Methods and compositions for the treatment of cancer

Abstract

Disclosed herein are methods and compositions for treating a condition in a patient in need thereof. In some embodiments, a method of treating cancer in a patient in need thereof comprises disposing a composition comprising an inhibitor of dihydroorotate dehydrogenase (DHODH) within a biological compartment of the patient. In some cases, the uridine salvage capability of the cancer is assessed. In some instances, the uridine salvage capability of the cancer is deficient. In some instances, the cancer is ovarian cancer.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U. S. C. § 119 to U. S. Provisional Patent Application No 63 / 760,170 filed February 19, 2025, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant / contract number HT9425-25-1-0217 awarded by the Department of Defense. The government has certain rights in the invention.FIELD

[0003] This disclosure is generally in the area of anti-cancer treatments, and more specifically, in the area of dihydroorotate dehydrogenase (DHODH) inhibitors for use in the treatment of cancer, wherein the cancer is deficient in uridine salvage capability.BACKGROUND

[0004] Cancer is a major public health burden that is the second leading cause of death in the United States, leading to 10 million deaths in 2020. A variety of cancer treatments are available and include surgery, radiation, and chemotherapy, alone or in combination.

[0005] Current treatments for cancer have numerous shortcomings, including limitations for application and significant side effects. For example, generally, surgery, which typically involves removing all or part of a tumor from the body, is most effective for treating early stages of cancer, and its application is limited to areas where tumors are accessible. Moreover, regarding radiation, radiation is typically applied to a defined area of the patient’s body that contains the cancerous tissue in order to maximize the amount of radiation absorbed by the cancerous tissue and minimize the amount absorbed by “normal” or non-cancerous tissue. However, it is difficult to selectively use radiation on only cancerous tissue without exposing “normal” or non-cancerous tissues. Additionally, chemotherapy, which involves the use of drugs or therapeutics to slow or stop the growth of cancer cells, often has severe side effects that impact the quality oflife of patients, such as vomiting, weight loss, hair loss, and low white blood cell count. These severe side effects can cause patients to discontinue treatment. Further, drug resistance to chemotherapies and metastasis may also occur. Thus, with the numerous shortcomings of current treatments for cancer, there is a need for improved anti-cancer therapies.SUMMARY

[0006] Disclosed herein are DHODH inhibitors and pharmaceutical compositions comprising DHODH inhibitors useful in the treatment of cancer.

[0007] In one aspect, disclosed herein is a method of treating cancer in a patient in need thereof, wherein the method comprises: disposing a composition comprising an inhibitor of dihydroorotate dehydrogenase (DHODH) within a biological compartment of the patient; wherein the cancer has a deficient uridine salvage capability. In some embodiments, the method further comprises assessing the uridine salvage capability of the cancer. In some embodiments, the cancer is ovarian cancer, such as for example epithelial ovarian cancer (EOC).BRIEF DESCRIPTION OF THE FIGURES

[0008] FIG. 1A shows that ovarian cancer cell lines exhibit differential sensitivity to a DHODH inhibitor, HOSU-53, after culturing in medium supplemented with 10% FBS and treatment with HOSU-53 for 7 days. Methylene blue staining assay was conducted to determine the cell viability The IC50 for each cell line was calculated using GraphPad Prism.

[0009] FIG. 1B is a tabulated summary of the data in FIG. 1 A, where N = 5, Bar: SD.

[0010] FIGS. 2A-J are graphs of relative cell viability for a range of ovarian cancer cell lines cultured in media supplemented with either 10% standard FBS or dialyzed FBS, from which all small molecules having a molecular weight less than 10 kDa have been removed via filtration, treated with HOSU-53 for 7 days.

[0011] FIGS. 3A-3G are graphs of the relative cell viability for a range of ovarian cancer cell lines cultured in medium supplemented with either standard FBS or dialyzed FBS, treated with brequinar for 7 days. N = 5, Bar: SD.

[0012] FIGS. 3H-3N are graphs of the relative cell viability for a range of ovarian cancer cell lines cultured in medium supplemented with either standard FBS or dialyzed FBS, treated with BAY-2402234 for 7 days. N = 5, Bar: SD. IC50 is identified.

[0013] FIGS. 4A-4H are graphs of relative cell viability for a range of ovarian cancer cell lines cultured in dialyzed FBS containing media, treated with HOSU-53 in the presence or absence of 20 pM uridine for 7 days, showing extracellular uridine contributes to the resistance to DHODH inhibitors in certain ovarian cancer cell lines. Methylene blue staining assay was conducted to determine the cell viability. N = 5, Bar: SD. **: P < 0.01. Linear mixed effects models were used to analyze cell viability trends across doses. IC50 is identified.

[0014] FIG. 5A is a volcano plot of metabolites, showing minimal restoration of pyrimidine derivatives in HOSU-53-sensitive Kuramochi cells following uridine treatment.

[0015] FIG. 5B is a volcano plot of metabolites, showing pronounced restoration of pyrimidine derivatives in HOSU-53-resistant OVCAR3 cells following uridine treatment.

[0016] FIG. 6A is a graph of relative uridine abundance in HOSU-53 resistant cells (PEO1, OVCAR3, and OVCAR4) cultured in media supplemented with dialyzed FBS, treated with HOSU-53 in the absence or presence of uridine for 40 h. LC-MS was used to determine the cellular level of uridine.

[0017] FIG. 6B is a graph of relative uridine abundance in HOSU-53 sensitive cells (Kuramochi, CAOV3, ES2) cultured in media supplemented with dialyzed FBS, treated with HOSU-53 in the absence or presence of uridine for 40 h. LC-MS was used to determine the cellular level of uridine.

[0018] FIGS. 7A-7E are graphs of relative abundance of labeled and unlabeled uridine in HOSU-53 resistant cells R127 (FIG. 7A) and PEO1 (FIG. 7B) and HOSU-53 sensitive cells R182 (FIG. 7C), Kuramochi (FIG. 7D), and ES2 (FIG. 7E) cultured in media supplemented with dialyzed FBS, treated with HOSU-53 (100 nM) for 24 hr to deplete endogenous uridine.13C-labeled uridine (20 pM) was added for different time periods, LC-MS / MS was used to measure unlabeled and13C-labeled uridine.

[0019] FIGS. 8A-8B are graphs of relative abundance of labeled and unlabeled uridine in HOSU-53 resistant cells R127 (FIG. 8A) and PEO1 (FIG. 8B) cultured in media supplemented with dialyzed FBS, treated with HOSU-53 (100 nM) for 24 hr to deplete endogenous uridine or mock treated.13C-labeled uridine (20 pM) was added for 30 min. LC-MS / MS was used to measure unlabeled and13C-labeled uridine.

[0020] FIG. 9 are graphs of relative cell viability of a panel of ovarian cancer cell lines cultured in medium supplemented with 10% FBS, treated with gemcitabine for 7 days.Methylene blue staining assay was conducted to determine the cell viability. The IC50 for each cell line was calculated using GraphPad Prism. N = 5, Bar: SD.

[0021] FIGS. 10A-F show that HOSU-53 efficiently blocks tumor growth in an ES2-derived orthotopic ovarian xenograft model. FIG. 10A is a schematic illustration of the animal study. FIG. 10B are representative bioluminescence images of intraperitoneal xenografts following two weeks of treatment. FIG. 10C is a graph of Bioluminescent Flux (x1011p / sec). FIG. 10D are photographs of ascites in xenograft bearing mice after euthanasia. FIG. 10E is a graph of ascitic fluid from ascites in xenograft bearing mice after euthanasia. FIG. 10F is a Kaplan-Meier survival curve depicting the survival of mice post-treatment.

[0022] FIGS. 11A-D show that HOSU-53 efficiently blocks tumor growth in an R182-derived orthotopic ovarian xenograft model. FIG. 11A is a schematic illustration of the animal study. FIG. 11B are representative bioluminescence images of intraperitoneal xenografts following 18 days of treatment. FIG. 11C is a graph of abdomen to chest ratio for tumor volumes. FIG. 11D is a graph of tumor volumes measured by Bioluminescence Imaging (BLI).

[0023] FIG. 12A are fluorescence microscope images of salvage proficient cells (OVCAR4 cells) used to evaluate the nucleoside-intake ability in ovarian cancer cells, visualizing 5-EU-incorporated RNA, using DAPI (4',6-diamidino-2-phenylindole) in colocalization assay for nuclear staining.

[0024] FIG. 12B are fluorescent microscope images of salvage deficient cells (Kuramochi cells) used to evaluate the nucleoside-intake ability in ovarian cancer cells, visualizing 5-EU-incorporated RNA and DAPI.

[0025] FIG. 13 A are fluorescent microscope images of a panel of salvage proficient (DHODHi resistant) cells (OVCAR3, OVCAR4, UWB1, PEO1, and R127) cultured in the presence of 50 pM 5-EU for 24 hrs, visualizing 5-EU-incorporated RNA and DAPI.

[0026] FIG. 13B are fluorescent microscope images of a panel of salvage deficient (DHODHi sensitive) cells (Kuramochi, ES2, OVSAHO, CAOV3, R182, OV35) cultured in the presence of 50 pM 5-EU for 24 hrs, visualizing 5-EU-incorporated RNA and DAPI.

[0027] FIG. 14A are fluorescent microscope images of ASC195 (EOC cells from ascites) evaluating the presence of EpCAM and PAX8 with DAPI.

[0028] FIG. 14B are fluorescent microscope images of ASC3 (EOC cells from ascites) evaluating the presence of EpCAM and PAX8 with DAPI.

[0029] FIGS. 15A-15D are fluorescent microscope images, from a 5-EU and DAPI in colocalization assay, and graphs comparing the relative cell viability in primary cancer cells: ASC 195 (FIG. 15A), M1250760A (FIG. 15B), M1250890B (FIG. 15C), and M1251951A (FIG.15D) upon treatment with HOSU-53 with and without uridine supplementation.

[0030] FIGS. 16A-16D are fluorescence microscope images, using 5-EU and DAPI in colocalization assay, and graphs of the relative cell viability comparing the relative cell viability in primary cancer cells ASC3 (FIG. 16A), M1251273A (FIG. 16B), M11242018A (FIG. 16C), and M1250835A (FIG. 16D) upon treatment with HOSU-53 with and without uridine supplementation.

[0031] FIGS. 17A-17E are graphs of relative mRNA levels from a qRT-PCR analysis in a panel of ovarian cancer cells (ES2, Kuramochi, OV2008, CAOV3, OVSAHO, OVCAR3, PEO1, COV362, OV90 and OVCAR4) with different sensitivity to HOSU-53: SLC28A1 (FIG. 17A) SLC28A2 (FIG. 17B) SLC28A3 (FIG.17C), SLC29A1 (FIG. 17D) and SLC29A2 (FIG.17E).

[0032] FIG. 17F are images of immunoblotting of tubulin (control), SLC28A1, and SLC28A3 in a panel of ovarian cancer cell lines- salvage deficient (Kuramochi, CAOV3, and OVSAHO) and salvage proficient (OVCAR3, OVCAR4, PEO1, OV90 and COV362).

[0033] FIG. 18A are images of immunoblotting (tubulin as control) showing CNT1 (SLC28A1) expression was knocked down in OVCAR3 cells using two distinct shRNAs targeting SLC28A1.

[0034] FIG. 18B are fluorescence microscope images showing uridine uptake in OVCAR3 cells following SLC28A1 knockdown using 5-EU assay with DAPI.

[0035] FIG. 18C are fluorescence microscope images showing uridine uptake after OVCAR3 cells were treated with the ENT inhibitor nitrobenzylthioinosine (NBMPR) (25 pM) using 5-EU assay with DAPI.

[0036] FIG. 18D is a graph of relative cell viability of OVCAR3 cells cultured in dialyzed FBS containing media and treated with HOSU-53 in the presence or absence of 20 pM uridine or / and NBMPR for 7 days.

[0037] FIG. 19A is a graph of relative FPKM (log2) expression levels of various concentrative nucleoside transporters (CNTs or SLC28A1 / 2 / 3), equilibrative nucleoside transporters (ENTs or SLC29A1 / 2), and uridine-cytidine kinases (UCK1 & UCK2) as analyzed from The Cancer Genome Atlas (TCGA) database for 308 serous ovarian cancer patients.

[0038] FIG. 19B are fluorescence microscope images from 5-EU assays showing different uridine uptake capacities in primary High-Grade Serous Ovarian Carcinoma (HGSOC) cells: OV35 cells were derived from an HGSOC tumor tissue, R182 and R127 cells were derived from the ascites of two HGSOC patients.DETAILED DESCRIPTION

[0039] Embodiments described herein can be understood more readily by reference to the following summary and its previous and following descriptions. Elements and methods described herein, however, are not limited to the specific embodiments presented in the summary and Appendix. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

[0040] In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.

[0041] When a range of integers is given, the range includes any number falling within the range and the numbers defining ends of the range. For example, when the terms “integer from 1 to 20” is used, the integers included in the range are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., up to and including 20. All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10” should generally be considered to include the end points 5 and 10.

[0042] Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.

[0043] Furthermore, the terms “substantially,” “approximately,” and “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. The term “consists essentially of’ (and grammatical variants) shall be given its ordinary meaning and shall also mean that thecomposition or method referred to can contain additional components as long as the additional components do not materially alter the composition or method. The term “consists of’ (and grammatical variants) shall be given its ordinary meaning and shall also mean that the composition or method referred to is closed to additional components. The term “comprising” (and grammatical variants) shall be given its ordinary meaning and shall also mean that the composition or method referred to is open to contain additional components.

[0044] It is also to be understood that the article “a” or “an” refers to “at least one,” unless the context of a particular use requires otherwise.

[0045] Also as used herein, “and / or” refers broadly to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0046] Compounds, pharmaceutical compositions including the compounds, and methods of preparation and uses thereof are disclosed. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. The terminology used in the description of the subject matter herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the subject matter. The present disclosure will be better understood with reference to the following definitions.Definitions

[0047] The term “effective amount,” as used herein, refers broadly to that amount of a recited compound effective to treat, prevent, or reduce the severity or progression of a disorder in a subject, such as a human subject. This includes improving the subject’s condition (e.g., in one or more symptoms), delaying or reducing the progression of the disease and / or disorder, preventing or delaying the onset of the disorder, and / or changing clinical parameters, disease or illness, etc., as would be well known in the art.

[0048] For example, an effective amount can refer to the amount of a composition, compound, or agent that improves a condition in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0049] The term “pharmaceutically acceptable salt” is used throughout the specification to describe any pharmaceutically acceptable form, such as an ester, which, upon administration to a patient, provides the compound. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as lithium, potassium and sodium and alkaline earth metals such as calcium and magnesium, among numerous other cations well known in the pharmaceutical art, including but not limited to ammonium, tetramethylammonium, and tetraethylammonium, or metal cations such as Fe+2, Cu+2, Zn+2, Al+3, Fe+3, and combinations thereof. Further such salts include those comprising a cation of one or more of L-arginine, L-lysine, or N-methyl-D-glucamine.

[0050] As used herein, the terms “treating,” “treatment,” and the like are used to mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and / or may be therapeutic in terms of a partial or complete cure for a disorder and / or adverse effect attributable to the disorder, or the relief or elimination of a symptom thereof. Thus, treatment includes preventing or protecting against the disease or disorder, that is, causing the clinical symptoms not to develop; and / or inhibiting the disease or disorder, that is, arresting or suppressing the development of clinical symptoms; and / or relieving the disease or disorder that is, causing the regression of clinical symptoms; and / or reducing the metastasis of the primary tumor or cancer.

[0051] The terms “DHODH inhibitor,” “inhibitor of DHODH,” “DHODHi,” or “DHODH inhibitor compound” refer broadly to a compound that inhibits the activity of dihydroorotate dehydrogenase.

[0052] The phrase “uridine salvage capability” refers to the ability of a cell to synthesize uridine from intermediates in its degradation pathway. The term “deficient” when used in reference to uridine salvage capability means the cell does cannot synthesize uridine from intermediates in its degradation pathway. In some instances, the cell cannot access enough uridine under physiological conditions to make needed amounts of RNA and / or DNA. In some instances, when the de novo pyrimidine synthesis pathway is blocked in a cell with deficient uridine capability, the cell may die.

[0053] The phrase “uridine uptake” refers to a step in the uridine salvage pathway, whereby cells use transporters (e.g. ENTs / CNTs) to import uridine. As used herein, “deficient uridineuptake” means that a cell has limited uptake in a physiological concentration of uridine. In some instances, the cell cannot uptake enough uridine under physiological conditions to make needed amounts of RNA and / or DNA. In some instances, when the de novo pyrimidine synthesis pathway is blocked in a cell with deficient uridine uptake, the cell may die.

[0054] The “patient” or “subject” treated as disclosed herein is, in some embodiments, a human patient, although it is to be understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective with respect to all vertebrate species, including mammals, which are intended to be included in the terms “subject” and “patient.” Suitable subjects are generally mammalian subjects. The subject matter described herein finds use in research as well as veterinary and medical applications. The term “mammal” as used herein includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dog, rabbits, rodents (e g., rats or mice), monkeys, etc. Human subjects include neonates, infants, juveniles, adults and geriatric subjects. The subject “in need of’ the methods disclosed herein can be a subject that is experiencing a disease state, such as cancer and / or is anticipated to experience a disease state, such as cancer, and the methods and compositions of the invention are used for therapeutic and / or prophylactic treatment.

[0055] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.I. General

[0056] Cancer cells, particularly in aggressive malignancies such as ovarian cancer, exhibit a high demand for nucleotides to support uncontrolled proliferation. In some embodiments, methods and compositions described herein comprise small molecule inhibitors of the enzyme dihydroorotate dehydrogenase (DHODH), a player in the de novo pyrimidine synthesis pathway, to impair pyrimidine production and limit cancer cell growth by specifically disrupting the de novo pyrimidine biosynthesis pathway. This approach is particularly effective in limiting the ability of cancer cells to synthesize pyrimidines needed for DNA and RNA synthesis, leading to reduced tumor proliferation.

[0057] Epithelial ovarian cancer (EOC) is a lethal malignancy with a poor survival rate. While initial treatments can be effective, recurrence and resistance remain major problems, suggesting the need for new therapies. One promising target is nucleotide metabolism, as EOC cells require elevated pyrimidine pools to support rapid proliferation and DNA repair.Pyrimidines are synthesized via de novo synthesis and salvage pathways. Dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pathway is necessary in both RNA and DNA synthesis, but DHODH inhibitors (DHODHi) remain underexplored in EOC.

[0058] Cancer cells use both de novo and salvage pathways to generate pyrimidine pools. SLC28A1 (CNT1) and SLC29 family (ENT) transporters import extracellular uridine and cytidine, which are then converted by Uridine-Cytidine Kinase (UCK) to UMP and CTP. The pyrimidine salvage pathway allows cancer cells to maintain necessary pyrimidine pools for DNA replication and RNA synthesis. It is important for slow-growing or nutrient-deprived cells, and it can become particularly important when a de novo pyrimidine synthesis pathway inhibitor, such as a DHODHi is used.

[0059] Not intending to be bound by theory, it is believed that extracellular uridine salvage may be a resistance mechanism in DHODH inhibitor-resistant ovarian cancer cells. Moreover, not intending to be bound by theory, it is believed that resistance is linked to the ability of cancer cells to uptake extracellular uridine, such as through one or more of the transporters, e.g.SLC29A1 / 2, and SLC28A1 / 2 / 3, to maintain pyrimidine pools. The transporters can also influence the efficacy of therapeutic agents, such as nucleoside analog drugs (e.g. gemcitabine). In one embodiment disclosed herein, DHODH inhibitor-resistant cells express high levels of one or more, two or more, three or more, or four or more of SLC29A1, SLC29A2, SLC28A1,SLC29A2, and SLC29A3. In one embodiment, DHODH inhibitor-resistant cells express high levels of each of SLC28A1, SLC28A2, SLC29A1, SLC29A2, and SLC29A3. As disclosed herein, DHODH inhibitor-resistant cells exhibit increased uridine uptake, while DHODH inhibitor-sensitive cells show defective or deficient uridine uptake.

[0060] As shown herein, cancer cells, such as for example, ovarian cancers, that are deficient in extracellular uridine salvage exhibit heightened sensitivity to DHODH inhibitors, enabling the identification of patients who can benefit most from therapeutic administration of a DHODHi. In one embodiment, as disclosed herein, a 5-EU assay, which measures uridine uptake, can be used to identify patients who are likely to respond to DHODH inhibitors based on their cancer cells’ extracellular uridine uptake capabilities.

[0061] Combining DHODH inhibition with an evaluation of uridine salvage mechanism(s) provides a targeted therapeutic option for cancer, especially for cancers with defective uridine uptake pathways, such as ovarian cancer. This invention provides personalized treatment strategies for cancers, including ovarian cancer and / or leukemia, and allows for the identification of patients with impaired uridine salvage who would respond favorably to therapeutic administration of DHODH inhibitors. It not only offers a therapeutic pathway but also serves as a diagnostic tool to predict treatment response, particularly for patients with cancer, including ovarian cancer, such as for example, advanced- stage EOC.

[0062] In some embodiments, this therapeutic strategy is useful for the pharmaceutical and biotech industries, particularly in the field of oncology drug development. However, in some cases, this approach is also useful for precision therapies for cancer patients and for use in companion diagnostics, such as using the 5-EU assay to assess pyrimidine uptake as a companion diagnostic tool to select patients who are likely to respond to DHODH inhibitors, allowing for tailored treatment strategies.IL Compounds and Compositions

[0063] Compounds and compositions disclosed herein may comprise and methods disclosed herein may use any DHODH inhibitor not inconsistent with technical objectives of the current disclosure.

[0064] In one embodiment, the DHODH inhibitor is selected from the group consisting of brequinar, leflunomide, redoxal, vidofludimas, S-2678, ASLAN003 (2-(3,5-difluoro-3'-methoxybiphenyl-4-ylamino)nicotinic acid), BAY-2402234 (N-(2-chloro-6-fluorophenyl)-4-(4-ethyl-3-(hydroxymethyl)-5-oxo-4,5-dihydro-lH-l,2,4-triazol-l-yl)-5-fluoro-2-((l,l,l-trifluoropropan-2-yl)oxy)benzamide), AG-636 (l-methyl-5-(2'-methyl-[l,r-biphenyl]-4-yl)-lH-benzo[d][l,2,3]triazole-7-carboxylic acid), PTC-299 (4-chlorophenyl (S)-6-chloro-l-(4-methoxyphenyl)-l,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate), JNJ-74856665, Meds433, RP7214, ML390, Laflunimus, Tenovin-1, Tenovin-6, hDHODH-IN-4, DHODH-IN-11, and teriflunomide.

[0065] In another embodiment, a DHODH inhibitor comprises one or more compounds disclosed in US Patent 11,312,686; US Patent Application Publication No. 2023 / 0303710; US Patent Application Publication No. 2023 / 0109418; US Patent Application Publication No.2023 / 0089524; US Patent Application Publication No. 2024 / 0018108; or US Patent Application Publication No. 2024 / 0325373, each of which is incorporated herein by reference.

[0066] In one embodiment, a DHODH inhibitor comprises a compound of Formula I:wherein R1is selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and-CF2CF3; one ofR5a, R5b, R5c, R5d, andR5eis selected from a group having formula represented by a structure: — R20, — R30— A1— R40, — A1— R40, — A1— R30— A2— R40, or— A1— R30— A2— R40— A3— R41; A1is selected from -O- and -NR50; R50is selected from -C 1-C 10 aminoalkyl, -Cl -CIO alkylamino, and -Cl -CIO hydroxy alkyl; A2is selected from -O-and -NR60; R60is selected from -Cl -CIO aminoalkyl, -Cl -CIO alkylamino, and -Cl -CIO hydroxyalkyl; A3is selected from -O- and -NR60; R70is selected from -Cl -CIO aminoalkyl, -Cl-C10 alkylamino, and -Cl -CIO hydroxy alkyl; R20is selected from halogen, -Cl -CIO alkylamino and -Cl -CIO alkoxy; R30is selected from -Cl -CIO alkanediyl, -Cl -CIO aminoalkanediyl, and -C1-C10 hydroxyalkanediyl; and each of R40and R41is independently selected from -Cl -CIO alkyl, -Cl - CIO aminoalkyl, -Cl -CIO hydroxy alkyl, and -(CH2)nAr1;n is an integer selected from 1, 2, and 3; and Ar1is a phenyl group substituted with 1, 2, or 3 groups independently selected from halogen, -SFs, -CN, -N3, -OH, -NH2, -C1-C3 alkyl, -Cl -C3 alkoxy, -C1-C3 haloalkyl, -C1-C3 aminoalkyl, -C1-C3 alkylamino, -Cl -C3 haloalkylamino, -C1-C3 hydro xyalkyl, -C1-C3 halohydro xyalkyl, cycloalkyl, and heterocycloalkyl; and four of R5a, R5b, R5c, R5d, and R5eis independently selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3; or a pharmaceutically acceptable salt thereof.

[0067] In one embodiment, R1is selected from halogen, —SFs, — CN, — N3, —OH, — NH2, — CF3, and — CF2CF3; each of R5band R5cis independently selected from -R20, hydrogen, halogen, —SFs, — CN, — N3, —OH, — NH2, — CF3, and — CF2CF3; R20is selected from halogen, — Cl -CIO alkylamino and — Cl -CIO alkoxy; provided that one of R5band R5cis -R20; and each of R5a, R5d, and R5eis independently selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3; or a pharmaceutically acceptable salt thereof. In one embodiment, R1is halogen; each of R5band R5cis independently selected from -R20and hydrogen; R20is selected from — Cl -CIO alkylamino and — Cl -CIO alkoxy; provided that one of R5band R5cis -R20; and each of R5a, R5d, and R5eis hydrogen; or a pharmaceutically acceptable salt thereof. In another embodiment, R1is -F; each of R5band R5cis independently selected from -R20and hydrogen; R20is selected from — C1-C4 alkylamino and — C1-C4 alkoxy; provided that one of R5band R5cis -R20; and each of R5a, R5d, and R5eis hydrogen; or a pharmaceutically acceptable salt thereof. In one embodiment, R1is -F; each of R5band R5cis independently selected from -R20and hydrogen; R20is selected from — C1-C4 alkoxy; provided that one of R5band R5eis -R20; and each of R5a, R5d, and R5eis hydrogen; or a pharmaceutically acceptable salt thereof. In one variation of any embodiment disclosed herein, each of each of R5band R5cis independently selected from -R20and hydrogen; R20is selected from — C2-C7 alkylamino and — C2-C7 alkoxy; provided that one of R5band R5cis -R20.

[0068] In one embodiment, a DHODH inhibitor is Compound 1:Compound 1 or a pharmaceutically acceptable salt thereof. Compound 1 is also referred herein as HOSU-53.

[0069] In another embodiment, a DHODH inhibitor comprises a compound of Formula II:HO^^ON^YZ’'Z^ R5aztzAR5eR5dFormula IIwherein each of Z1, Z2, Z3, and Z4is independently selected from CH and N, optionally provided that at least one of Z1, Z2, Z3, and Z4is N; R1is selected from hydrogen, halogen, — SF5, — CN, — N3, —OH, — NH2, — CF3, and — CF2CF3; one of R5a, R5b, R5c, R5d, and R5eis selected from a group having formula represented by a structure: — R20, — R30— A1— R40, — A1— R40,— A1— R30— A2— R40, or — A1— R30— A2— R31— A3— R40; A1is selected from — O— and —NR50—; R50is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxyalkyl; A2is selected from — O— and —NR60—; R60is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — C1-C10 hydroxyalkyl; A3is selected from — O— and —NR70—; R70is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxyalkyl; R20is selected from halogen, — Cl -CIO alkyl, — Cl -CIO haloalkyl, — Cl -CIO hydroxyalkyl, — Cl -CIO alkylamino, and — C1-C10 alkoxy; each of R30and R31is independently selected from — C1-C10 alkanediyl, — Cl -CIO haloalkanediyl, — Cl -CIO aminoalkanediyl, and — Cl -CIOhydroxy alkanediyl; and R40is selected from — Cl -CIO alkyl, — Cl -CIO haloalkyl, — Cl -CIO aminoalkyl, — C1-C10 hydroxyalkyl, and — (CH2)nAr1; n is an integer selected from 1, 2, and 3; and Ar1is a phenyl group substituted with 0,1, 2, 3, 4, or 5 groups independently selected fromhalogen, — SF5, — CN, — N3, —OH, — NH2, — C1-C4 alkyl, — C1-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — C1-C4 alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl; and four of R5a, R5b, R5c, R5d, and R5eare independently selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3; or a pharmaceutically acceptable salt thereof.

[0070] In yet another embodiment, a DHODH inhibitor comprises a compound of Formula III:Formula III wherein Z5is a five-membered heterocyclic diyl; R1is selected from hydrogen, halogen, — SF5, — CN, — N3, —OH, — NH2, — CF3, and — CF2CF3; one of R5a, R5b, R5c, R5d, and R5eis selected from a group having formula represented by a structure: — R20, — R30— A1— R40, — A1— R40,-A1-R30-A2-R40, or — A1— R30— A2— R31— A3— R40; A1is selected from -O- and -NR50-; R50is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxyalkyl; A2is selected from — O— and —NR60—; R60is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxyalkyl; A3is selected from — O— and —NR70—; R70is selected from hydrogen, — C1-C10 alkyl, — C1-C10 aminoalkyl, and — C1-C10 hydroxyalkyl; R20is selected from halogen, — Cl -CIO alkyl, — Cl -CIO alkylamino and — Cl -CIO alkoxy; each ofR30and R31is independently selected from — C1-C10 alkanediyl, — C1-C10 aminoalkanediyl, and — Cl -CIO hydroxyalkanediyl; and R40is selected from — Cl -CIO alkyl, — Cl -CIO aminoalkyl, — C1-C10 hydroxyalkyl, and — (CH2)nAr1; n is an integer selected from 1, 2, and 3; and Ar1is a phenyl group substituted with 0,1, 2, 3, 4, or 5 groups independently selected from halogen, -SF5, -CN, -N3, -OH, -NH2, from -C1-C4 alkyl, -C1-C4 alkoxy, -C1-C4 haloalkyl, -Cl-C4 aminoalkyl, — C1-C4 alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl; and four of R5a, R5b, R5c, R5d, and R5eisindependently selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3; or a pharmaceutically acceptable salt thereof.

[0071] In yet another embodiment, a DHODH inhibitor comprises a compound of Formula IV:Formula IV wherein R1is selected from hydrogen, halogen, —SFs, — CN, — N3, —OH, — NH2, — CF3, and — CF2CF3; one of R5a, R5b, R5c, R5d, and R5eis selected from a group having formula represented by a structure: — R20, — R30— A1— R40, — A1— R40, — A1— R30— A2— R40, or— A1— R30— A2— R31— A3— R40; A1is selected from — O— and —NR50—; R50is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxyalkyl; A2is selected from — O— and —NR60—; R60is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxyalkyl; A3is selected from — O— and —NR70—; R70is selected from hydrogen, — C1-C10 alkyl, — C1-C10 aminoalkyl, and — C1-C10 hydroxyalkyl; R20is selected from halogen, — Cl -CIO alkyl, — Cl -CIO haloalkyl, — Cl -CIO hydroxyalkyl, — Cl -CIO alkylamino, — C1-C10 alkoxy, — (CH2)nCy1, and — (CH2)nAr1; n is an integer selected from 1, 2, and 3; and Cy1is a C3-C10 cycloalkyl group or a C2-C9 heterocycloalkyl group substituted with 0, 1, 2, 3, 4, or 5 groups independently selected from halogen, —SFs, — CN, — N3, —OH, — NH2, from — C1-C4 alkyl, — C1-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — C1-C4 alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl; Ar1is a phenyl group substituted with 0, 1, 2, 3, 4, or 5 groups independently selected from halogen, —SFs, — CN, — N3, —OH, — NH2, from — C1-C4 alkyl, — Cl-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — C1-C4 alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl; each of R30and R31is independently selected from — C1-C10 alkanediyl, — Cl-C10 haloalkanediyl, — Cl -CIO aminoalkanediyl, and — Cl -CIO hydroxy alkanediyl; and R40isselected from —Cl -CIO alkyl, —Cl -CIO haloalkyl, — Cl -CIO aminoalkyl, —Cl -CIO hydroxyalkyl, — (—(CH2)nCy1, and —(CH2)nAr1; n is an integer selected from 1, 2, and 3; and Cy1is a C3-C10 cycloalkyl group or a C2-C9 heterocycloalkyl group substituted with 0, 1, 2, 3, 4, or 5 groups independently selected from halogen, — SF5, — CN, — N3, —OH, — NH2, from — C1-C4 alkyl, — C1-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — C1-C4 alkylamino, — C1-C4 haloalkyl ami no, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl; Ar1is a phenyl group substituted with 0, 1, 2, 3, 4, or 5 groups independently selected from halogen, — SF5, — CN, — N3, —OH, — NH2, from — C1-C4 alkyl, — C1-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — C1-C4 alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl; and four of R5a, R5b, R5c, R5d, and R5eare independently selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3; each of R6a, R6b, R6c, and R6dis independently selected from hydrogen, halogen, — SF5, — CN, — N3, —OH, — NH2, Cl -CIO alkyl, Cl -CIO alkoxy, Cl -CIO haloalkyl, Cl -CIO aminoalkyl, and Cl -CIO hydroxyalkyl, provided that at least one of R6a, R6b, R6c, and R6dis not hydrogen; or a pharmaceutically acceptable salt thereof. In one embodiment, R1is selected from halogen, — SF5, — CN, — N3, — OH, — NH2, — CF3, and — CF2CF3; R5b= R20and R20is selected from — Cl -CIO alkylamino and — Cl -CIO alkoxy; and wherein each of R5a, R5c, R5d, and R5eis independently selected from hydrogen, halogen, — SF5, — CN, — N3, — OH, — NH2, — CF3, and — CF2CF3; or R5eis -R20and R20is selected from — C1-C10 alkylamino and — Cl -CIO alkoxy; and wherein each of R5a, R5b, R5d, and R5eis independently selected from hydrogen, halogen, — SF5, — CN, — N3, — OH, — NH2, — CF3, and — CF2CF3; and wherein each of R6a, R6b, R6c, and R6dis independently selected from hydrogen, halogen, — SF5, — CN, — N3, —OH, — NH2, — Cl -CIO alkyl, — Cl -CIO alkoxy, — Cl -CIO haloalkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxyalkyl, provided that at least one of R6a, R6b, R6c, and R6dis not hydrogen; or a pharmaceutically acceptable salt thereof. In another embodiment each of R6a, R6b, R6c, and R6dis independently selected from hydrogen, halogen, — OH, — NH2, — Cl-C3 alkyl, — C1-C3 alkoxy, — C1-C3 haloalkyl, — C1-C3 aminoalkyl, and — C1-C3 hydroxyalkyl, provided that at least one of R6a, R6b, R6c, and R6dis not hydrogen. In one variation of any embodiment disclosed herein, R1is -F; each of R5band R5cis independently selected from -R20and hydrogen; R20is selected from — C1-C4 alkylamino and — C1-C4 alkoxy; provided that one of R5band R5cis -R20; and each of R5a, R5d, and R5eis hydrogen; or a pharmaceuticallyacceptable salt thereof. In another variation, R1is -F; each of R5band R5cis independently selected from -R20and hydrogen; R20is selected from — C1-C4 alkoxy; provided that one of R5band R5cis -R20; and each of R5a, R5d, and R5eis hydrogen; or a pharmaceutically acceptable salt thereof. In yet another variation, each of each of R5band R5cis independently selected from -R20and hydrogen; R20is selected from — C2-C7 alkylamino and — C2-C7 alkoxy; provided that one of R5band R5cis -R20.

[0072] In another embodiment, the pharmaceutically acceptable salt comprises the conjugate base of compound of Formula I, Formula II, Formula III, Formula IV or the free acid of Compound 1, and a counter ion selected from Li+, K+, Na+, ammonium, tetramethyl ammonium, tetraethylammonium, Fe+2, Cu+2, Zn+2, Mg+2, Ca+2, Al+3, Fe+3, a cation of one or more of L-arginine, L-lysine, or N-methyl-D-glucamine, and combinations thereof. In yet another embodiment, the counter ion is selected from Li+, K+, Na+, and combinations thereof. In one embodiment, the counter ion is Na+In one embodiment, the counter ion is selected from ammonium, tetramethylammonium, tetraethylammonium, and combinations thereof. In one embodiment, the counter ion is selected from, Fe+2, Cu+2, Zn+2, Mg+2, Ca+2, Al+3, Fe+3, and combinations thereof. In one embodiment, the counter ion is selected from a cation of one or more of L-arginine, L-lysine, or N-methyl-D-glucamine, and combinations thereof.

[0073] In another aspect, provided are compositions comprising a compound described herein, such as a compound of Formula I, Formula II, Formula III, Formula IV, or the free acid of Compound I or a pharmaceutically acceptable salt thereof. A compound used in a pharmaceutical composition described herein may be any compound described herein. In general, a compound described herein is mixed with a suitable carrier or excipient in a therapeutically effective amount. By a “therapeutically effective dose”, “therapeutically effective amount”, or, interchangeably, “pharmacologically acceptable dose” or “pharmacologically acceptable amount”, it is meant that a sufficient amount of the compound and a pharmaceutically acceptable carrier, will be present in order to achieve a desired result, e.g., treating cancer.

[0074] In general, the compounds described herein will be administered in a therapeutically effective amount by any of the accepted modes of administration. The actual amount of the compound will depend upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors. The compound can be administered according to anysuitable dosage regimes, such as once, twice, three times, or four times, etc. a day, or as needed. All of these factors are within the skill of the attending clinician. In some embodiments, the compound is administered one or more times during a treatment cycle. In further embodiments, the treatment cycle is 21 days. In other embodiments, the treatment cycle is 28 days. In some embodiments, the compound is administered one or more times during a treatment cycle for up to four treatment cycles.

[0075] Therapeutically effective amounts of the compound may range from approximately 0.03 to 50 mg per kilogram body weight of the recipient per day; for example, about 0.1-25 mg / kg / day, or from about 0.5 to 10 mg / kg / day. Thus, for administration to a 70 kg person, the dosage range can be about 1-3,500 mg per day.

[0076] In some of the embodiments of the technology described herein, the pharmaceutical compositions are packaged in unit dosage form. The unit dosage form is effective in treating a disease, such as cancer. Generally, a unit dosage including a compound of the present technology will vary depending on patient considerations. Such considerations include, for example, age, protocol, condition, sex, extent of disease, contraindications, concomitant therapies, and the like. An exemplary unit dosage based on these considerations can also be adjusted or modified by a physician skilled in the art. For example, a unit dosage for a patient comprising a compound of the present technology can vary from 3 x 10-5g / kg to 1 g / kg, preferably, 1 x 10-3g / kg to 1.0 g / kg. Dosage of a compound of the present technology can also vary from 0.01 mg / kg to 100 mg / kg or, preferably, from 0.1 mg / kg to 10 mg / kg.

[0077] In some embodiments, the unit dosage comprises 0.01 mg / kg to 0.5 g / kg. In some embodiments, the unit dosage comprises 0.01 mg / kg to 100 mg / kg. In some embodiments, the unit dosage comprises 0.01 mg / kg to 50 mg / kg. In some embodiments, the unit dosage comprises 0.01 mg / kg to 10 mg / kg. In some embodiments, the unit dosage comprises 0.01 mg / kg to 5 mg / kg. In some embodiments, the unit dosage comprises 0.1 mg / kg to 0.5 g / kg. In some embodiments, the unit dosage comprises 0.1 mg / kg to 100 mg / kg. In some embodiments, the unit dosage comprises 0.1 mg / kg to 50 mg / kg. In some embodiments, the unit dosage comprises 0.01 mg / kg to 10 mg / kg. In some embodiments, the unit dosage comprises 0.1 mg / kg to 5 mg / kg.

[0078] In general, compounds described herein will be administered as pharmaceutical compositions by any one of the following routes: oral, transdermal, intranasal, by suppository, parenteral (e.g., intramuscular, intravenous or subcutaneous), or intrathecal administration.Compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions. The choice of formulation depends on various factors such as the mode of drug administration and bioavailability of the drug substance.

[0079] In some embodiments, pharmaceutical compositions described herein are comprised of, in general, a compound described herein in combination with at least one pharmaceutically acceptable excipient. In some instances, acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic benefit of the compound. Such excipient may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.

[0080] Solid pharmaceutical excipients include but are not limited to starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, and the like. Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferred liquid carriers, particularly for injectable solutions, include water, saline, phosphate buffered saline, citrate buffer, aqueous dextrose, glycols, etc.

[0081] The amount of a compound described herein in a pharmaceutical formulation or composition can vary within the full range employed by those skilled in the art. In some embodiments, the formulation will contain from about 0.01-99.99 wt.% of the active ingredient compound based on the total weight of the formulation, with the balance being one or more suitable pharmaceutical excipients. Preferably, in some instances, a compound described herein is present at a level of about 1-80 wt.%.

[0082] A pharmaceutical composition described herein may be made in any manner not inconsistent with the technical objectives of the current disclosure. For example, in some embodiments, a compound may be mixed (such as with a high shear mixer) with one or more additional components (such as a pharmaceutically acceptable excipient) to form a pharmaceutical composition described herein.III. Methods

[0083] In one aspect, methods and compositions described herein pertain to a therapeutic approach for the treatment of cancer, in a patient or subject in need thereof, particularly a human subject. In some cases, cancer may comprise ovarian cancer, such as epithelial ovarian cancer (EOC). In some instances, methods and compositions described herein target de novo pyrimidine synthesis.

[0084] Disclosed herein is a method for treating ovarian cancer by targeting de novo pyrimidine synthesis through the inhibition of dihydroorotate dehydrogenase (DHODH). By understanding the role of extracellular uridine salvage in drug resistance, this approach offers a therapeutic advantage for patients with defective uridine uptake pathways. Additionally, diagnostic tools to assess uridine salvage status enhance the precision medicine capabilities of this technology, making it a valuable tool for the pharmaceutical and oncology industries.

[0085] In one aspect, disclosed herein is a method of treating cancer in a patient in need thereof, wherein the method comprises: disposing a composition comprising an inhibitor of DHODH within a biological compartment of the patient; wherein the cancer has a deficient uridine salvage capability. In one embodiment, the method further comprises assessing the uridine salvage capability of the cancer, such as the cancer cells. In one embodiment, the method comprises assessing before disposing the composition comprising the inhibitor of DHODH. In one embodiment, assessing the uridine salvage capability comprises testing cells from the cancer in a 5-ethynyl uridine (5-EU) assay as disclosed herein. In one embodiment, a 5-EU assay can be used to identify patients who can benefit from administration of a DHODH inhibitor. In one embodiment, the uptake of 5-EU is evaluated, optionally by analyzing intensity and localization of the 5-EU signal in the cancer cells, such as with fluorescence microscopy. In another embodiment, assessing the uridine salvage capability comprises measuring the expression in the cancer cells of one or more, two or more, three or more, four or more or all of SLC28A1, SLC28A2, SLC28A3, SLC29A1, and SLC29A2. In one embodiment, the method further comprises obtaining a sample of cancer cells from the patient.

[0086] In another aspect, disclosed herein is a method of treating cancer in a subject comprising: obtaining a sample of cancer cells from the subject; testing cells from the cancer in a 5-ethynyl uridine (5 EU) assay; and administering an inhibitor of DHODH to the subject if thecancer cells are negative for 5-EU staining. In one embodiment, the 5-EU assay is an assay disclosed herein.

[0087] In one embodiment of any aspect disclosed herein, the concentration of 5-EU in the 5-EU assay is a concentration that selectively labels salvage-proficient cancer cell lines but does not selectively label salvage-deficient cancer cell lines. In one embodiment, the 5-ethynyl uridine concentration is 100 - 10 pM, 90 - 20 pM, 80 - 30 pM, 70 - 40 pM, or 65 - 45 pM. In another embodiment, 5-ethynyl uridine concentration is no more than 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, or no more than 40 pM. In another embodiment, the 5-ethynyl uridine concentration is 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, or 40 pM. In another embodiment, the 5-ethynyl uridine concentration is 50 pM.

[0088] In one embodiment, the 5-EU assay comprises measuring basal uridine salvage activity in cancer cells. In another embodiment, the 5-EU assay comprises measuring uridine uptake in cancer cells in response to treatment with a DHODH inhibitor. In another embodiment, the method further comprises analyzing the intensity and localization of the 5-EU signal in the cancer cells.

[0089] In one embodiment, the cancer cells in the 5-EU assay are exposed to a DHODH inhibitor. In yet another embodiment of any aspect disclosed herein, the 5-EU assay comprises use of 5-EU and a DHODH inhibitor. In one embodiment, the concentration of DHODH inhibitor used is 200 - 10 pM, 190 - 20 pM, 180 - 30 pM, 170 -40 pM, 160 - 50 pM, 150 - 60 pM, 140 - 70 pM, 130 - 80 pM, or 120 - 90 pM. In another embodiment, the concentration of DHODH inhibitor is no more than 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, or 50 pM. In another embodiment, the concentration of DHODH inhibitor is 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, or 50 pM. In one embodiment, the concentration of DHODH inhibitor is 100 pM. In one embodiment, the cancer cells are exposed to a DHODH inhibitor before being exposed to 5-EU. In another embodiment, the cancer cells are exposed to a DHODH inhibitor and 5-EU simultaneously.

[0090] In one embodiment, cancer cells tested, such as in the 5-EU assay, are conditionally reprogrammed. In one embodiment, the cancer cells are conditionally reprogrammed in a medium supplemented with estradiol and / or progesterone.

[0091] In one embodiment of any method disclosed herein, cancer cells having a deficient uridine salvage capability are negative for 5-EU staining in a 5-EU assay disclosed herein.Negative staining for 5-EU uptake indicates the cancer is DHODHi-sensitive and positive staining for 5-EU indicates the cancer is DHODHi-resistant. In one embodiment, negative staining comprises no detectable 5-EU signal in the 5 EU assay disclosed herein. In another embodiment, negative staining comprises a negligible or weak signal in the 5-EU assay disclosed herein. In another embodiment, negative staining of cancer cells in a 5-EU assay disclosed herein is equivalent to the staining of one or more, two or more, three or more, or four or more known salvage deficient cell lines treated in the same manner, including but not limited to Kuramochi, ES2, OVSAHO, CAOV3, R182 and OV35. In another embodiment, positive staining of cancer cells in a 5-EU assay disclosed herein corresponds to the staining of one or more, two or more, three or more, or four or more known salvage proficient cell lines treated in the same manner, including but not limited to OVCAR2, OVCAR4, UWB1, PEO1, and R127.

[0092] In one embodiment of any method disclosed herein, the cancer has or the cells of the cancer have deficient uridine uptake. In another embodiment, cancer cells having a deficient uridine salvage capability show less than 50%, 40%, 30%. 20%, 10%, or less than 5% uptake of radiolabeled uridine in 30 minutes when exposed to a DHODHi under physiological conditions as disclosed herein. In another embodiment, cancer cells having a deficient uridine salvage capability have an uptake of radiolabeled uridine in 30 minutes when exposed to a DHODHi under physiological conditions as disclosed herein, wherein the uptake is within 25%, 20%, 15% or 10% of the update of radiolabeled uridine of one or more, two or more, three or more, or four or more known salvage deficient cell lines treated in the same manner, including but not limited to Kuramochi, ES2, OVSAHO, CAOV3, R182 and OV35. In another embodiment, cancer cells having an uptake of radiolabeled uridine in 30 minutes when exposed to a DHODHi under physiological conditions as disclosed herein, wherein the uptake is within 25%, 20%, 15% or 10% of the update of radiolabeled uridine of one or more, two or more, three or more, or four or more known salvage proficient cell lines treated in the same manner, including but not limited to OVCAR2, OVCAR4, UWB1, PEO1, and R127 are excluded from administration of a DHODH inhibitor.

[0093] In one embodiment of any method disclosed herein, cancer cells having a deficient uridine salvage capability when cultured in media supplemented with dialyzed FBS and treatedwith 100 nM HOSU-53 in the presence of 20 pM uridine for 40 hr as disclosed herein show less than 75% or less than 50% relative uridine abundance compared to control, using LC-MS to determine the cellular level of uridine. In another embodiment of any method disclosed herein, cancer cells having a proficient uridine salvage capability when cultured in media supplemented with dialyzed FBS and treated with 100 nM HOSU-53 in the absence or presence of 20 pM uridine for 40 hr as disclosed herein show at least about 90%, 100%, or at least about 150% relative uridine abundance compared to control, using LC-MS to determine the cellular level of uridine. In some embodiments, uridine salvage proficient cancer is excluded from treatment according to the methods disclosed herein; in some embodiments, uridine salvage deficient cancer is treated according to the methods disclosed herein.

[0094] In yet another embodiment, the cancer is ovarian cancer, such as for example, EOC.

[0095] In one variation, the cancer cells to be tested, such as in the 5-EU assay, are conditionally reprogrammed, optionally in a medium supplemented with estradiol and / or progesterone.

[0096] In yet another aspect, disclosed herein is a method of treating cancer in a subject comprising administering an inhibitor of DHODH to the subject if the cancer cells are negative for 5-EU staining. In one embodiment, the method further comprises analyzing intensity and localization of the 5-EU signal in the cancer cells. In another embodiment, the method further comprises culturing the cancer cells with 5-ethynyl uridine (5-EU), optionally in combination with the inhibitor of DHODH. In another embodiment, the method further comprises conditionally reprogramming the cancer cells. In another embodiment, the method further comprises obtaining a sample of cancer cells from the subject. In yet another aspect, disclosed herein is a method of treating cancer in a subject comprising: obtaining a sample of cancer cells from the subject; optionally conditionally reprogramming the cancer cells; culturing the cancer cells with 5-ethynyl uridine (5-EU), optionally with a DHODH inhibitor; and administering an inhibitor of DHODH to the subject if the cancer cells are negative for 5-EU staining. In one embodiment, the method further comprises analyzing intensity and localization of the 5-EU signal in the cancer cells.

[0097] In one embodiment of any method disclosed herein, the inhibitor of DHODH is selected from the group consisting of brequinar, leflunomide, redoxal, vidofludimas, S-2678, ASLAN003 (2-(3,5-difluoro-3'-methoxybiphenyl-4-ylamino)nicotinic acid), BAY-2402234 (N-(2-chloro-6-fluorophenyl)-4-(4-ethyl-3-(hydroxymethyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-5-fluoro-2-((1,1,1-trifluoropropan-2-yl)oxy)benzamide), AG-636 (1-methyl-5-(2'-methyl-[1,1'-biphenyl]-4-yl)-1H-benzo[d][1,2,3]triazole-7-carboxylic acid), PTC-299 (4-chlorophenyl (S)-6-chloro-1-(4-methoxyphenyl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate), JNJ-74856665, Meds433, RP7214, ML390, Laflunimus, Tenovin-1, Tenovin-6, hDHODH-IN-4, DHODH-IN-11, and teriflunomide.

[0098] In one embodiment of any method disclosed herein, the inhibitor of DHODH may be any compound described in Section II. In some embodiments, the DHODH inhibitor is a compound of Formula I, and / or a pharmaceutically acceptable salt and / or solvate thereof. In some embodiments, the DHODH inhibitor is a compound of Formula II, and / or a pharmaceutically acceptable salt and / or solvate thereof. In some other embodiments, the DHODH inhibitor is a compound of Formula III, and / or a pharmaceutically acceptable salt and / or solvate thereof. In another embodiment of any method disclosed herein, the inhibitor of DHODH comprises HOSU-53 and / or a pharmaceutically acceptable salt and / or solvate thereof. In another embodiment, the pharmaceutically acceptable salt comprises the conjugate base of compound of Formula I, Formula II, Formula III, Formula IV or Compound I, and a counter ion selected from Li, K+, Na+, ammonium, tetramethylammonium, tetraethylammonium, Fe+2, Cu+2, Zn+2, Mg+2, Ca+2, Al+3, Fe+3, a cation of one or more of L-arginine, L-lysine, or N-methyl-D-glucamine and combinations thereof. In yet another embodiment, the counter ion is Na+In yet another embodiment, the counterion is a cation of one or more of L-arginine, L-lysine, or N-methyl-D-glucamine.

[0099] In some embodiments, the DHODH inhibitor disclosed herein is combined with one or more additional therapeutic agents for treating a subject in need thereof. A compound used in combination therapy may comprise any compound described herein. In some embodiments, the one or more additional therapeutic agents are selected from anti-cancer compounds. When a combination therapy is used, the one or more additional therapeutic agents may be administered sequentially or simultaneously with a compound described herein. In some embodiments, the one or more additional therapeutic agents is administered prior to the administration of a compound described herein. In some embodiments, the one or more additional therapeutic agents is administered after the administration of a compound described herein. In some embodiments, theone or more additional therapeutic agents is administered concurrently with the administration of a compound described herein.

[0100] In some embodiments, the one or more additional therapeutic agents is an antiangiogenesis agent (e.g., an agent that stops tumors from developing new blood vessels). In some embodiments, the anti-cancer agent is a so called “signal transduction inhibitor” (e.g., inhibiting the means by which regulatory molecules that govern the fundamental processes of cell growth, differentiation, and survival communicate within the cell). In some embodiments, a compound disclosed herein is used together with classical antineoplastic agents. Classical antineoplastic agents include but are not limited to hormonal modulators such as hormonal agents, anti-hormonal agents, androgen agonist agents, androgen antagonist and anti-estrogen therapeutic agents, histone deacetylase (HD AC) inhibitors, gene silencing agents or gene activating agents, ribonucleases, proteosomics, Topoisomerase I inhibitors, Camptothecin derivatives, Topoisomerase II inhibitors, alkylating agents, antimetabolites, poly(ADP -ribose) polymerase-I (P ARP-1) inhibitor, microtubulin inhibitors, antibiotics, plant derived spindle inhibitors, platinum-coordinated compounds, gene therapeutic agents, antisense oligonucleotides, vascular targeting agents (VTAs), and statins.

[0101] Further, in some cases, a compound disclosed herein is used in combination with a compound used for therapies for ovarian cancer. In some cases, for example, a compound used for treatment of ovarian cancer used with a compound disclosed herein includes, but is not limited to, a combination of cyclophosphamide and bevacizumab, docetaxel, etoposide, gemcitabine, liposomal doxorubicin alone or in combination with bevacizumab, paclitaxel alone or in combination with bevacizumab, topotecan alone or in combination with bevacizumab, carboplatin, olaparib, niraparib, rucaparib, aromatase inhibitors, such as leuprolide, megestrol, or tamoxifen, or a combination thereof.

[0102] In some embodiments, the one or more additional therapeutic agents is selected from 3 -deazauridine, 4-thiouracil, 5-benzylacyclouridine, 5-fluorouracil, 6-hydroxy-4-methyl-lH-pyridin-2-one-3 -carbonitrile, 8-MDP, benzylacyclouridine, capci tabine, cyclopentenyl uracil, cytarabine, decitabine, DI-87, dilazep, dipyridamole, draflamine, floxuridine, gemcitabine, N-(phosphonacetyl)-L-aspartate, nitrobenzylmercaptopurine riboside, nitrobenzylthioinosine, tegafur, ticagrelor, and combinations thereof.

[0103] Moreover, in some embodiments, a DHODH inhibitor described herein may be used with different types of therapies. For example, in some cases, a compound disclosed herein is used in combination with radiotherapy. In some embodiments, the method disclosed herein further comprises surgery, wherein all or part of a cancerous tumor is removed from the body of a patient. In one embodiment, surgery is used to remove all or part of a tumor, cells from which are evaluated using a 5-EU assay disclosed herein, and a DHODH inhibitor is administered to the subject if the cancer cells have deficient uridine salvage capability or deficient uridine uptake.

[0104] Some embodiments are further illustrated in the following non-limiting Examples.EXAMPLES

[0105] Embodiments described herein can be understood more readily by reference to the following summary and its previous and following descriptions. Elements and methods described herein, however, are not limited to the specific embodiments presented in the summary and Appendix. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

[0106] The efficacy of a DHODH inhibitor, HOSU-53, was evaluated and the molecular mechanisms for refractory to treatment in EOC were investigated. A panel of human EOC cell lines was tested and half were hyper-susceptible to HOSU-53 in vitro (FIG. 1A-1B). The ovarian cancer cell lines were cultured in medium supplemented with 10% FBS, treated with HOSU-53 (0-100 nM) for 7 days. Methylene blue staining assay was conducted to determine the cell viability. The IC50 for each cell line was calculated using GraphPad Prism. (N = 5, Bar: SD). DHODHi resistance

[0107] Small molecules in FBS were shown to contribute to the resistance of ovarian cancer cells to treatment with DHODH inhibitors. A panel of ovarian cancer cell lines was cultured in media supplemented with either 10% standard FBS or dialyzed FBS and treated with 0, 1, 5, 20, 100, or 500 nM HOSU-53 (FIGS. 2A-J), 0, 10, 50, 100, 200, or 500 nM Brequinar (FIGS. 3A-3G) or 0.1-20 nM BAY- 2402234 (FIGS. 3H-3N) for 7 days. Methylene blue staining assay wasconducted to assess the cell viability. Dialyzed FBS had all small molecules (M. W < 10 kDa) removed by fdtration.

[0108] Without being bound by theory, comparison of uridine-free and uridine-supplemented conditions showed that the vulnerability to DHODH inhibition was driven by deficiency in extracellular uridine salvage. Physiological uridine level varies: FBS@ 5 pM; Human plasma @ ~5 pM; organs, such as mouse liver & kidney @ 30 pM; Mouse@ 5 pM. A panel of ovarian cancer cell lines (OVCAR2, OVCAR4, COV362, PEO1, Kuramochi, OVSAHO, CAOV3, ES2) were cultured in dialyzed FBS containing media, treated with 0, 1, 5, 20, 100, or 500 nM HOSU-53 in the presence or absence of 20 pM uridine for 7 days. (FIGS. 4A-4H) Methylene blue staining assay was conducted to determine the cell viability. N = 5, Bar: SD. **: P < 0.01. Linear mixed effects models were used to analyze cell viability trends across doses. IC50 is indicated.

[0109] Uridine was shown to differentially rescue pyrimidine synthesis in ovarian cancer cells treated with HOSU-53. HOSU-53-sensitive (Kuramochi) and HOSU-53-resistant (OVCAR3) cells were cultured in media supplemented with dialyzed FBS and treated with 100 nM HOSU-53 in the absence or presence of 20 pM uridine for 40 h. LC-MS was used to profile pyrimidine derivatives. Volcano plots of metabolites show minimal restoration of pyrimidine derivatives in HOSU-53-sensitive Kuramochi cells (FIG. 5A), while pronounced restoration is observed in HOSU-53 -resistant OVCAR3 cells following uridine treatment (FIG. 5B).

[0110] Extracellular uridine was shown to restore the uridine pool depleted by HOSU-53 in resistant cells but not in sensitive cells. HOSU-53-resistant cells (PEO1, OVCAR3, OVCAR4) (FIG. 6A) and HOSU-53-sensitive cells (Kuramochi, CAOV3, ES2) (FIG. 6B) were cultured in media supplemented with dialyzed FBS, treated with 100 nM HOSU-53 in the absence or presence of 20 pM uridine for 40 h. LC-MS was used to determine the cellular level of uridine. In HOSU-53-resistant cells, extracellular uridine restores uridine pool depleted by HOSU-53. In HOSU-53-sensitive cells, extracellular uridine was unable to restore uridine pool depleted by HOSU-53.

[0111] Isotope-labeled uridine tracing confirmed proficient uptake in resistant cells, but not uptake in sensitive ones. HOSU-53 resistant cells R127 (FIG. 7A) and PEO1 (FIG. 7B) and HOSU-53 sensitive cells R182 (FIG. 7C), Kuramochi (FIG. 7D), and ES2 (FIG. 7E) were cultured in media supplemented with dialyzed FBS, treated with 100 nM HOSU-53 for 24 hr to deplete endogenous uridine. Then 20 pM13C-labeled uridine was added for different timeperiods, LC-MS / MS was used to measure unlabeled and13C-labeled uridine. Lines were defined as salvage-proficient or salvage-deficient, with salvage-proficient cells showing higher intracellular levels of isotope-labeled uridine than salvage-deficient cells, suggesting a stronger uridine uptake capacity in salvage-proficient cells.

[0112] DHODH inhibition was shown to activate the uptake of extracellular uridine in DHODHi resistant cells. HOSU-53-resistant cells R127 (FIG. 8A) and PEO1 (FIG. 8B) were cultured in media supplemented with dialyzed FBS, treated with HOSU-53 (100 nM) for 24 hr to deplete endogenous uridine or mock treated.13C-labeled uridine (20 pM) was then added for 30 min, LC-MS / MS was used to measure unlabeled and13C-labeled uridine. Comparing intracellular levels of isotope-labeled uridine with and without HOSU-53 treatment showed that13C-uridine levels were increased after HOSU-53 treatment. For example, in R127 cells, deuridine accounted for -20% without HOSU-53 treatment, whereas it exceeded 100% after HOSU-53 treatment. Without being bound by theory, these results indicate that HOSU-53 treatment enhanced uridine uptake.

[0113] DHODHi-resistant cells were sensitive to gemcitabine. A panel of ovarian cancer cell lines (PEO1, OVCAR4, OVCAR3, UWB1.289, Kuramochi, OVSAHO, CAOV3, ES2) were cultured in medium supplemented with 10% FBS, treated with 0, 0.5, 2, 10, 50, or 200 nM gemcitabine for 7 days. Methylene blue staining assay was conducted to determine the cell viability. The IC50 (inset) for each cell line was calculated using GraphPad Prism (N = 5, Bar: SD). (FIG. 9) HOSU-53-resistant cells exhibit high sensitivity to gemcitabine, while HOSU-53-sensitive cells exhibit resistance to gemcitabine. Given that gemcitabine sensitivity relies on nucleoside transporters, without being bound by theory, this result indicates that DHODHi resistant cells may contain activated nucleotide transporters.Xenograft models

[0114] In vivo studies using xenograft models derived from salvage-deficient cells demonstrated strong tumor suppression upon treatment significant tumor suppression upon treatment. In particular, the DHODHi, HOSU-53, efficiently blocked tumor growth in the ES2-derived orthotopic ovarian xenograft model (FIGS. 10A-10F). HOSU-53 significantly suppressed the progression of salvage-deficient tumors, prolonged mouse survival, and reduced ovarian cancer-induced ascites accumulation. FIG. 10A is a schematic illustration of the animal study, in which mice were treated orally with HOSU-53 at doses of vehicle, 3 mg / kg, or6 mg / kg, five times per week. Tumor progression was monitored using an in vivo imaging system (IVIS). Upon euthanasia, ascitic fluid was collected from the abdominal cavity, and ascites volume was measured. FIG. 10B are representative bioluminescence images of intraperitoneal xenografts following 2 weeks of treatment according to FIG. 10A. FIG. 10C is a graph of Bioluminescent Flux (x1011p / sec). FIG. 10D are photographs of ascites in xenograft bearing mice after euthanasia. FIG. 10E is a graph of ascitic fluid from ascites in xenograft bearing mice after euthanasia following treatment with vehicle, 3 mg / kg HOSU-53 or 6 mg / kg HOSU-53. FIG. 10F is a Kaplan-Meier survival curve depicting the survival of mice posttreatment. HOSU-53 also efficiently blocked tumor growth in the R182-derived orthotopic ovarian xenograft model (FIGS. 11A-11D). FIG. 11A is a schematic illustration of the animal study, modeled on the study in FIG. 10A. FIG. 11B are representative bioluminescence images of intraperitoneal xenografts following 18 days of treatment according to FIG. 11A. FIG. 11C is a graph of abdomen to chest ratio for tumor volumes. FIG. 11D is a graph of tumor volumes measured by BLI.5-ethynyl uridine assay

[0115] To facilitate clinical stratification, a functional assay was developed to assess uridine uptake using 5-ethynyl uridine (5-EU). A total of 1.2 x 106cells (OVCAR4 or Kuramochi) were seeded in glass-bottom dishes. Two days later, after the cells had adhered and displayed normal morphology, the medium was replaced with medium containing 10% dialyzed FBS. To determine the optimal conditions for assessing nucleoside uptake in cancer cells, the cells were treated with 100 nM HOSU-53 in combination with 0, 25, 50, 75, 100, or 200 pM EU for 24 h. After 24 hr of treatment, the cells were fixed and permeabilized at room temperature for 20 min using buffer containing 3.7% formaldehyde, 0.1% Triton X-100, 10 mM EGTA, 1 mM MgCl2, and 200 mM HEPES. The click reaction was then performed using 0.2% CuSO4, 25 mg / mL ascorbate, and 10 pM Alexa Fluor 488-azide in 100 mM Tris-HCl (pH 8.0) at 40 °C for 2 hr. Finally, 5-EU incorporation was detected using a fluorescence microscope. Images of OVCAR4 cells and Kuramochi cells revealed minimal 5-EU signal (minimal labeling) in salvage-deficient cells (Kuramochi, FIG. 12B) and strong 5-EU signal (strong labeling) in salvage-proficient ones (OVCAR4, FIG. 12A).

[0116] High concentration of 5-EU, such as 200 pM, was found to label both salvageproficient and salvage-deficient cells (see for example, the last panel in each of FIG. 12A andFIG. 12B). In contrast, a moderate concentration of 5-EU, such as 50 pM, was found to selectively label salvage-proficient cells, allowing distinction between these two cell types and enabling stratification. Therefore, for subsequent experiments, 50 pM 5-EU was used as the test condition, with 0 pM 5-EU as the negative control and 1,000 pM 5-EU as the positive control for setting microscope parameters and gating.

[0117] FIG. 13A are fluorescence microscope images of a panel of salvage proficient (DHODHi resistant) cells (OVCAR3, OVCAR4, UWB1, PEO1, and R127) cultured in the presence of 50 pM 5-EU for 24 hrs. FIG. 13B are images of a panel of salvage deficient (DHODHi sensitive) cells (Kuramochi, ES2, OVSAHO, CAOV3, R182, OV35) cultured in the presence of 50 pM 5-EU for 24 hrs. Cells were permeabilized and fixed and click chemistry reagents were added as described herein to visualize 5-EU-incorporated RNA. As shown, DHODHi-sensitive cells exhibit deficient nucleoside-uptake ability, evaluated at 50 pM 5-EU. The 5-EU assay as disclosed herein thus offers a scalable platform to identify tumors with impaired uridine salvage that may be responsive to DHODH inhibition.Conditional reprogramming

[0118] Additionally, the cells to be tested, including primary HGSOC cells, can conditionally reprogrammed, as generally described in Zhao, Ruihua, et al. " Conditional cell reprogramming in modeling digestive system diseases." Frontiers in Cell and Developmental Biology 9 (2021): 669756, modified as noted herein with additional hormones, such as for example, estradiol and / or progesterone. In particular, tumor tissue was isolated from a patient and diced into small pieces with a diameter of approximately 2-3 mm. The tissue fragments were transferred into a 50 mL conical tube containing 10 mL digestion buffer (5 mg / mL [625 U / mL] collagenase type II in PBS). Samples were incubated at 37 °C for 1 hr in a CO2 incubator on an orbital shaker set to 80-100 rpm. The digestion mixture was triturated every 15 min using a 5 or 10 mL pipette. After digestion, 10 mL PBS was added, and samples were centrifuged at 1,500 × g for 5 min to collect the cell pellet. The pellet was washed once with 10 mL PBS and centrifuged again at 1,500 × g for 5 min. Cells were resuspended in 1 mL PBS and transferred to a 15 mL tube containing 4 mL ACK lysis buffer, followed by incubation at room temperature for 5 min to remove red blood cells. Cells were then centrifuged at 1,500 × g for 5 min. The pellet was resuspended in 1 mL 0.25% trypsin and incubated at 37 °C for 5-10 min. Trypsinization was stopped by adding 9 mL PBS, followed by centrifugation at 1,500 × g for 5 min. Cells were subsequently resuspended in1 mL PBS containing 100 pg / mL DNase I and incubated at 37 °C for 5-10 min. The reaction was stopped by adding 9 mL PBS. The cell suspension was filtered through a 70 pm cell strainer into a 50 mL conical tube, and the strainer was rinsed with an additional 10 mL PBS. Cells were collected by centrifugation at 1,500 x g for 5 min. Finally, cells were seeded in complete CRC medium consisting of DMEM / Ham’s F-12 (3:1), 5% dialyzed FBS, insulin (5 pg / mL), hydrocortisone (0.8 pg / mL), cholera toxin (8.4 ng / mL), epidermal growth factor (20 ng / mL), penicillin-streptomycin (I*), Y-27632 (10 pM), and estradiol (10 nM). EpCAM and PAX 8, both HGSOC markers, were evaluated. FIG. 14A are fluorescence microscope images of ASCI 95 (EOC cells from ascites) evaluating the presence of EpCAM and PAX8, using DAPI. FIG. 14B are fluorescence microscope images of ASC3 (EOC cells from ascites) evaluating the presence of EpCAM and PAX8 using DAPI.5-EU assay combined with CRC can be used to measure cells ’ uridine salvage ability

[0119] The uridine salvage capacity in primary cancer cells (ASC 195, M1250760A, M1250890B, M1251951A, ASC3, M1251273A, M11242018A and M1250835A) was assessed using the 5-EU assay described above. Briefly, cells were seeded in glass-bottom dishes for 48 h, after which the medium (CRC medium) was replaced and cells were treated with 100 nM HOSU-53 and 50 pM 5-EU. Negative (0 pM 5-EU) and positive (1,000 pM 5-EU) controls were included for microscope parameter setup. After 24 hr of treatment, cells were fixed, permeabilized, and subjected to the click reaction. The 5-EU signal was then detected using a fluorescence microscope (FIG. 15 and FIG. 16). The dose-response curves (FIG. 15 and FIG. 16) show the IC50 of primary EOC cells under uridine-free and uridine-supplemented conditions. In particular, a total of 25,000 cells per well were seeded in 96-well plates using CRC medium containing estradiol. After 24 h, cells were treated with 0, 10, 50, 200, 1,000, or 5,000 nM HOSU-53, with or without 20 pM uridine (n = 5). After 7 days of treatment, cells were fixed with 3.7% formaldehyde at room temperature for 1 hr and stained with 0.5% methylene blue for 1 hr. Excess dye was removed and plates were washed with water. The dye was then dissolved in methanol: acetic acid (1:2), and absorbance at 609 nm was measured to calculate relative cell viability. IC₅₀ values were determined using GraphPad Prism 10. Four primary HGSOC samples ASC 195 (FIG. 15A), M1250760A (FIG. 15B), M1250890B (FIG. 15C), and M1251951A (FIG.15D) were found to be negative for 5-EU staining, indicating a deficiency in uridine salvage capacity. Consistent with this defect, supplementation of the culture medium with uridine did notalter their sensitivity to HOSU-53. Four primary HGSOC samples ASC3 (FIG. 16A), M1251273A (FIG. 16B), M11242018A (FIG. 16C), and M1250835A (FIG. 16D) were found to be positive for 5-EU staining, indicating proficient uridine salvage capacity (FIG. 16). Consistent with this phenotype, supplementation of the culture medium with uridine reduced their sensitivity to HOSU-53.Transporters

[0120] Since uridine uptake is mediated by SLC28A and SLC29A transporter families, their function, expression, and localization were investigated in several salvage-proficient EOC cell lines. Substantial intertumoral heterogeneity was observed in transporter expression and subcellular distribution, suggesting that different EOC cells may rely on distinct transporter subtypes or localization mechanisms to support uridine salvage. Without being bound by theory, such heterogeneity may account for the inconsistent responses in prior attempts to combine DHODH and SLC29A inhibitors.

[0121] ENT1 expression was shown to be lower in DHODHi-sensitive cells compared to DHODHi-resistant cells. A qRT-PCR was conducted to determine mRNA levels of various CNTs (SLC28A) and ENTs (SLC29A) in a panel of ovarian cancer cells with different sensitivity to HOSU-53 (SLC28A1 (FIG. 17A) SLC28A2 (FIG. 17B) SLC28A3 (FIG.17C), SLC29A1 (FIG. 17D) and SLC29A2 (FIG.17E)). Total RNA was extracted from ovarian cancer cells using a standard RNA isolation kit according to the manufacturer’s instructions. cDNA was synthesized from equal amounts of RNA using a reverse transcription kit. Quantitative real-time PCR was performed using SYBR Green master mix on a real-time PCR system to measure mRNA levels of CNTs (SLC28A family) and ENTs (SLC29A family). Gene expression was normalized to a housekeeping gene, and relative expression levels were calculated using the ΔΔCt method.

[0122] Immunoblotting was conducted to determine the protein level of SLC28A1 and SLC28A3 in a panel of ovarian cancer cell lines (FIG. 17F). Briefly, cells were lysed in SDS sample buffer and boiled at 100 °C for 15 min. Equal amounts of protein were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked and incubated with primary antibodies against SLC28A1 or SLC28A3, followed by HRP-conjugated secondary antibodies. Protein signals were detected using enhanced chemiluminescence and loading was normalized to a housekeeping protein (tubulin).

[0123] CNT1 was shown to play an essential role in uridine uptake in DHODH inhibitor-resistant OVCAR3 cells. ACNT1 (SLC28A1) expression was knocked down in OVCAR3 cells using two distinct shRNAs targeting SLC28A1 and knockdown efficiency was validated by Western blotting (FIG. 18A). The 5-EU assay was performed at 50 pM to assess uridine uptake in OVCAR3 cells following SLC28A1 knockdown (FIG. 18B). The results indicated that SLC28A1 knockdown significantly impaired 5-EU uptake, indicating that uridine uptake in OVCAR3 cells is largely dependent on SLC28A1. OVCAR3 cells were also treated with the ENT (pan-SLC29A) inhibitor NBMPR (25 pM) to further validate reliance on SLC28A1.NBMPR treatment did not alter 5-EU labeling in OVCAR3 cells (FIG. 18C) and did not affect uridine-mediated rescue of cell viability following HOSU-53 treatment (FIG. 18D). OVCAR3 cells were cultured in dialyzed FBS containing media, treated with HOSU-53 in the presence or absence of 20 pM Uridine or / and NBMPR for 7 days. Methylene blue staining assay was conducted to determine the cell viability. N = 5, Bar: SD.

[0124] HGSOC patients exhibit variable levels of CNT expression and uridine uptake capacity. The expression levels of various concentrative nucleoside transporters (CNTs, or SLC28A1 / 2 / 3) and equilibrative nucleoside transporters (ENTs, or SLC29A1 / 2) were analyzed from The Cancer Genome Atlas (TCGA) database for 308 serous ovarian cancer patients (FIG.19A). The 5-EU assay disclosed herein demonstrated different uridine uptake capacities in primary HGSOC cells (FIG. 19B); OV35 cells were derived from an HGSOC tumor tissue, while R182 and R127 cells were derived from the ascites of two HGSOC patients.

[0125] These results highlight DHODH inhibition as a strategy for treating cancer, such as ovarian cancer, particularly EOC, exemplified in tumors with impaired uridine salvage.Metabolic heterogeneity in uridine uptake was identified as a variable related to therapeutic response to DHODH inhibition. The 5-EU assay presented herein is a practical tool for stratifying patients based on metabolic phenotype. Together, these findings support the disclosed development of targeted and combination therapies to improve clinical outcomes in cancer, particularly ovarian cancer.

[0126] Additional exemplary embodiments contemplated herein are as follows:

[0127] Embodiment 1. A method of treating cancer in a patient in need thereof, wherein the method comprises: disposing a composition comprising an inhibitor of dihydroorotate dehydrogenase (DHODH) within a biological compartment of the patient; wherein the cancer has a deficient uridine salvage capability.

[0128] Embodiment 2. The method of Embodiment 1, further comprising assessing the uridine salvage capability of the cancer, optionally before disposing the composition comprising the inhibitor of DHODH.

[0129] Embodiment 3. The method of Embodiments 1 or 2, wherein assessing the uridine salvage capability comprises: testing cells from the cancer in a 5-ethynyl uridine (5-EU) assay.

[0130] Embodiment 4. The method of Embodiment 3, further comprising analyzing the intensity of the 5-EU signal in the cancer cells.

[0131] Embodiment 5. The method of Embodiment 3 or 4, further comprising analyzing the localization of the 5-EU signal in the cancer cells;

[0132] Embodiment 6. A method of treating cancer in a subject comprising: obtaining a sample of cancer cells from the subject; testing cells from the cancer in a 5-ethynyl uridine (5 EU) assay at a 5-EU concentration; and administering an inhibitor of DHODH to the subject if the cancer cells are negative for 5-EU staining.

[0133] Embodiment 7. The method of any of Embodiments 3-6, wherein the 5-ethynyl uridine concentration is 100 - 10 pM, 90 - 20 pM, 80 - 30 pM, 70 - 40 pM, or 65 - 45 pM.

[0134] Embodiment 8. The method of any of Embodiments 3-6, wherein the 5-ethynyl uridine concentration is no more than 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, or no more than 40 pM.

[0135] Embodiment 9. The method of any of Embodiments 3-6, wherein the 5-ethynyl uridine concentration is 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, or 40 pM.

[0136] Embodiment 10. The method of any of Embodiments 3-6, wherein the 5-ethynyl uridine concentration is 50 pM.

[0137] Embodiment 11. The method of any of Embodiments 3-10, wherein the 5-EU assay comprises measuring uridine uptake in cancer cells in response to treatment with a DHODH inhibitor rather than basal salvage activity.

[0138] Embodiment 12. The method of any of Embodiments 3-11, wherein the cancer cells are treated with a DHODH inhibitor before the 5-EU assay.

[0139] Embodiment 13. The method of any of Embodiments 3-11, wherein the 5-EU assay comprises exposing the cancer cells to 5-EU and a DHODH inhibitor.

[0140] Embodiment 14. The method of Embodiment 12 or 13, wherein the DHODH inhibitor concentration is 200 - 10 pM, 190 - 20 pM, 180 - 30 pM, 170-40 pM, 160 - 50 pM, 150 - 60 pM, 140 - 70 pM, 130 - 80 pM, or 120 - 90 pM.

[0141] Embodiment 14. The method of Embodiment 12 or 13, wherein the DHODH inhibitor concentration is no more than 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, or 50 pM.

[0142] Embodiment 15. The method of Embodiment 12 or 13, wherein the DHODH inhibitor concentration is 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, or 50 pM.

[0143] Embodiment 16. The method of Embodiment 12 or 13, wherein the DHODH inhibitor concentration is 100 pM.

[0144] Embodiment 17. The method of any of Embodiments 3-16, wherein the cancer cells are conditionally reprogrammed.

[0145] Embodiment 18. The method of Embodiment 17, wherein the cancer cells are conditionally reprogrammed in a medium supplemented with estradiol and / or progesterone.

[0146] Embodiment 19. The method of any of Embodiments 3-18, wherein cancer cells having a deficient uridine salvage capability are negative for 5-EU staining in the 5-EU assay.

[0147] Embodiment 20. The method of any of Embodiments 1-19, further comprising obtaining a sample of cancer cells.

[0148] Embodiment 21. The method of any of Embodiments 1-20, wherein the cancer has deficient uridine uptake.

[0149] Embodiment 22. The method of any of Embodiments 1-21, wherein the cancer is ovarian cancer, optionally epithelial ovarian cancer.

[0150] Embodiment 23. The method of any of Embodiments 1-22, wherein the inhibitor of DHODH is selected from the group consisting of brequinar, leflunomide, redoxal, vidofludimas, S-2678, ASLAN003 (2-(3,5-difluoro-3'-methoxybiphenyl-4-ylamino)nicotinic acid), BAY-2402234 (N-(2-chloro-6-fluorophenyl)-4-(4-ethyl-3-(hydroxymethyl)-5-oxo-4,5-dihydro-lH-1,2,4-triazol- 1 -yl)-5-fluoro-2-(( 1,1,1 -trifluoropropan-2-yl)oxy)benzamide), AG-636 ( 1 -methyl-5-(2'-methyl-[l,r-biphenyl]-4-yl)-lH-benzo[d][l,2,3]triazole-7-carboxylic acid), PTC-299 (4-chlorophenyl (S)-6-chloro-l-(4-methoxyphenyl)-l,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate), JNJ-74856665, Meds433, RP7214, ML390, Laflunimus, Tenovin-1, Tenovin-6, hDHODH-IN-4, DHODH-IN-11, and teriflunomide.

[0151] Embodiment 24. The method of any of Embodiments 1-22, wherein the inhibitor of DHODH is a compound of Formula I:wherein R1is selected from halogen, — SF5, — CN, —Ns, —OH, — NH2, — CF3, and -CF2CF3;wherein each of R5band R5cis independently selected from — R20, hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3; wherein R20is selected from — Cl -CIO alkylamino and — Cl -CIO alkoxy;provided that one of R5band R5cis — R20; andwherein each R5a, R3d, and R3eis independently selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3;or a pharmaceutically acceptable salt thereof.

[0152] Embodiment 25. The method of any of Embodiments 1-22, wherein the inhibitor of DHODH comprises a compound with the following structure:

[0153] Embodiment 26. The method of any of Embodiments 1-22, wherein the inhibitor of DHODH is a compound of Formula II:(Formula II)wherein each of Z1, Z2, Z3, and Z4is independently selected from CH and N; wherein R1is selected from hydrogen, halogen, — SF5, — CN, — N3, —OH, — NH2, — CF3, and — CF2CF3;wherein one of R5a, R5b, R5c, R3d, and R5eis selected from a group having formula represented by a structure:— R20, -R30-A1-R40, -A'-R40, — A1— R30— A2— R40, or — A1— R30— A2— R31— A3— R40; wherein A1is selected from — O— and —NR30—;wherein R50is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxy alkyl;wherein A2is selected from — O— and —NR60—;wherein R60is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxyalkyl;wherein A3is selected from — O— and —NR70—;wherein R70is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and —Cl -CIO hydroxyalkyl;wherein R20is selected from halogen, — C1-C10 alkyl, — C1-C10 haloalkyl, — C1-C10 hydroxyalkyl, — Cl -CIO alkylamino, and — Cl -CIO alkoxy;wherein each of R30and R31is independently selected from — C1-C10 alkanediyl, — Cl- C 10 haloalkanediyl, — C 1 -C 10 aminoalkanediyl, and — C 1 -C 10 hydroxyalkanediyl; and wherein R40is selected from — Cl -CIO alkyl, — Cl -CIO haloalkyl, — Cl -CIO aminoalkyl, — C1-C10 hydroxyalkyl, and — (CH₂)nAr1;wherein n is an integer selected from 1, 2, and 3; andwherein Ar1is a phenyl group substituted with 0,1, 2, 3, 4, or 5 groups independently selected from halogen, — SF5, — CN, — N3, —OH, — NH2, — C1-C4 alkyl, — C1-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — C1-C4alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl;and wherein four of R5a, R5b, R5c, R5d, and R5care independently selected from hydrogen, halogen, –SF₅, –CN, –N₃, –OH, –NH₂, –CF₃, and –CF₂CF₃;or a pharmaceutically acceptable salt thereof.

[0154] Embodiment 27. The method of any of Embodiments 1-22, wherein the inhibitor of DHODH is a compound of Formula III:wherein Z5is a five-membered heterocyclic diyl;wherein R1is selected from hydrogen, halogen, — SF5, — CN, — N3, —OH, — NH2, — CF3, and — CF2CF3;wherein one of R5a, R5b, R5c, R5d, and R5eis selected from a group having formula represented by a structure:— R20, — R30— A1— R40, — A1— R40, — A1— R30— A2— R40, or — A1— R30— A2— R31— A3— R40;wherein A1is selected from — O— and —NR50—;wherein R50is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — C1-C10 hydroxyalkyl;wherein A2is selected from — O— and —NR60—;wherein R60is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxy alkyl;wherein A3is selected from — O— and —NR70—;wherein R70is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxy alkyl;wherein R20is selected from halogen, — Cl -CIO alkyl, — Cl -CIO alkylamino and — Cl -CIO alkoxy;wherein each of R30and R31is independently selected from — C1-C10 alkanediyl, — Cl -CIO aminoalkanediyl, and — Cl -CIO hydroxy alkanediyl; andwherein R40is selected from — Cl -CIO alkyl, — Cl -CIO aminoalkyl, — Cl -CIO hydroxyalkyl, and —(CH2)nAr1;wherein n is an integer selected from 1, 2, and 3; andwherein Ar1is a phenyl group substituted with 0,1, 2, 3, 4, or 5 groups independently selected from halogen, — SF5, — CN, — N3, —OH, — NH2, from — C1-C4 alkyl, — C1-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — Cl- C4 alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl;and wherein four of R5a, R5b, R5c, R5d, and R5eis independently selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3;or a pharmaceutically acceptable salt thereof.

[0155] Embodiment 28. The method of any of Embodiments 1-22, wherein the inhibitor of DHODH is a compound of Formula IV:(Formula IV) wherein R1is selected from hydrogen, halogen, — SF5, — CN, — N3, —OH, — NH2, — CF3, and -CF2CF3;wherein one of R5a, R5b, R5c, R5d, and R5eis selected from a group having formula represented by a structure:— R20, — R30— A1— R40, -A'-R40, -A1-R30-A2-R40, or -A1-R30-A2-R31-A3-R40; wherein A1is selected from — O— and —NR30—;wherein R50is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and —Cl -CIO hydroxyalkyl;wherein A2is selected from — O— and —NR60—;wherein R60is selected from hydrogen, — C1-C10 alkyl, — C1-C10 aminoalkyl, and — Cl -CIO hydroxy alkyl;wherein A3is selected from — O— and —NR70—;wherein R70is selected from hydrogen, — Cl -CIO alkyl, — Cl -CIO aminoalkyl, and — Cl -CIO hydroxy alkyl;wherein R20is selected from halogen, — Cl -CIO alkyl, — Cl -CIO haloalkyl, — Cl -CIO hydroxyalkyl, — Cl -CIO alkylamino, — Cl -CIO alkoxy, — (CH2)nCy1, and— (CH2)nAr1;wherein n is an integer selected from 1, 2, and 3; andwherein Cy1is a C3-C10 cycloalkyl group or a C2-C9 heterocycloalkyl group substituted with 0, 1, 2, 3, 4, or 5 groups independently selected from halogen, -SF5, -CN, -N3, -OH, -NH2, from -C1-C4 alkyl, -C1-C4 alkoxy, -C1-C4 haloalkyl, — C1-C4 aminoalkyl, — C1-C4 alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl;wherein Ar1is a phenyl group substituted with 0, 1, 2, 3, 4, or 5 groups independently selected from halogen, — SF5, — CN, — N3, —OH, — NH2, from — C1-C4 alkyl, — C1-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — Cl- C4 alkylamino, — C1-C4 haloalkylamino, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl;wherein each of R30and R31is independently selected from — C1-C10 alkanediyl, — Cl -CIO haloalkanediyl, — Cl -CIO aminoalkanediyl, and— Cl -CIOhydroxy alkanediyl; andwherein R40is selected from — C1-C10 alkyl, — C1-C10 haloalkyl, — C1-C10 aminoalkyl, — C1-C10 hydroxyalkyl, — (CH2)nCy1, and — (CH2)nAr1;wherein n is an integer selected from 1, 2, and 3; andwherein Cy1is a C3-C10 cycloalkyl group or a C2-C9 heterocycloalkyl group substituted with 0, 1, 2, 3, 4, or 5 groups independently selected from halogen, -SF5, -CN, -N3, -OH, -NH2, from -C1-C4 alkyl, -C1-C4 alkoxy, -C1-C4 haloalkyl, — C1-C4 aminoalkyl, — C1-C4 alkylamino, — C1-C4 haloalkylamino,— C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl;wherein Ar1is a phenyl group substituted with 0, 1, 2, 3, 4, or 5 groups independently selected from halogen, — SF5, — CN, — N3, —OH, — NH2, from — C1-C4 alkyl, — C1-C4 alkoxy, — C1-C4 haloalkyl, — C1-C4 aminoalkyl, — Cl- C4 alkylamino, — C1-C4 haloalkyl ami no, — C1-C4 hydroxyalkyl, — C1-C4 halohydroxyalkyl, cycloalkyl, and heterocycloalkyl;and wherein four of R5a, R5b, R5c, R5d, and R5eare independently selected from hydrogen, halogen, -SF5, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3;wherein each of R6a, R6b, R6c, and R6dis independently selected from hydrogen, halogen, -SF5, -CN, — N3, -OH, -NH2, Cl -CIO alkyl, Cl -CIO alkoxy, Cl -CIO haloalkyl, Cl- C10 aminoalkyl, and Cl -CIO hydroxyalkyl, provided that at least one of R6a, R6b, R6c, and R6dis not hydrogen;or a pharmaceutically acceptable salt thereof.

[0156] Embodiment 29. The method of any of Embodiments 24-28 wherein the pharmaceutically acceptable salt comprises a counter ion selected from Li+, K+, Na+, ammonium, tetramethylammonium, tetraethylammonium, Fe+2, Cu+2, Zn+2, Mg+2, Ca+2, Al+3, Fe+3, a cation of one or more of L-arginine, L-lysine, or N-methyl-D-glucamine, and combinations thereof.

[0157] Embodiment 30. The method of Embodiment 29 wherein the counter ion is selected from Li+, K+, Na+, and combinations thereof.

[0158] Embodiment 31. The method of Embodiment 29 wherein the counter ion is selected from ammonium, tetramethylammonium, tetraethylammonium, and combinations thereof.

[0159] Embodiment 32. The method of Embodiment 29 wherein the counter ion is selected from, Fe+2, Cu+2, Zn+2, Mg+2, Ca+2, Al+3, Fe+3, and combinations thereof.

[0160] Embodiment 33. The method of Embodiment 29 wherein the counter ion is selected from a cation of L-arginine, L-lysine, N-methyl-D-glucamine, and combinations thereof.

[0161] Embodiment 34. The method of any of Embodiment 1-33, further comprising disposing a composition comprising a second therapeutic agent within a biological compartment of the patient.

[0162] Embodiment 35. The method of Embodiment 34, wherein the second therapeutic agent is selected from an anti-angiogenesis agent, a signal transduction inhibitor, or a classical antineoplastic agent.

[0163] Embodiment 36. The method of Embodiment 34, wherein the second therapeutic agent is selected from a hormonal modulator, an HD AC inhibitor, a gene silencing agent, a gene activating agent, a ribonuclease, a proteosomic, a Topoisomerase I inhibitor, a camptothecin derivative, a Topoisomerase II inhibitor, an alkylating agent, an antimetabolite, a PARP-1 inhibitor, a microtubulin inhibitor, a plant derived spindle inhibitor, a platinum-coordinated compound, a gene therapeutic agent, an antisense oligonucleotide, a vascular targeting agents, a statin and combinations thereof.

[0164] Embodiment 37. The method of Embodiment 34, wherein second therapeutic agent is selected from cyclophosphamide, bevacizumab, docetaxel, etoposide, gemcitabine, liposomal doxorubicin, paclitaxel, topotecan, carboplatin, olaparib, niraparib, rucaparib, and an aromatase inhibitor and combinations thereof.

[0165] Embodiment 38. The method of Embodiment 34, wherein the second therapeutic agent is selected from 3 -deazauridine, 4-thiouracil, 5-benzylacyclouridine, 5-fluorouracil, 6-hydroxy-4-methyl-lH-pyridin-2-one-3-carbonitrile, 8-MDP, benzyl acy cl ouri dine, capcitabine, cyclopentenyl uracil, cytarabine, decitabine, DI-87, dilazep, dipyridamole, draflamine, floxuridine, gemcitabine, N-(phosphonacetyl)-L-aspartate, nitrobenzylmercaptopurine riboside, nitrobenzylthioinosine, tegafur, ticagrelor, and combinations thereof.

[0166] All patent documents referred to herein are incorporated by reference in their entireties. Various embodiments of the invention have been described in fulfillment of the various objectives of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

Claims

1. CLAIMS1. A method of treating cancer in a patient in need thereof, wherein the method comprises:disposing a composition comprising an inhibitor of dihydroorotate dehydrogenase (DHODH) within a biological compartment of the patient;wherein the cancer has a deficient uridine salvage capability.

2. The method of claim 1, further comprising assessing the uridine salvage capability of the cancer.

3. The method of claim 2, wherein assessing the uridine salvage capability comprises testing cells from the cancer in a 5-ethynyl uridine (5-EU) assay.

4. The method of claim 3, wherein the cancer cells tested in the 5-EU assay are conditionally reprogrammed.

5. The method of claim 3, wherein cancer cells having a deficient uridine salvage capability are negative for 5-EU staining in the 5-EU assay.

6. The method of claim 2, further comprising obtaining a sample of cancer cells.

7. The method of claim 1, wherein the cancer has deficient uridine uptake.

8. The method of claim 1, wherein the cancer is ovarian cancer.

9. The method of any of claims 1-8, wherein the inhibitor of DHODH is selected from the group consisting of brequinar, leflunomide, redoxal, vidofludimas, S-2678, ASLAN003 (2-(3,5-difluoro-3'-methoxybiphenyl-4-ylamino)nicotinic acid), BAY-2402234 (N-(2-chloro-6-fluorophenyl)-4-(4-ethyl-3-(hydroxymethyl)-5-oxo-4,5-dihydro-lH-l,2,4-triazol-l-yl)-5-fluoro-2-(( 1,1,1 -trifluoropropan-2-yl)oxy)benzamide), AG-636 ( 1 -methyl-5-(2'-methyl-[ 1, 1 '-biphenyl]-4-yl)-lH-benzo[d][l,2,3]triazole-7-carboxylic acid), PTC-299 (4-chlorophenyl (S)-6-chloro-l-(4-methoxyphenyl)-l,3,4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate), JNJ-74856665, Meds433, RP7214, ML390, Laflunimus, Tenovin-1, Tenovin-6, hDHODH-IN-4, DHODH-IN-11, and teriflunomide.

10. The method of any of claims 1-8, wherein the inhibitor of DHODH is a compound of Formula I:(Formula I)wherein R1is selected from halogen, —SFs, — CN, — N3, —OH, — NH2, — CF3, and -CF2CF3;wherein each of R5band R5cis independently selected from — R20, hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3; wherein R20is selected from — C1-C10 alkylamino and — C1-C10 alkoxy;provided that one of R5band R5cis — R20; andwherein each R5a, R5d, and R5eis independently selected from hydrogen, halogen, -SFs, -CN, -N3, -OH, -NH2, -CF3, and -CF2CF3;or a pharmaceutically acceptable salt thereof.

11. The method of claim 10, wherein the inhibitor of DHODH is a compound having the structure:(Compound 1)or a pharmaceutically acceptable salt thereof.

12. The method of claim 10 wherein the pharmaceutically acceptable salt comprises the conjugate base of a compound of Formula I and a counter ion selected from Li+, K+, Na+, ammonium, tetramethylammonium, tetraethylammonium, Fe+2, Cu+2, Zn+2, Mg+2, Ca+2, Al+3, Fe+3, a cation of one or more of L-arginine, L-lysine, or N-methyl-D-glucamine, and combinations thereof.

13. The method of claim 1, further comprising disposing a composition comprising a second therapeutic agent within a biological compartment of the patient.

14. The method of claim 13, wherein the second therapeutic agent is selected from3 -deazauridine, 4-thiouracil, 5-benzylacyclouridine, 5-fluorouracil, 6-hydroxy-4-methyl-lH-pyridin-2-one-3 -carbonitrile, 8-MDP, benzylacyclouridine, capcitabine, cyclopentenyl uracil, cytarabine, decitabine, DI-87, dilazep, dipyridamole, draflamine, floxuridine, gemcitabine, N-(phosphonacetyl)-L-aspartate, nitrobenzylmercaptopurine riboside, nitrobenzylthioinosine, tegafur, ticagrelor, and combinations thereof.

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