combination therapy, the FOLFIRI dose and schedule is irinotecan 180 mg / m2 intravenously over 90 minutes, concomitant folinic acid 400 mg / m2 intravenously over 2 hours, followed by 5-FU 2400 mg / m2 intravenously over 46 hours on days 1 and 15 of each 28-day cycle, and the bevacizumab dose is 5 mg / kg on days 1 and 15 of each 28-day cycle.

[0309] During treatment, patients are evaluated on an outpatient basis. After discontinuation of therapy, patients are evaluated within 21 days after the last dose of therapy. Follow-up for disease status and survival after discontinuation of therapy can continue for up to 12 months after initiation of therapy.

[0310] Safety will be assessed during the study by documentation of adverse events (AEs), clinical laboratory tests, physical examinations, vital sign measurements, electrocardiograms (ECGs), and Eastern Cooperative Oncology Group (ECOG) performance status (PS). Serial blood samples for PK and pharmacodynamic analyses will be collected from all patients.

[0311] Prior to treatment, imaging (chest / abdominal / pelvic computed tomography [CT] scan or magnetic resonance imaging [MRI], if indicated) and tumor biopsy will be performed. Patients with cutaneous, subcutaneous, or lymph node metastases will also have tumor assessment by physical exam (including ruler measurements). Tumor measurements and disease response assessments will also be performed at the end of Cycle 2 (approximately 8 weeks after the first study therapy administration) and approximately every 8 weeks thereafter until PD develops. For patients with evidence of disease control (stable disease or better) at Week 24, tumor measurements and disease response assessments will be performed less frequently thereafter (approximately every 16 weeks).

[0312] Tumor measurements and disease response assessments will also be performed at the end of therapy. Additionally, measurement of tumor markers (i.e., CEA, CA19-9, CA15-3) is highly recommended in situations where tumor markers are used in the clinical management of patients with a given malignancy and the patient has a known elevation of the given marker.

[0313] Example 16: Single Agent Dose Escalation Following pre-treatment evaluation, subjects will receive RGX-202-01 orally (PO) twice daily (BID) at a total daily dose of 1200 mg / day (600 mg BID) on days 1-28 of a 28-day cycle. Dose escalation will follow a standard 3+3 design.

[0314] Example 17: Combination Dose Escalation and Expansion Combination therapy dose escalation of RGX-202-01, FOLFIRI, and bevacizumab will begin after the maximum tolerated dose (MTD) of single-agent RGX-202-01 has been identified or at least two cohorts of RGX-202-01 monotherapy have been completed.

[0315] The starting dose of RGX-202-01 in combination with FOLFIRI in the subject is at least one dose level below the last dose of RGX-202-01 evaluated and not considered the MTD. The starting dose of RGX-202-01 in combination with FOLFIRI and bevacizumab is at least one dose level below the last dose of RGX-202-01 evaluated in combination with FOLFIRI and not considered the MTD. A total of approximately 27 patients will be treated at the MTD of RGX-202-01 in combination with FOLFIRI and bevacizumab.

[0316] Study population During the dose-escalation phase, 24-36 patients with gastrointestinal tumors that are relapsed / refractory to currently available therapies will be treated. During the expansion phase, approximately 24 patients in a single-dose expansion cohort with malignant colorectal tumors will be treated.

[0317] Example 18: Treatments, Dosages, and Modes of Administration RGX-202-01 RGX-202-01 capsules, 200 mg, or tablets, 400 mg, are administered PO. The single-agent starting dose regimen for RGX-202-01 (i.e., the dose regimen in Cohort 1) is 1200 mg / day in divided doses of 600 mg BID on days 1-28 every 28 days.

[0318] Additional creatine transporter inhibitors that may be used are shown in FIG.

[0319] FOLFIRI The FOLFIRI chemotherapy regimen, consisting of irinotecan, folinic acid (or leucovorin), and 5-fluorouracil, is administered as follows: on days 1 and 15 of each 28-day cycle, irinotecan is administered intravenously at 180 mg / m over 90 minutes, concomitantly with folinic acid (leucovorin) at 400 mg / m over 2 hours, followed by 5-FU at 2400 mg / m over 46 hours.

[0320] Bevacizumab Bevacizumab is a vascular endothelial growth factor inhibitor administered at a dose of 5 mg / kg on days 1 and 15 of each 28-day cycle.

[0321] Efficacy assessments, including disease response and progression, duration of response, and survival, will be assessed using the following methods.

[0322] Tumor tissue (at least 5 and up to 15 unstained slides or paraffin blocks) obtained from archival tissue samples or fresh tissue biopsies will be assessed for expression of the CKB biomarker and potentially other markers associated with the phosphocreatine transport pathway.

[0323] Serum and / or plasma will be collected both prior to the first dose of RGX-202-01 and at selected time points after initiation of RGX-202-01 to assess levels of creatine, creatinine, and other pharmacodynamic biomarkers.

[0324] In patients, plasma concentrations of RGX-202-01 and metabolites will be measured on Days 1 and 15 of Cycle 1. Limited plasma concentrations will be assessed at other time points to measure RGX-202-01 concentrations and identify metabolites at steady state in these patients. Plasma concentration data over time will be used to characterize pharmacokinetic (PK) trends of RGX-202-01 and metabolites, evaluate changes in RGX-202-01 PK characteristics between the first dose and steady state and between treatment cycles, and relate RGX-202-01 PK characteristics to toxicity and anticancer activity. Plasma concentrations will be related to QTc interval and interval changes. For patients enrolled in the dose-escalation phase, pooled urine will be collected during the 12-hour dosing interval after the first dose on both Days 1 and 15 of Cycle 1 to measure RGX-202-01 excretion and identify potential metabolites in urine. A pretreatment spot urine sample will be collected prior to Day 1 of Cycle 1.

[0325] Statistical methods: The results obtained in relation to RGX-202-01 as a single agent and in combination therapy will be evaluated using the following statistical methods: Demographic characteristics (e.g., sex, age, race) and baseline characteristics (e.g., Eastern Cooperative Oncology Group (ECOG) performance status, height, weight, and prior therapy) will be summarized by RGX-202-01 dose group and tumor type using descriptive statistics.

[0326] Treatment-emergent AEs up to 30 days after the last single-agent dose will be summarized by MedDRA™ version 21.0 (or higher) system organ class and preferred term. The incidence and percentage of patients experiencing each AE will be summarized using descriptive statistics. AEs will be summarized by National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE), version 5 (or higher) grade and causality (attributed to study treatment). Dose-limiting toxicities, grade 3-4 AEs, serious adverse events (SAEs), and AEs leading to dose modification or treatment discontinuation will be summarized by preferred term.

[0327] Test results will be classified according to NCI-CTCAE, version 5 (or higher). Test results that do not correspond to NCI-CTCAE terminology will not be graded. The incidence of test abnormalities will be summarized using descriptive statistics.

[0328] Vital signs and physical examination results will be summarized using descriptive statistics.

[0329] ORR is defined as the proportion of patients achieving a best response of complete response (CR) or partial response (PR) according to RECIST version 1.1. Duration of response (DoR) is defined as the time from the date when a criterion for PR or CR was first met to the date when a criterion for PD was first met. DoCR is defined as the time from the date when a criterion for CR was first met to the date when a criterion for PD was first met. PFS is defined as the time from the start of study treatment to the date when a criterion for PD or death from any cause, whichever occurs first. OS is defined as the time from the start of study treatment to the date of death from any cause. Tests for ORR are performed using a one-sample binomial test with a one-sided type I error rate of 10%. Distributions for PFS, DoR, DoCR, and OS are estimated using the Kaplan-Meier method.

[0330] Multiple doses of orally administered RGX-202-01, with or without FOLFIRI ± bevacizumab (monotherapy or combination therapy), will be evaluated in patients with advanced gastrointestinal tumors (i.e., locally advanced and unresectable, or metastatic) who have had progressive disease (PD) on available standard systemic therapy or for whom there is no standard systemic therapy with relevant clinical impact.

[0331] Seventeen patients are being treated in the single-agent dose-escalation phase of the study, and 10 patients are being treated in the FOLFIRI dose-escalation phase of the study.

[0332] Table 4. Patient and disease characteristics, Study RGX-202-01 TIFF2024528705000079.tif90155

[0333] Treatment administration by dose and incidence of DLTs for monotherapy and combination therapy during the dose-escalation phase are summarized in Error! Reference source not found. 5. No DLTs were observed among the 13 evaluable patients in the monotherapy cohort and the 9 evaluable patients in the combination with FOLFIRI cohort as of the data cutoff date.

[0334] Table 5. Treatment Regimen, DLTs, and Response, Mono- and Combination Therapy Dose Escalation Phase, Study RGX-202-001 TIFF2024528705000080.tif97164

[0335] Patients who are deemed eligible based on screening assessments will be enrolled in the study and receive their first dose of study therapy on Day 1 of Cycle 1. Treatment cycles are 28 days long. All patients will receive RGX-202-01 orally (PO) on a continuous daily schedule. The RGX-202-01 dose regimen will depend on the cohort in which the patient is enrolled. For all RGX-202-01 dosing cohorts receiving combination therapy, the FOLFIRI dose and schedule will consist of irinotecan at 180 mg / m on Days 1 and 15 of each 28-day cycle. 2 intravenously over 90 minutes, concomitantly with folinic acid 400 mg / m 2 intravenously over 2 hours, followed by 5-FU at 2400 mg / m 2 over 46 hours, and the bevacizumab dose is 5 mg / kg on days 1 and 15 of each 28-day cycle.

[0336] Dose Escalation Scheme Dose escalation will begin with single-agent RGX-202-01. When at least two cohorts of patients treated with single-agent RGX-202-01 have been evaluated, dose escalation will begin with RGX-202-01 in combination with fixed-dose FOLFIRI. The following text describes dose escalation for monotherapy but also applies to RGX-202-01 in combination therapy.

[0337] The starting dose regimen for single-agent RGX-202-01 is 600 mg BID on days 1-28 of a 28-day cycle. The starting dose of RGX-202-01 in combination with FOLFIRI is at least one dose level below the last dose of RGX-202-01 completed as monotherapy and not considered the MTD. The starting dose of RGX-202-01 in combination with FOLFIRI plus bevacizumab is at least one dose level below the last dose of RGX-202-01 evaluated in combination with FOLFIRI and not considered the MTD.

[0338] Dosage and Administration of FOLFIRI and Other Concomitant Therapies All three cytotoxic therapies are administered parenterally on a 14-day schedule in the FOLFIRI regimen, which is commonly used to treat patients with advanced colorectal and gastric cancer and is being studied in patients with pancreatic and bile duct cancer.

[0339] For all RGX-202-01 dosing cohorts receiving combination therapy, the FOLFIRI dose and schedule was irinotecan 180 mg / m on days 1 and 15 of each 28-day cycle. 2 intravenously over 90 minutes, concomitantly with folinic acid 400 mg / m 2 intravenously over 2 hours, followed by 5-FU at 2400 mg / m 2 The dose is administered intravenously over 46 hours.

[0340] The following methods and materials were used to obtain the results given in Examples 1-7.

[0341] Experimental design The sample size for each group was selected based on knowledge of within-group variability and expected effect size. Sample sizes for in vitro experiments were selected based on prior knowledge of within-group variability. Data were collected until a predetermined endpoint (in vitro assays, PDX studies, and clinical sample analysis) was reached or a tumor burden of 2000 mm 3 Data were collected until >10 ...

[0342] animal lineage All mouse experiments and procedures were approved by the Institutional Animal Care and Use Committees (IACUC) of the New York Blood Center (NYBC), The Rockefeller University, and Crown Biosciences. C57BL / 6 (JAX stock #000664, RRID:IMSR_JAX:000664), NOD-SCID (JAX stock #001303), athymic nude (J:NU, JAX stock #007850), NOD scid gamma (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, JAX stock #005557), and B6129SF1 / J (JAX stock #101043) mice were purchased from The Jackson Laboratory. BALB / c mice (stock #028) were purchased from Charles River. SLC6A8 knockout mice were originally obtained from the laboratory of Dr. Skelton (34) and bred in-house. BALB / c nude mice were purchased from Beijing Anikeeper Biotech Co., Ltd. (Beijing, China).

[0343] Primary tumor growth studies For primary tumor growth experiments, cells (suspended in 50 μl of PBS) were mixed 1:1 with Matrigel (356231, BD Biosciences, Bedford, MA) and injected subcutaneously unilaterally or bilaterally into the lower flanks of 6- to 8-week-old, gender-matched mice. Upon detecting tumor volumes reaching the sizes indicated in each figure, mice were randomly assigned to drug-treated or control cohorts. RGX-202 was administered at the indicated doses through a formulated drug diet (Purina 5001, Research Diet, New Brunswick, NJ) or formulated in sterile drinking water for oral gavage. Control cohorts received either a regular diet (Purina 5001) or vehicle control. When xenograft models were tested for biomarker analysis, 10 to 15 animals were inoculated with the indicated cell lines. Of these animals, 7 to 10 mice were assigned to the efficacy portion and treated for the duration of the experiment. The remaining 3 to 5 animals had tumors approximately 500 mm 3 Control tumors were sacrificed when they reached a median age of 100. Protein and RNA analysis of CKB expression was performed on control tumors. Tumor growth measurements were performed using digital calipers on the indicated days throughout the course of the experiment. Tumor volume was calculated using the formula: Volume = (longest diameter) / 2 × (shortest diameter). 2 For survival analysis, the total tumor burden was calculated as 2000 mm in volume. 3 Mice were euthanized when IACUC guidelines were met with a tumor burden exceeding 100 mg / kg. Tumor growth inhibition (TGI) was calculated using the formula TGI (%) = (((ΔC i -ΔC0)-(ΔT i -ΔT0)) / (ΔC i Calculated using -ΔC0))*100%.

[0344] Patient-derived xenograft (PDX) research PDX transplantation was performed as previously described (22). Briefly, 20–30 mm 3Tumor fragments were implanted subcutaneously bilaterally into the flanks of 6- to 8-week-old age-matched athymic nude mice under general anesthesia with 100 mg / kg ketamine (995-2949-100 mg / mL, Henry Schein Animal Health, Melville, NY) and 10 mg / kg xylazine (X1251, Sigma Aldrich, St. Louis, MO).

[0345] Metastasis assay The experiment was performed using 5 x 10 cells suspended in 50 μL of PBS. 5 Luciferase-labeled Lvm3b or CKB CRISPR knockout or CRISPR control Lvm3b cells were injected intrasplenicly into 6-8 week-old NOD SCID or NSG mice anesthetized with an IP injection of ketamine / xylazine solution (100 mg / kg ketamine, 10 mg / kg xylazine). The day after tumor cell inoculation, mice were randomly assigned to either the control or RGX-202 treatment groups. Control mice received an IP injection of 200 μL of PBS, and treated mice received 200 μL of 0.5 M RGX-202 (approximately 650 mg / kg). Treatment continued daily until the end of the experiment. For PANC1 experiments, cells grown in D10F complete medium were pretreated in vitro for 48 hours with or without RGX-202 at a dose of 10 mM (1.31 mg / mL) RGX-202 before injection into mice. After cell injection, the mice received no treatment. Bioluminescence measurements were performed once a week for the duration of the experiment. 100 μL of D-luciferin (88292, Thermofisher, Waltham, MA, 1 g in 60 mL of sterile DPBS) was injected into the venous sinus, and the bioluminescence signal on the liver was measured using an IVIS Spectrum in vivo imaging system (Perkin Elmer). The photon flux ratio is the ratio of the bioluminescence signal at a given time point to the signal on day 0.

[0346] Drug treatment 5-Fluorouracil (5-FU) (F6627, Millipore Sigma, St. Louis, MO) was administered once weekly by intraperitoneal injection (ip) in sterile 0.9% NaCl as indicated. Gemcitabine (G6423, Millipore Sigma, St. Louis, MO) was administered ip at 100 mg / kg / week in PBS. Irinotecan (I1406-50 mg, Millipore Sigma, St. Louis, MO) was administered ip at 15 mg / kg / week in 2.5% DMSO 97.5% NaCl 0.9%. Leflunomide was formulated in DMSO and administered by daily ip injection at 2.5 or 7.5 mg / kg in 0.5 mL / kg. Uridine was formulated in sterile water at 800 mg / kg and administered by daily ip injection.

[0347] cell culture UN-KPC-961 cells were obtained from Professor S.K. Batra at the Eppley Institute for Research in Cancer (Omaha, Nebraska). COLO 205 (CCL-222), HCT-15 (CCL-225), HT-29 (HTB-38), SW480 (CCL-228), HCT116 (CCL-247), HCT-8 (CCL-244), NCI-H508 (CCL-253), HepG2 (HB-8065), Hs746T (HTB-135), CT26 (CRL-2638), LS-174T (CL-88), PANC1 (CRL-1469), and NCI-N87 (CRL-5822) cell lines were purchased from ATCC (Baltimore, MD) and maintained under standard conditions according to the supplier's instructions. Lvm3b was generated by in vivo selection from the parental cell line LS-174T (ATCC, Baltimore, MD) ( 15 ). UN-KPC-961 cells were maintained in DMEM (11960-044, Gibco, Langley, OK), 7.5% sodium bicarbonate (25080-094, Gibco, Langley, OK), 1% penicillin-streptomycin (Lonza, 17-745E), 10% fetal bovine serum (F4135, Sigma, St. Louis, MO), 200 mM L-glutamine (25030081, Gibco, Langley, OK), 1 mM HEPES (15630080, Gibco, Langley, OK), and 50 mg / ml gentamicin (15750078, Gibco, Langley, OK). MC38 cells were cultured in DMEM, 10% fetal bovine serum, and 1 mM HEPES.

[0348] 1×1 PDX trial (HuTrial) Model details can be found in Figure 9. PDX models were inoculated with PDX cell suspensions or tumor fragments as follows: A cryovial containing PDX tumor cells was thawed, and the cells were washed in RPMI, counted, and resuspended in cold RPMI at a concentration of 50,000–100,000 viable cells / 50 mL. The cell suspension was mixed with an equal volume of Cultrex ECM. 100 μL of the cell suspension in ECM medium was injected subcutaneously into the hind flank of five female NOD-SCID mice per model. Alternatively, tumor fragments from stock mice were harvested and used to inoculate the mice. Five 6- to 8-week-old female BALB / c nude mice per model were inoculated subcutaneously into the right flank with primary human tumor fragments (2–3 mm in diameter) for tumor development. Two of these five mice were grown to a mean tumor volume of 100–150 mm. 3 When the mice reached 21 days of age, they were randomized into two groups (one mouse per group), and treatment with either a control diet (Purina 5001) or an RGX-202-formulated diet (Purina 5001, Research Diet, New Brunswick, NJ) at approximately 400 mg / kg was initiated within 24 hours of randomization. Randomization was performed based on the "Matched distribution" method (StudyDirector™ software, version 3.1.399.19). Tumor growth inhibition (TGI) at day 21 was assessed. 21 ) is the formula TGI 21 =((C 21 -C0) / C 21 )-((T 21 The PDX study was performed at Crown Biosciences (San Diego, CA and Taicang Jiangsu Province, China).

[0349] Urine and plasma creatine analysis Six- to eight-week-old female CD-1 mice were fed 100, 400, or 1200 mg / kg of control or RGX-202-01 formulated diets (Purina 5001, Research Diet, New Brunswick, NJ) for 10 days. Blood was collected into EDTA-coated blood collection tubes (02-669-33, Fisher Scientific), and plasma was separated by centrifugation. Plasma and urine samples were collected from both the control and RGX-202-01-treated cohorts on days 10 (240, 246.5, and 254.5 hours) and 11 (264 hours). Samples were flash-frozen and stored at -80°C at Pharmaron (Beijing, China) until bioanalysis for RGX-202 and creatine levels. Analysis of mouse plasma and urine was performed as a non-GLP study according to standard operating procedures at Pharmaron (Beijing, China). Briefly, the concentration of RGX-202-001 was determined by liquid chromatography tandem mass spectrometry (LC-MS / MS) using the surrogate analyte RGX-202- 13 C1 15 The lower limit of quantitation (LLOQ) was determined to be 50.0 ng / mL. The in-life portion of this study was performed at L2P Research.

[0350] Creatine-(methyl-d3) uptake in tumors and the heart Three to four male B6129SF1 / J mice bearing UN-KPC-961 tumors treated with either a control diet or an RGX-202-formulated diet at approximately 800 mg / kg for 35 days were given a single dose of 1 mg / kg creatine-(methyl-d3) (DLM-1302-0.25, Cambridge Isotope Laboratories, Tewksbury, MA) via i.p. injection. After 1.5 hours, the animals were euthanized, and the tumors and hearts were extracted, flash-frozen in liquid nitrogen, and subjected to biological analysis.

[0351] Three to four 6- to 9-week-old C57BL / 6J wild-type male mice were administered a single dose of RGX-202 at 100, 250, or 500 mg / kg in sterile 0.9% NaCl. Seven minutes after the injection of RGX-202 or vehicle control, a single dose of d3-creatine was administered at 1 mg / kg via i.p. injection in sterile 0.9% NaCl. SLC6A8 knockout mice also received a single dose of d3-creatine. One hour later, mice were anesthetized using 2.5% isoflurane, and the animals were perfused with 5 to 10 mL of DPBS, which was injected into the left ventricle. Hearts were then extracted and flash-frozen in liquid nitrogen. Heart samples were stored at -80°C until submitted to Seventh Wave Laboratories for analysis. Heart and tumor tissues were homogenized in a 70 / 30 methanol / water solution at a 3:1 (v:w) ratio. 20 μL of homogenate was diluted with 1 mL of internal standard solution (creatine-d5 in methanol). Samples were centrifuged, and the supernatant was analyzed. A fit-for-purpose semi-quantitative liquid chromatography with tandem mass spectrometry (LC-MS / MS) method was developed at Seventh Wave Laboratories for the measurement of d3-creatine and creatinine in heart and tumor tissue homogenates. Creatinine concentration was used to normalize the d3-creatine signal across samples.

[0352] Real-time PCR analysis of tumor samples RNA was extracted from 10 mg of flash-frozen vehicle- or RGX-202-01-treated tumors using a total RNA purification kit (37500, Norgen Biotek, Thorold, Canada) according to the manufacturer's instructions. Genomic DNA was removed using RNase-Free DNase I (25710, Norgen Biotek, Thorold, Canada). cDNA synthesis was performed with 600 ng of total RNA using the Verso cDNA Synthesis Kit (AB-1453 / B, Fisher Scientific, Waltham, MA) according to the protocol. qPCR was performed with 1 μL of 1:5 diluted cDNA using TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG (Fisher Scientific, Cat#: 4366073), and TaqMan probes (20X).

[0353] Reactions were performed on a Step 1+ real-time PCR system (Applied Biosystems) using predesigned Taqman gene expression assays for CKB, SLC6A8, CKM, CKMT1, CKMT2, and GUSB (Hs00176484_m1, Hs00940515_m1, Hs00176490_m1, Hs00179727_m1, Hs00176502_m1, Hs00939627_m1, Fisher Scientific, Waltham, MA). GUSB was used as an internal control.

[0354] A standard curve for normalization was generated by preparing serial dilutions as follows: Using undiluted cDNA from the HCT116 cell line at a stock concentration of 30 ng / μL, dilutions of 12.5 ng, 4.17 ng, 1.25 ng, 0.42 ng, 0.125 ng, and 0.013 ng were prepared. A 1:5 diluted cDNA sample of the HCT116 cell line was used as a reference run on all qPCR plates to account for plate-to-plate variability. All standards and samples were tested in triplicate or quadruplicate.

[0355] Histology, immunohistochemistry and immunofluorescence Tumors were fixed overnight in 4% PFA (50-980-497, EMS, Hatfield, PA) at 4°C. After two washes with phosphate-buffered saline (PBS) (10010023, Gibco), tissues were embedded in paraffin according to standard protocols. Tumors were sectioned using a microtome (Leica), and 5-μm-thick sections were mounted on Superfrost Plus Microscope Slides (22-037-246, Fisher Scientific). Tissue sections were first blocked for 30 minutes in background blocking reagent (NB306, Innovex). Rabbit monoclonal anti-creatine kinase type B antibody (ab92452, Abcam) was used at a dilution of 1:400. Incubation with the primary antibody was performed for 5 hours, followed by 60 minutes with 5.75 μg / ml biotinylated goat anti-rabbit IgG (PK6101, Vector Labs) using blocker D, streptavidin-HRP, and a DAB detection kit (Ventana Medical Systems) according to the manufacturer's instructions. Slides were counterstained with hematoxylin and coverslipped with Permount (Fisher Scientific). Antibody specificity was determined by staining HCT116 cells transiently transfected with CKB siRNA. A tumor microarray (TMA) containing 10 human healthy tissues in duplicate was purchased (CT565861, Origene).

[0356] Immunofluorescence detection of Ki67 Al 488+ and cleaved caspase-3 CF 594 was performed as follows. After 32 minutes of heating and CC1 (Cell Conditioning 1, 950-500, Ventana Cat.) recovery, tissue sections were first blocked for 30 minutes in background blocking reagent (NB306, Innovex). Mouse monoclonal anti-Ki67 antibody (M7240, DAKO) was used at a concentration of 0.5 μg / mL. Incubation with the primary antibody was performed for 5 hours, followed by 5.75 μg / mL biotinylated goat anti-mouse secondary (MOM kit BMK-2202, Vector Labs). Blocker D, streptavidin-HRP D (part of the DAB Map kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 488 (T20922, Invitrogen) prepared at 1:150 for 16 minutes according to the manufacturer's instructions. Rabbit polyclonal anti-cleaved caspase-3 (9661, Cell Signaling) was used at a concentration of 0.1 mg / ml. Incubation with the primary antibody was performed for 5 hours, followed by incubation with biotinylated goat anti-rabbit IgG (PK6101, Vector Lab) at 5.75 μg / ml for 60 minutes. Blocker D, streptavidin-HRP, and Tyramide-CF594 (92174, Biotium) were prepared at a dilution of 1:1500 for 16 minutes according to the manufacturer's instructions. After staining, slides were counterstained with DAPI (D9542, Sigma Aldrich, 5 mg / ml) for 10 min and mounted with Mowiol. All staining was performed at the Memorial Sloan Kettering Cancer Center Molecular Cytology Core Facility using a Discovery XT processor (Ventana Medical System, Roche-Indianapolis, IN).

[0357] Analysis of tumor samples Six to eight tumors per xenograft model were assigned a score of 0 to 3 based on the intensity of CKB staining. A score of 0 was interpreted as negative for protein expression, while scores of 1, 2, and 3 were interpreted as positive staining for each core, with 3 being maximal intensity. For each positive sample, the area (% of tumor) corresponding to each intensity of staining was recorded to allow for calculation of the tumor positivity rate (tumor percentage score). The weighted overall staining score (H score) was calculated as (percentage area of ​​1 + staining × 1) + (percentage area of ​​2 + staining × 2) + (percentage area of ​​3 + staining × 3). Tumor sections were excluded from analysis if they were primarily necrotic. In these cases, new tumor sections were stained to reach a minimum of 3 to 4 sections per tumor and 3 tumors per cohort.

[0358] Image quantification Quantification of the number of Ki67-positive cells and the percentage of CC3 per area was performed using Image J (version 1.50i). Three tumors per group were selected, and three to five fields per tumor were selected for quantification.

[0359] Hypoxic cell growth assay Lvm3b cells were seeded at 300,000 cells / well in 6-well plates. After 24 hours of incubation under normoxia, cells were cultured under hypoxia (0.5% oxygen, 5% CO2, 37°C) in the presence of 10 μM RGX-202, creatine (Cr) (Sigma-Aldrich #C3630), or phosphocreatine (PCr) (Sigma-Aldrich #237911) for 96 hours. Incubation continued for up to 120 hours, at which time the medium was replaced and cells were counted.

[0360] Metabolite extraction and liquid chromatography Lvm3b cells were cultured at 3 × 10 in RPMI 1640 in the presence of dialyzed FBS, 2 mM glutamine, and 6 mM glucose. 5Cells were seeded in triplicate at 10000 cells / well and allowed to attach to the plate for 24 hours. Cells were treated with either control or 10 mM RGX-202 for 24 hours in 0.5% O2. Cells were washed with ice-cold 0.9% NaCl and recovered in ice-cold 80:20 LC-MS methanol:water (v / v). Samples were vortexed vigorously and centrifuged at 20,000 g at maximum speed for 10 minutes at 4°C. The supernatant was transferred to a new tube. Samples were then completely dried using a nitrogen dryer. All samples were reconstituted in 30 μl of 2:1:1 LC-MS water:methanol:acetonitrile. The injection volume for polar metabolite analysis was 5 μL. Metabolite extraction and subsequent liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) for cellular polar metabolites were performed using a Q Exactive Plus.

[0361] Liquid chromatography A ZIC-pHILIC 150 × 2.1 mm (5 μm particle size) column (EMD Millipore) was used on a Vanquish Horizon UHPLC system for compound separation at 40 °C. The autosampler tray was maintained at 4 °C. Mobile phase A was water containing 20 mM ammonium carbonate, 0.1% ammonium hydroxide, pH 9.3, and mobile phase B was 100% acetonitrile. The gradient was linear: 0 min, 90% B; 22 min, 40% B; 24 min, 40% B; 24.1 min, 90% B; 30 min, 90% B. The flow rate was 0.15 ml / min. All solvents were LC-MS grade and purchased from Fisher Scientific.

[0362] mass spectrometry The Q Exactive Plus MS (Thermo Scientific) was equipped with a heated electrospray ionization probe (HESI), and the relevant parameters included a heated capillary at 250 °C, a HESI probe at 350 °C, a sheath gas at 40, an auxiliary gas at 15, a sweep gas at 0, and a spray voltage at 3.0 kV. A full scan was used in the range of 55 to 825 (m / z). The resolution was set to 70,000. The maximum injection time was 80 milliseconds (ms). The automatic gain control (AGC) was set to 1 × 10. 6 ions were targeted. The maximum injection time was 20 ms. Raw data collected from the LC-Q Exactive Plus MS was analyzed using Skyline (https: / / <skyline.ms / project / home / software / Skyline / begin.view> The data were processed using the ELISA kit (available on the World Wide Web at ). An output file containing the detected m / z and relative intensities in different samples was obtained after data processing. Quantification and statistics were calculated using Microsoft Excel, GraphPad Prism 8.1, and Rstudio 1.0.143.

[0363] Clinical specimen analysis On Day 15 of Cycle 1, serum samples were collected from patients at 0, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 hours after administration of RGX-202-01 and analyzed for RGX-202 using an analytically validated LC-MS / MS method at Syneos Health. Urine samples were collected on Day 15 of Cycle 1 at 0, 4, and 12 hours after administration of RGX-202. Mean values ​​are shown in the figure. Serum samples for creatine analysis were collected 12 hours after administration. Creatine levels were analyzed by Q2 Solutions at a referral laboratory.

[0364] Patient details The RGX-202-01 Phase 1a / b human study enrolled adult patients, both male and female, aged 18 years and older. Data from 13 patients were analyzed. The study is currently accruing and ongoing.

[0365] clinical research design This Phase 1a / b study is an open-label, multicenter, single-arm study with the primary objective of determining the maximum tolerated dose of RGX-202-01 or the maximum test dose at which multiple dose-limiting toxicities (DLTs) are not observed. Inclusion and exclusion criteria are specified, and all patients must have pathological confirmation of locally advanced or metastatic solid tumors or lymphomas deemed refractory to standard therapy. Patients may be free of other active malignancies that could confound study endpoints. Patients must not have a history of pancreatitis, active hepatitis B or C, or any other medical or social conditions that, in the investigator's opinion, could limit compliance with study requirements. Patients are not permitted to be treated with other anti-tumor therapies during the study. Typical Phase 1 study parameters include performance status, hematological, and other organ function measurements. Patients are required to use an acceptable method of contraception during the study and for a specified period thereafter. Concomitant medications are restricted only if they pose a clinical risk of drug-drug interactions. Treatment of any condition with corticosteroids is not permitted unless the dose is less than 10 mg prednisone equivalent per day. Patients were treated with RGX-202 at 600 mg, 1200 mg, 2400 mg, or 3600 mg twice daily, depending on the enrolled patient cohort. Patients continued treatment with RGX-202-01 until treatment intolerance or disease progression. The primary endpoint was the incidence of DLTs, which was assessed by a medical monitor in collaboration with all treating clinical investigators. Secondary endpoints included pharmacokinetic measurements of RGX-202 and its metabolites in plasma and urine. Exploratory endpoints included measurements of serum and urinary levels of creatine. Finally, efficacy endpoints were obtained with a large sample size of patients in disease-specific expansion cohorts.Dose-limiting toxicity is defined as any of the following toxicities occurring during the first 4 weeks of treatment that is not clearly related to another cause (i.e., disease progression): Grade 3 or higher non-hematologic AEs, except for Grade 3 nausea, vomiting, diarrhea, constipation, fever, fatigue, or skin rash in the presence of suboptimal prophylaxis and management that resolves to Grade 2 within 72 hours; Grade 4 thrombocytopenia or Grade 3 thrombocytopenia with greater than Grade 1 bleeding or need for platelet transfusion; Grade 4 neutropenia; Grade 3 or higher febrile neutropenia; Grade 3 or higher transaminase (AST / ALT) elevations; any toxicity resulting in more than 25% held / skipped doses during a cycle; or any other significant toxicity considered dose-limiting by the trial sponsor's medical personnel.

[0366] statistical analysis Significance of tumor growth curve comparisons was determined using a two-tailed t-test. Metastasis assays were analyzed using the nonparametric Mann-Whitney test. Statistical comparisons of survival analyses were performed using the Mantel-Cox log-rank test. Statistical analysis of creatine and RGX-202 AUC correlations in human patients and mice was performed using simple linear regression with Prism8. Statistical comparisons of cleaved caspase-3 and Ki67 signals, in vitro and in vivo d3-creatine assays, qPCR, and IHC quantification were performed using a two-tailed t-test. T-tests were used to compare metabolite abundances with Bonferroni's multiple testing correction, where indicated. Throughout all figures, *p<0.05, **p<0.01, and ***p<0.001, ****p<0.0001. Significance was concluded at p<0.05.

[0367] Example 18: Tumor growth inhibition achieved using RGX-202 in combination with low-dose KRAS inhibitors We conducted a study to evaluate the inhibition of tumor growth in a mouse KRAS G12C RAS ​​mutant syngeneic pancreatic cancer model (MIA PaCA-2) using RGX-202 and MRTX849 (adagrasib), alone or in combination. Mice were treated with RGX-202 (800 mg / kg), MRTX849 (3 mg / kg starting on day 16, then 10 mg / kg starting on day 23), control, or a combination of RGX-202 and MRTX849 (n=6-7 / cohort). Xenograft growth in mice was measured over time. Administration of RGX-202 in combination with MRTX849 was the most effective treatment for inhibiting tumor growth (Figures 17A and 17B).

[0368] Example 19: RGX-202 was effective in inhibiting the growth of non-small cell lung cancer (NSCLC) xenografts. Experiments were conducted using RGX-202 to evaluate the inhibition of non-small cell lung cancer (NSCLC) xenografts in mice.

[0369] Administration of RGX-202 resulted in tumor growth inhibition in NCL-H460 KRASQ61H xenografts in mice (Figure 18). 4x10 6 NCI-H460 cells were injected into 6- to 8-week-old female nude mice. Tumors grew to approximately 200 mm 3 Treatment with RGX-202-formulated diets was initiated when the mice reached a normal age. NCI-H460 is a non-small cell lung cancer cell line harboring the KRAS Q61H mutation. Mice were administered RGX-202 at a dose of 800 mg / kg.

[0370] Administration of RGX-202 also resulted in tumor growth inhibition in NCL-H358 KRASG12C xenografts in mice (Figure 19). 6 NCI-H358 cells were injected into 6- to 8-week-old female nude mice. Tumors grew to approximately 80 mm 3Treatment with RGX-202-formulated diets was initiated when the mice reached a 90-fold increase in serotonin concentration (90%). H358 is a non-small cell lung cancer cell line harboring the KRAS G12C mutation. Mice were administered RGX-202 at a dose of 800 mg / kg.

[0371] Other embodiments From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein and that it can be adapted to various usages and conditions, and such embodiments also fall within the scope of the following claims.

[0372] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or as any combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0373] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

Claims

1. Use of a creatine transporter inhibitor and a KRAS inhibitor in the preparation of a medicament for inhibiting the proliferation or survival of cancer cells.

2. The creatine transporter is SLC6A8, and the creatine transporter inhibitor is selected from the group consisting of β-guanidinopropionic acid, N-methylamidinomethylglycine, 1-carboxymethyl-2-imino-hexahydropyrimidine (cyclocreatine), DL-α-guanidinopropionic acid, N-methyl-N-amidinobeta-alanine, N-ethyl-N-amidinoglycine, DL-α-guanidinobutyric acid, DL-β-guanidinobutyric acid, gamma-guanidinobutyric acid, and guanidinoacetic acid, or a pharmaceutically acceptable salt thereof, the use according to claim 1.

3. The creatine transporter inhibitor is β-guanidinopropionic acid (β-GPA) or a pharmaceutically acceptable salt thereof, the use according to claim 2.

4. The KRAS inhibitor is selected from the group consisting of sotorasib, adagrasib, MRTX1133, RMC-6291, and RMC-6236, the use according to claim 3.

5. The cancer cells are colorectal cancer cells, the KRAS inhibitor is adagrasib, and the creatine transporter inhibitor is β-GPA, the use according to claim 1.

6. Use of β-GPA and adagrasib in the preparation of a medicament for inhibiting the proliferation or survival of cancer cells.

7. Use of a creatine transporter inhibitor; FOLFIRI comprising leucovorin (folic acid - FOL), 5-fluorouracil (F), and irinotecan hydrochloride (IRI); and / or bevacizumab in the preparation of a medicament for treating cancer in a subject.

8. The medicament comprises β-GPA, FOLFIRI, and bevacizumab, the use according to claim 7.

9. Use of β-GPA and 5-fluorouracil in the preparation of a medicament for treating cancer in a subject.

10. Use of a creatine transporter inhibitor, and FOLFOX comprising leucovorin (folic acid - FOL), 5-fluorouracil (5-FU), and oxaliplatin (OX) in the preparation of a medicament for treating cancer in a subject.

11. The use according to claim 10, wherein the agent comprises β-GPA and FOLFOX.

12. Use of β-GPA or a pharmaceutically acceptable salt thereof, and folinic acid-fluorouracil-irinotecan regimen (FOLFIRI) in the preparation of an agent for treating cancer, wherein the agent comprising β-GPA or its salt form is administered twice a day (BID) at a dosage of 1000 mg to 3600 mg, or at a dosage of 2400 mg or 3000 mg.

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