WO2020242773A1

WO2020242773A1 - Methods of promoting differentiation of myeloid-derived suppressor cells using inhibitors of dihydroorotate dehydrogenase

Info

Publication number: WO2020242773A1
Application number: PCT/US2020/032794
Authority: WO
Inventors: Vikram S. Kumar; David P. Hesson
Original assignee: Clear Creek Bio, Inc.
Priority date: 2019-05-31
Filing date: 2020-05-14
Publication date: 2020-12-03

Abstract

The invention provides methods of using an inhibitor of dihydroorotate dehydrogenase (DHODH) to promote differentiation of myeloid-derived suppressor cells (MDSC) in a subject. The methods are useful for treating cancer. The DHODH inhibitor may be provided in combination with, or to improve the effects of, an agent that inhibits immunosuppression.

Description

METHODS OF PROMOTING DIFFERENTIATION OF MYELOID-DERIVED

SUPPRESSOR CELLS USING INHIBITORS OF DIHYDROOROTATE DEHYDROGENASE

Cross-Reference to Related Applications

This application claims the benefit of, and priority to, U.S. Provisional Patent Application No. 62/855,342, filed May 31, 2019, the contents of which are incorporated by reference.

Field of the Invention

The invention relates generally to therapeutic methods, including methods of treating cancer.

Background

Each year over 8 million people die worldwide from cancer or cancer-related illnesses. Cancer results from unchecked cell growth, and the unregulated cells invade other parts of the body, hijack nutritional resources, and impair the function of other tissues. Cancer is an indiscriminate killer, and nearly two in five people will be diagnosed with cancer at some point in their lives.

A characteristic of many types of cancer is suppression of activity of cells of the immune system that would otherwise eliminate cancer cells from the body. Immunosuppression may result from the activity of tumor cells and/or other cell types that support tumor progression. In tumor-cell mediated immunosuppression, tumor cells engage proteins on the surface of T cells to trigger regulatory checkpoints that halt the anti-tumor response. Among the non-tumor cells that play a key role in immunosuppression are myeloid-derived suppressor cells (MDSC), immature myeloid cells that display phenotypic heterogeneity. MDSC produce both diffusible and surface- bound signals that inhibit T cell proliferation and activation and block destruction of tumor cells.

Several current therapies for treating cancer seek to unlock immunosuppression and allow the body's immune system destroy the cancer cells. Such approaches, however, have met with only modest success. For example, immune checkpoint inhibitors, which block the checkpoint-triggering interactions between tumor cells and T cells, have no effect in many patients due in part to the inability of the checkpoint inhibitors to fully release the anti-tumoral immunosuppression such as by affecting MDSC-mediated mechanisms of immunosuppression. Therapies that deplete MDSC activity by causing differentiation of MDSC into mature macrophages or dendritic cells through differentiating agents such as all-trans retinoic acid (ATRA) are under investigation. Consequently, existing strategies for combating

immunosuppression to treat cancer are inadequate, and millions of people continue to die from cancer each year.

Summary

The invention provides methods for treating cancer by promoting differentiation of MDSC (e.g., into macrophages/dendritic cells, e.g., terminal differentiation) using inhibitors of dihydroorotate dehydrogenase (DHODH). DHODH inhibitors, such as brequinar, efficiently drive differentiation of MDSC into more mature cell types that lack the immunosuppressive properties of MDSC. Thus, by providing a DHODH inhibitor to cancer patients, methods of the invention reduce or prevent MDSC-mediated immunosuppression.

The invention provides a variety of methods for the use of DHODH inhibitors (e.g., Brequinar) to treat cancer in subjects. Because DHODH inhibitors (e.g., Brequinar) have a specific pharmacodynamic marker that can be used for intra-individual dose adjustment, such inhibitors may be used in lieu and/or may be superior to other therapies for suppressing MDSCs, such as ATRA. In preferred embodiments, the DHODH inhibitor is Brequinar. As an example, and without being limited by any mechanism of action, Brequinar is believed to be an ideal agent for suppressing MDSCs because Brequinar can be dose-adjusted and provided in a very precise manner to each patient. In certain embodiments, it is believed that a degree of MDSC

differentiation may be related to the DHO level (i.e. dose of the drug), which is a powerful approach for precise dosing. In that manner, thresholds are provided that can be correlated to safety and efficacy, such as thresholds of safety and efficacy (or MDSC pro-differentiation extent) in solid tumors.

In certain embodiments, DHODH inhibitors may also be used on conjunction with other agents that counter other mechanisms of immunosuppression. For example, the invention provides methods of using DHODH inhibitors in conjunction with agents, such as immune checkpoint inhibitors, that alleviate immunosuppression mediated directly by tumor cells.

In an aspect, the invention provides methods of promoting differentiation-of myeloid- derived suppressor cells (MDSC) in a subject by providing to the subject an inhibitor of dihydroorotate dehydrogenase (DHODH) in a therapeutically effective amount to promote differentiation of MDSC in the subject. The subject may have cancer.

In another aspect, the invention provides methods of treating cancer in a subject by providing to the subject an inhibitor of dihydroorotate dehydrogenase (DHODH) in a

therapeutically effective amount to promote differentiation of myeloid-derived suppressor cells (MDSC) in the subject. The methods may include providing to the subject an agent that inhibits immunosuppression in the subject, such as any of those described above.

In another aspect, the invention provides methods of treating cancer in a subject by providing to the subject an inhibitor of dihydroorotate dehydrogenase (DHODH) and an agent that inhibits immunosuppression in the subject.

In another aspect, the invention provides methods of amplifying an effect of an agent that inhibits immunosuppression in a subject by providing to a subject that has been given an agent that inhibits immunosuppression in the subject an inhibitor of dihydroorotate dehydrogenase (DHODH). The subject may have cancer.

The following details may pertain to any of the methods described above.

The DHODH inhibitor may be brequinar, leflunomide, teriflunomide, or a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation of any of the aforementioned compounds.

The cancer may be any cancer associated with MDSC activity. For example, the cancer may be bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer (e.g., squamous cell carcinoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, and B cell lymphoma), melanoma, Merkel cell carcinoma, myeloma (e.g., multiple myeloma), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, or uterine cancer.

The agent that inhibits immunosuppression in the subject may inhibit immunosuppression mediated by MDSC. For example, the agent that inhibits MDSC-mediated inhibition may be all- trans retinoic acid, axitinib, CFS-1R inhibitor, entinostat, gemcitabine, or phenformin. The agent that inhibits immunosuppression in the subject may be an immune checkpoint inhibitor. For example, the immune checkpoint inhibitor may be a CTLA-4 inhibitor, PD- 1 inhibitor, or a PD- L1 inhibitor. The CTLA-4 inhibitor may be ipilimumab. The PD-1 inhibitor may be cemiplimab, nivolumab, or pembrolizumab. PD-L1 inhibitor may be atezolizumab, avelumab, or durvalumab.

Detailed Description

A focus of many current cancer therapies is prevention of immunosuppression, a process by which tumor cells and other cell types block the immune system from attacking and destroying tumor cells. The rationale is that interfering with immunosuppression allows the immune system to eradicate cancer cells and/or increases the efficacy of drugs that target cancer cells directly. Studies have shown that inhibition of immunosuppression can improve the clinical outcomes of cancer patients, but the improvements are modest. In general, current therapeutic approaches are unable to achieve long-term success in treating cancer by combating

immunosuppression.

A major barrier to eliminating immunosuppression is the multifaceted nature of the phenomenon. Immunosuppression is driven by multiple cell types, each of which may use multiple molecular pathways. For example, tumor cells themselves provide immunosuppressive signals that prevent T cells from attacking and destroying them. In addition, in many cancers, immunosuppression is also mediated by myeloid-derived suppressor cells (MDSC).

Consequently, therapies that disrupt specific molecular pathways may show promise in initial treatments but eventually lose their potency due to compensating mechanisms of

immunosuppression.

The invention provides methods that address the aforementioned problems by reducing or eliminating MDSC from the body. The methods involve providing inhibitors of dihydroorotate dehydrogenase (DHODH), which promote the differentiation MDSC. Thus, the methods restore the natural cancer-fighting activity of the immune system and increase the effectiveness of other anti-cancer therapeutics, including agents that prevent other forms of immunosuppression and agents that inhibit cancer cell proliferation.

Myeloid-derived suppressor cells (MDSC) in cancer

Myeloid-derived suppressor cells (MDSC) are critical mediators of immunosuppression and tumor progression in many types of cancer. MDSC are immune cells that regulate the function of other types of immune cells, such as T cells, dendritic cells, macrophages, and natural killer (NK) cells. As a group, MDSC are heterogeneous, but they share immunosuppressive activity. MDSC expand in pathological situations, such as chronic infections and cancer, due to altered hematopoiesis. Specifically, myeloid differentiation is shifted from production of dendritic cells, macrophages, and neutrophils toward generation of MDSC. MDSC and their role in cancer are in detail in, for example, Veglia, et al., Myeloid- derived suppressor cells coming of age, Nat Immunol. 2018 February; 19(2): 108-119.

doi: 10.1038/s41590-017-0022-x; and Weber, et al., Myeloid-Derived Suppressor Cells Hinder the Anti-Cancer Activity of Immune Checkpoint inhibitors, Front Immunol. 2018 Jun 11 ;9: 1310. doi: 10.3389/fimmu.2018.01310, the contents of each of which are incorporated herein by reference.

In humans, MDSC fall into three phenotypic categories. Monocytic MDSC (M-MDSC) are defined as LhT CD1 lb⁺ CD14⁺ CD15 HLA DR ^{/ ,w}. Polymorphonuclear MDSC (PMN- MDSC) are defined LhT CD1 lb⁺ CD14 CD15⁺ HLAT>R or LhT CD1 lb⁺ CD14 CD66b⁺. Early stage MDSC (eMDSC) are defined as HLA-DR- CD33⁺ CD15 CD14 .

When MDSC are induced to differentiate to more mature cell types in the myeloid lineage, they lose their immunosuppressive and tumor- supporting properties. The retinoid all- trans retinoic acid (ATRA) promotes differentiation of MDSC. See, e.g., Nefdova, et al., Mechanism of All-Trans Retinoic Acid Effect on Tumor-Associated Myeloid-Derived

Suppressor Cells, Cancer Res. 2007 Nov 15;67(22): 11021-8, the contents of which are incorporated herein by reference. Consequently, ATRA has been used to target MDSC in melanoma patients treated with the immune checkpoint inhibitor ipilimumab. Tobin, et al., Targeting myeloid-derived suppressor cells using all-trans retinoic acid in melanoma patients treated with Ipilimumab, Int Immunopharmacol. 2018 Oct;63:282-291. doi:

10.1016/j.intimp.2018.08.007.

MDSC contribute to immunosuppression and tumor progression in a variety of cancers, including bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer (e.g., squamous cell carcinoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, and B cell lymphoma), melanoma, Merkel cell carcinoma, myeloma (e.g., multiple myeloma), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer. DHODH inhibitors

The invention recognizes that DHODH inhibitors stimulate MDSC differentiation. DHODH is a key enzyme in the pyrimidine biosynthesis pathway. Pyrimidine biosynthesis involves a sequence of step enzymatic reactions that result in the conversion of glutamine to uridine monophosphate as shown below:

Several of the enzymes in the pyridine synthesis pathway are targets of drugs or drug candidates. For example, aspartate carbamoyltransferase (also known as aspartate transcarbamoylase or ATCase), which catalyzes the conversion of carbamoyl phosphate to carbamoyl aspartate, is inhibited by PALA (N-phosphoacetyl-L-aspartate); dihydroorotate dehydrogenase (DHODH), which catalyzes conversion of dihydroorotate (DHO) to orotate, is inhibited by brequinar, leflunomide, and teriflunomide; and orotidine monophosphate decarboxylase (OMPD), which catalyzes conversion of orotidine monophosphate (OMP) to uridine monophosphate (UMP), is inhibited by pyrazofurin.

Exemplary DHODH inhibitors useful for embodiments of the invention are provided below. Brequinar, 6-fluoro-2-(2’-fluoro-l,l’ biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid, has the following structure:

Brequinar and related compounds are described in, for example, U.S. Patent Nos. 4,680,299 and 5,523,408, the contents of which are incorporated herein by reference. The use of brequinar to treat leukemia is described in, for example, U.S. Patent No. 5,032,597 and International

Publication No. WO 2017/037022, the contents of which are incorporated herein by reference.

Lefhmomide, N-(4'-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide (I), is described in, for example, U.S. Patent No. 4,284,786, the contents of which are incorporated herein by reference.

Terifhmomide, 2-cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]-2-butenamide, is described in, for example, U.S. Patent No. 5,679,709, the contents of which are incorporated herein by reference.

The DHODH inhibitor, such as any of the aforementioned compounds, may be provided as an analog, derivative, prodrug, micellar formulation, sustained release formulation, or salt of the pharmacologically active compound.

Because many of the enzymes involved in pyrimidine or purine biosynthesis are the targets of known inhibitors, metabolites in these pathways can serve as indicators of engagement of therapeutic agents with their targets. For example, the utility of DHO as an indicator of target engagement by DHODH inhibitors has been described in co-owned, co-pending U.S.

Application No. 62/682,419, the contents of which are incorporated herein by reference. One advantage of DHO is that cell membranes are permeable to the molecule. DHODH is localized to the mitochondrial inner membrane within cells, making direct measurement of enzyme activity difficult. However, DHO, which accumulates when DHODH is inhibited, diffuses out of cells and into the blood, which can be easily sampled. DHO is also sufficiently stable that levels of the metabolite can be measured reliably. Thus, by analyzing levels of DHO in blood or blood products, one can readily assess target engagement of a DHODH inhibitor.

Agents that inhibit immunosuppression

In certain embodiments, methods of the invention include providing one or more agents that inhibit immunosuppression in conjunction with a DHODH inhibitor. The agent may inhibit immunosuppression mediated by MDSC. For example, the agent that inhibits MDSC-mediated inhibition may be all-trans retinoic acid, axitinib, CFS-1R inhibitor, entinostat, gemcitabine, or phenformin. The agent may be an immune checkpoint inhibitor. For example, the immune checkpoint inhibitor may be a CTLA-4 inhibitor, PD-1 inhibitor, or a PD-L1 inhibitor. The CTLA-4 inhibitor may be ipilimumab. The PD-1 inhibitor may be cemiplimab, nivolumab, or pembrolizumab. PD-L1 inhibitor may be atezolizumab, avelumab, or durvalumab. Immune checkpoint inhibitors are described in detail in, for example, Darvin, et ah, Immune checkpoint inhibitors: recent progress and potential biomarkers, Exp Mol Med. 2018 Dec 13;50(12): 165. doi: 10.1038/sl2276-018-0191-l, the contents of which are incorporated herein by reference.

Determining a dosing regimen for the DHODH inhibitor

Methods of the invention may include determining a dosing regimen of a DHODH inhibitor for a subject. The dosing regimen may include a dose, i.e., an amount, of the DHODH inhibitor that should be administered. The dosing regimen may include a time point for administration of a dose of the DHODH inhibitor to the subject. Because the dosing regimen is based on one or more measured levels of DHO in a sample obtained from the subject, the dosing regimen is tailored to an individual subject, e.g., a patient. Consequently, the methods of the invention provide customized dosing regimens that account for variability in pharmacokinetic properties, i.e., metabolism of the active pharmaceutic ingredient (API) by the subject, and pharmacodynamics properties, effect of the API on its target, among individuals.

The dosing regimen may be determined by comparing a measured level of DHO in a sample obtained from a subject to a reference that provides an association between the measured level and a recommended dosage adjustment of the DHODH inhibitor. For example, the reference may provide a relationship between administration of the DHODH inhibitor and levels of DHO in the subject. The relationship can be empirically determined from a known dose and time of administration of the DHODH inhibitor and measured levels of DHO at one or more subsequent time points. The reference may include a relationship between measured levels of the DHODH inhibitor or a metabolic product of the DHODH inhibitor and measured levels of DHO.

From the comparison between the measured level of DHO and the reference, a dosing regimen may then be determined. The dosing regimen may include a dosage of the DHODH inhibitor, a time for administration of the dosage, or both. The dosing regimen may be determined de novo, or it may comprise an adjustment to a previous dosing regimen, such as an adjustment in the dosage, the interval between administration of dosages, or both.

The dosing regimen is designed to deliver the DHODH inhibitor to the subject in an amount that achieves a therapeutic effect. The therapeutic effect may be a sign or symptom of a disease, disorder, or condition. The therapeutic effect may be inhibition of an enzyme in the metabolic pathway, or it may be a change in an indicator of inhibition of an enzyme in a metabolic pathway. The indicator may be DHO in the pathway, and the therapeutic effect may be an increase or decrease in levels of DHO. The therapeutic effect may be a decrease in number of cancer cells, a decrease in proliferation of cancer cells, an increase in differentiation of pre- cancerous cells, such as myeloblasts, complete remission of cancer, complete remission with incomplete hematologic recovery, morphologic leukemia-free stat, or partial remission.

Increased differentiation of myeloblasts may be assessed by one or more of expression of CD 14, expression of CD1 lb, nuclear morphology, and cytoplasmic granules. The therapeutic effect may be differentiation of MDSC or a sub-population of MDSC.

The dosing regimen may ensure that levels of DHO are raised or maintained a minimum threshold required to achieve a certain effect. For example, the dosing regimen may raise or maintain levels of DHO above a threshold level in the subject for a certain time period. The time period may include a minimum, a maximum, or both. For example, the dosing regimen may raise or maintain levels of DHO above the threshold level for at least 6 hours, 12, hours, 24 hours, at least 48 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, or more. The dosing regimen may raise or maintain levels of DHO above the threshold level for not more than 24 hours, not more than 36 hours, not more than 48 hours, not more than 60 hours, not more than 72 hours, not more than 84 hours, not more than 96 hours, not more than 5 days, not more than 6 days, not more than 7 days, not more than 10 days, or not more than 2 weeks. The dosing regimen may raise or maintain levels of DHO above the threshold level for at least 72 hours but not more than 96 hours, for at least 72 hours but not more than 5 days, for at least 72 hours but not more than 6 days, for at least 72 hours but not more than 7 days, for at least 96 hours but not more than 7 days.

The dosing regimen may ensure that levels of DHO do not exceed or are maintained below a maximum threshold that is associated with toxicity. Levels of DHO above a maximum threshold may indicate that the DHODH inhibitor is causing or is likely to cause an adverse event in the subject. For example and without limitation, adverse events include abdominal pain, anemia, anorexia, blood disorder, constipation, diarrhea, dyspepsia, fatigue, fever,

granulocytopenia, headache, infection, leukopenia, mucositis, nausea, pain at the injection site, phlebitis, photosensitivity, rash, somnolence, stomatitis, thrombocytopenia, and vomiting.

The dosing regimen may include a time point for administration of one or more subsequent doses to raise or maintain levels of DHO above a threshold level for a certain time period. The time point for administration of a subsequent dose may be relative to an earlier time point. For example, the time point for administration of a subsequent dose may be relative to a time point when a previous dose was administered or a time point when a sample was obtained from a subject.

The dosing regimen may include a schedule for administration of doses. For example, doses may be administered at regular intervals, such as every 24 hours, every 36 hours, every 48 hours, every 60 hours, every 72 hours, every 84 hours, every 96 hours, every 5 days, every 6 days, every week, every 2 weeks, every 3 weeks, or every 4 weeks. Alternatively, doses may be administered according to a schedule that does not require precisely regular intervals. For example, doses may be administered once per week, twice per week, three times per week, four times per week, once per month, twice per month, three times per month, four times per month, five times per month, or six times per month.

For example and without limitation, a dosing regimen for administration of a DHODH inhibitor, such brequinar, e.g., brequinar sodium, to a human subject may be as follows: 100 mg/m , administered intravenously twice weekly; 125 mg/m , administered intravenously twice weekly; 150 mg/m 2 , administered intravenously twice weekly; 200 mg/m 2 , administered intravenously twice weekly; 250 mg/m 2 , administered intravenously twice weekly; 275 mg/m 2 , administered intravenously twice weekly; 300 mg/m , administered intravenously twice weekly;

350 mg/m 2 , administered intravenously twice weekly; 400 mg/m 2 , administered intravenously twice weekly; 425 mg/m 2 , administered intravenously twice weekly; 450 mg/m 2 , administered intravenously twice weekly; 500 mg/m 2 , administered intravenously twice weekly; 550 mg/m 2 , administered intravenously twice weekly; 600 mg/m , administered intravenously twice weekly;

650 mg/m 2 , administered intravenously twice weekly; 700 mg/m 2 , administered intravenously twice weekly; 750 mg/m 2 , administered intravenously twice weekly; 800 mg/m 2 , administered intravenously twice weekly; 100 mg/m 2 , administered intravenously every 72 hours; 125 mg/m 2 , administered intravenously every 72 hours; 150 mg/m , administered intravenously every 72 hours; 200 mg/m 2 , administered intravenously every 72 hours; 250 mg/m 2 , administered intravenously every 72 hours; 275 mg/m , administered intravenously every 72 hours; 300 mg/m 2 , administered intravenously every 72 hours; 350 mg/m 2 , administered intravenously every

72 hours; 400 mg/m 2 , administered intravenously every 72 hours; 425 mg/m 2 , administered intravenously every 72 hours; 450 mg/m , administered intravenously every 72 hours; 500 mg/m 2 , administered intravenously every 72 hours; 550 mg/m 2 , administered intravenously every

72 hours; 600 mg/m 2 , administered intravenously every 72 hours; 650 mg/m 2 , administered intravenously every 72 hours; 700 mg/m , administered intravenously every 72 hours; 750 mg/m 2 , administered intravenously every 72 hours; 800 mg/m 2 , administered intravenously every

72 hours; 100 mg/m 2 , administered intravenously every 84 hours; 125 mg/m 2 , administered intravenously every 84 hours; 150 mg/m , administered intravenously every 84 hours; 200 mg/m 2 , administered intravenously every 84 hours; 250 mg/m 2 , administered intravenously every

84 hours; 275 mg/m 2 , administered intravenously every 84 hours; 300 mg/m 2 , administered intravenously every 84 hours; 350 mg/m , administered intravenously every 84 hours; 400 mg/m 2 , administered intravenously every 84 hours; 425 mg/m 2 , administered intravenously every

84 hours; 450 mg/m 2 , administered intravenously every 84 hours; 500 mg/m 2 , administered intravenously every 84 hours; 550 mg/m , administered intravenously every 84 hours; 600 mg/m 2 , administered intravenously every 84 hours; 650 mg/m 2 , administered intravenously every

84 hours; 700 mg/m 2 , administered intravenously every 84 hours; 750 mg/m 2 , administered intravenously every 84 hours; 800 mg/m , administered intravenously every 84 hours; 100 mg/m 2 , administered intravenously every 96 hours; 125 mg/m 2 , administered intravenously every

96 hours; 150 mg/m 2 , administered intravenously every 96 hours; 200 mg/m 2 , administered intravenously every 96 hours; 250 mg/m , administered intravenously every 96 hours; 275 mg/m 2 , administered intravenously every 96 hours; 300 mg/m 2 , administered intravenously every

96 hours; 350 mg/m 2 , administered intravenously every 96 hours; 400 mg/m 2 , administered intravenously every 96 hours; 425 mg/m , administered intravenously every 96 hours; 450 mg/m 2 , administered intravenously every 96 hours; 500 mg/m 2 , administered intravenously every

96 hours; 550 mg/m 2 , administered intravenously every 96 hours; 600 mg/m 2 , administered intravenously every 96 hours; 650 mg/m , administered intravenously every 96 hours; 700 mg/m 2 , administered intravenously every 96 hours; 750 mg/m 2 , administered intravenously every

96 hours; 800 mg/m 2 , administered intravenously every 96 hours; 100 mg/m 2 , administered orally twice weekly; 125 mg/m 2 , administered orally twice weekly; 150 mg/m 2 , administered orally twice weekly; 200 mg/m 2 , administered orally twice weekly; 250 mg/m 2 , administered orally twice weekly; 275 mg/m 2 , administered orally twice weekly; 300 mg/m 2 , administered orally twice weekly; 350 mg/m 2 , administered orally twice weekly; 400 mg/m 2 , administered orally twice weekly; 425 mg/m 2 , administered orally twice weekly; 450 mg/m 2 , administered orally twice weekly; 500 mg/m 2 , administered orally twice weekly; 550 mg/m 2 , administered orally twice weekly; 600 mg/m 2 , administered orally twice weekly; 650 mg/m 2 , administered orally twice weekly; 700 mg/m 2 , administered orally twice weekly; 750 mg/m 2 , administered orally twice weekly; 800 mg/m 2 , administered orally twice weekly; 100 mg/m 2 , administered orally every 72 hours; 125 mg/m 2 , administered orally every 72 hours; 150 mg/m 2 , administered orally every 72 hours; 200 mg/m 2 , administered orally every 72 hours; 250 mg/m 2 , administered orally every 72 hours; 275 mg/m 2 , administered orally every 72 hours; 300 mg/m 2 , administered orally every 72 hours; 350 mg/m 2 , administered orally every 72 hours; 400 mg/m 2 , administered orally every 72 hours; 425 mg/m 2 , administered orally every 72 hours; 450 mg/m 2 , administered orally every 72 hours; 500 mg/m 2 , administered orally every 72 hours; 550 mg/m 2 , administered orally every 72 hours; 600 mg/m 2 , administered orally every 72 hours; 650 mg/m 2 , administered orally every 72 hours; 700 mg/m 2 , administered orally every 72 hours; 750 mg/m 2 , administered orally every 72 hours; 800 mg/m 2 , administered orally every 72 hours; 100 mg/m 2 , administered orally every 84 hours; 125 mg/m 2 , administered orally every 84 hours; 150 mg/m 2 , administered orally every 84 hours; 200 mg/m 2 , administered orally every 84 hours; 250 mg/m 2 , administered orally every 84 hours; 275 mg/m 2 , administered orally every 84 hours; 300 mg/m 2 , administered orally every 84 hours; 350 mg/m 2 , administered orally every 84 hours; 400 mg/m 2 , administered orally every 84 hours; 425 mg/m 2 , administered orally every 84 hours; 450 mg/m 2 , administered orally every 84 hours; 500 mg/m 2 , administered orally every 84 hours; 550 mg/m 2 , administered orally every 84 hours; 600 mg/m 2 , administered orally every 84 hours; 650 mg/m 2 , administered orally every 84 hours; 700 mg/m 2 , administered orally every 84 hours; 750 mg/m 2 , administered orally every 84 hours; 800 mg/m 2 , administered orally every 84 hours; 100 mg/m 2 , administered orally every 96 hours; 125 mg/m 2 , administered orally every 96 hours; 150 mg/m 2 , administered orally every 96 hours; 200 mg/m 2 , administered orally every 96 hours; 250 mg/m 2 , administered orally every 96 hours; 275 mg/m 2 , administered orally every 96 hours; 300 mg/m 2 , administered orally every 96 hours; 350 mg/m 2 , administered orally every 96 hours; 400 mg/m 2 , administered orally every 96 hours; 425 mg/m 2 , administered orally every 96 hours; 450 mg/m 2 , administered orally every 96 hours; 500 mg/m 2 , administered orally every 96 hours; 550 mg/m 2 , administered orally every 96 hours; 600 mg/m 2 , administered orally every 96 hours; 650 mg/m 2 , administered orally every 96 hours; 700 mg/m 2 , administered orally every 96 hours; 750 mg/m 2 , administered orally every 96 hours; or 800 mg/m , administered orally every 96 hours.

Minimum and maximum threshold levels of a metabolite depend on a variety of factors, such as the type of subject, metabolite, therapeutic agent, and type of sample. Minimum and maximum threshold levels may be expressed in absolute terms, e.g., in units of concentration, or in relative terms, e.g., in ratios relative to a baseline or reference value. For example, the minimum threshold (below which a patient may receive a dose increase or additional dose) could also be calculated in terms of increase from a pre-treatment DHO level or baseline level.

Minimum threshold levels of DHO in a human plasma sample may be about 0 ng/ml, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/mL, about 450 ng/mL, about 500 ng/mL, about 550 ng/mL, about 600 ng/mL, about 650 ng/mL, about 700 ng/mL, about 750 ng/mL, about 800 ng/mL, about 850 ng/mL, about 900 ng/mL, about 950 ng/mL, about 1000 ng/mL, about 1250 ng/ml, about 1500 ng/ml, about 1750 ng/ml, about 2000 ng/ml, about 2500 ng/ml, about 3000 ng/ml, about 3500 ng/ml, about 4000 ng/ml, about 4500 ng/ml, about 5000 ng/ml, about 6000 ng/ml, about 8000 ng/ml, about 10,000 ng/ml, about 12,000 ng/ml, about 15,000 ng/ml, about 20,000 ng/ml, about 25,000 ng/ml, about 30,000 ng/ml, about 40,000 ng/ml, about 50,000 ng/ml, about 75,000 ng/ml, about 100,000 ng/ml, about 150,000 ng/ml, about 200,000 ng/ml, about 300,000 ng/ml, or about 400,000 ng/ml. The minimum threshold may include any value that falls between the values recited above. Thus, the minimum threshold may include any value between 0 ng/ml and 400,00 ng/ml.

Maximum threshold levels of DHO in a human plasma sample may be about 50 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/mL, about 450 ng/mL, about 500 ng/mL, about 550 ng/mL, about 600 ng/mL, about 650 ng/mL, about 700 ng/mL, about 750 ng/mL, about 800 ng/mL, about 850 ng/mL, about 900 ng/mL, about 950 ng/mL, about 1000 ng/mL, about 1250 ng/ml, about 1500 ng/ml, about 1750 ng/ml, about 2000 ng/ml, about 2500 ng/ml, about 3000 ng/ml, about 3500 ng/ml, about 4000 ng/ml, about 4500 ng/ml, about 5000 ng/ml, about 6000 ng/ml, about 8000 ng/ml, about 10,000 ng/ml, about 12,000 ng/ml, about 15,000 ng/ml, about 20,000 ng/ml, about 25,000 ng/ml, about 30,000 ng/ml, about 40,000 ng/ml, about 50,000 ng/ml, about 75,000 ng/ml, about 100,000 ng/ml, about 150,000 ng/ml, about 200,000 ng/ml, about 300,000 ng/ml, about 400,000 ng/ml, or about 500,000 ng/ml. The maximum threshold may include any value that falls between the values recited above. Thus, the maximum threshold may include any value between 50 ng/ml and 500,00 ng/ml.

The minimum threshold of DHO may be about 1.5 times the baseline level, about 2 times the baseline level, about 2.5 times the baseline level, about 3 times the baseline level, about 4 times the baseline level, about 5 times the baseline level, about 10 times the baseline level, about 20 times the baseline level, about 50 times the baseline level, about 100 times the baseline level, about 200 times the baseline level, about 500 times the baseline level, about 1000 times the baseline level, about 2000 times the baseline level, or about 5000 times the baseline level. The minimum threshold may include any ratio that falls between those recited above. Thus, the minimum threshold may be any ratio between 1.5 times the baseline level and 5000 times the baseline level.

The maximum threshold of DHO may be about 2 times the baseline level, about 2.5 times the baseline level, about 3 times the baseline level, about 4 times the baseline level, about 5 times the baseline level, about 10 times the baseline level, about 20 times the baseline level, about 50 times the baseline level, about 100 times the baseline level, about 200 times the baseline level, about 500 times the baseline level, about 1000 times the baseline level, about 2000 times the baseline level, about 5000 times the baseline level, or about 10,000 times the baseline level. The maximum threshold may include any ratio that falls between those recited above. Thus, the maximum threshold may be any ratio between 2 times the baseline level and 10,000 times the baseline level.

The DHODH inhibitor may be any DHODH inhibitor, such as those described above.

Dosing of the DHODH inhibitor may account for the formulation of the DHODH inhibitor. For example, DHODH inhibitors, such as brequinar, leflunomide, and teriflunomide, may be provided as prodrugs, analogs, derivatives, or salts. Any of the aforementioned chemical forms may be provided in a pharmaceutically acceptable formulation, such as a micellar formulation.

Dosage of the DHODH inhibitor also depends on factors such as the type of subject and route of administration. The dosage may fall within a range for a given type of subject and route of administration, or the dosage may be adjusted by a specified amount for a given type of subject and route of administration. For example, dosage of brequinar for oral or intravenous administration to a subject, such as human or mouse, may be about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, or about 100 mg/kg. Dosage of brequinar for oral or intravenous administration to a subject, such as a human or mouse, may be adjusted by about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 50 mg/kg. Dosage of brequinar for oral or intravenous administration to an animal subject, such as a human or mouse, may be about 50 mg/m , about 100 mg/m², about 200 mg/m², about 300 mg/m², about 350 mg/m², about 400 mg/m², about 500 mg/m , about 600 mg/m , about 700 mg/m , about 800 mg/m , or about 1000 mg/m . Dosage of brequinar for oral or intravenous administration to an animal subject, such as a human or mouse, may be adjusted by about 50 mg/m , about 100 mg/m , about 200 mg/m , about 300 mg/m , about 350 mg/m , or about 400 mg/m .

The methods are useful for providing guidance on dosing of therapeutic agents for individuals. Therefore, the methods may be performed by any party that wishes to provide such guidance. For example and without limitation, the methods may be performed by a clinical laboratory; a physician or other medical professional; a supplier or manufacturer of a therapeutic agent; an organization that provides analytical services to a physician, clinic, hospital, or other medical service provider; or a healthcare consultant.

Measuring the level of a metabolite in a sample

Methods of the invention may include analysis of a measured level of a metabolite, such as DHO in a sample from a subject. The methods may include measurement of the level of the metabolite.

In some embodiments, the metabolite is measured by mass spectrometry, optionally in combination with liquid chromatography. Molecules may be ionized for mass spectrometry by any method known in the art, such as ambient ionization, chemical ionization (Cl), desorption electrospray ionization (DESI), electron impact (El), electrospray ionization (ESI), fast-atom bombardment (FAB), field ionization, laser ionization (LIMS), matrix-assisted laser desorption ionization (MALDI), paper spray ionization, plasma and glow discharge, plasma-desorption ionization (PD), resonance ionization (RIMS), secondary ionization (SIMS), spark source, or thermal ionization (TIMS). Methods of mass spectrometry are known in the art and described in, for example, U.S. Patent No. 8,895,918; U.S. Patent No. 9,546,979; U.S. Patent No. 9,761,426; Hoffman and Stroobant, Mass Spectrometry: Principles and Applications (2nd ed.). John Wiley and Sons (2001), ISBN 0-471-48566-7; Dass, Principles and practice of biological mass spectrometry, New York: John Wiley (2001) ISBN 0-471-33053-1; and Lee, ed., Mass

Spectrometry Handbook, John Wiley and Sons, (2012) ISBN: 978-0-470-53673-5, the contents of each of which are incorporated herein by reference.

In certain embodiments, a sample can be directly ionized without the need for use of a separation system. In other embodiments, mass spectrometry is performed in conjunction with a method for resolving and identifying ionic species. Suitable methods include chromatography, capillary electrophoresis-mass spectrometry, and ion mobility. Chromatographic methods include gas chromatography, liquid chromatography (LC), high-pressure liquid chromatography (HPLC), and reversed -phase liquid chromatography (RPLC). In a preferred embodiment, liquid chromatography-mass spectrometry (LC-MS) is used. Methods of coupling chromatography and mass spectrometry are known in the art and described in, for example, Holcapek and Bry dwell, eds. Handbook of Advanced Chromatography/Mass Spectrometry Techniques, Academic Press and AOCS Press (2017), ISBN 9780128117323; Pitt, Principles and Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry, The Clinical Biochemist

Reviews. 30(1): 19-34 (2017) ISSN 0159-8090; Niessen, Liquid Chromatography-Mass

Spectrometry, Third Edition. Boca Raton: CRC Taylor & Francis pp. 50-90. (2006) ISBN 9780824740825; Ohnesorge et al., Quantitation in capillary electrophoresis-mass spectrometry, Electrophoresis. 26 (21): 3973-87 (2005) doi:10.1002/elps.200500398; Kolch et al., Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery, Mass Spectrom Rev. 24 (6): 959-77. (2005) doi:10.1002/mas.20051; Kanu et al., Ion mobility-mass spectrometry, Journal of Mass Spectrometry, 43 (1): 1-22 (2008)

doi:10.1002/jms.l383, the contents of which are incorporated herein by reference.

A sample may be obtained from any organ or tissue in the individual to be tested, provided that the sample is obtained in a liquid form or can be pre-treated to take a liquid form. For example and without limitation, the sample may be a blood sample, a urine sample, a serum sample, a semen sample, a sputum sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a plasma sample, a pus sample, an amniotic fluid sample, a bodily fluid sample, a stool sample, a biopsy sample, a needle aspiration biopsy sample, a swab sample, a mouthwash sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a synovial fluid sample, a phlegm sample, a saliva sample, a sweat sample, or a combination of such samples. The sample may also be a solid or semi-solid sample, such as a tissue sample, feces sample, or stool sample, that has been treated to take a liquid form by, for example, homogenization, sonication, pipette trituration, cell lysis etc. For the methods described herein, it is preferred that a sample is from plasma, serum, whole blood, or sputum.

The sample may be kept in a temperature-controlled environment to preserve the stability of the metabolite. For example, DHO is more stable at lower temperatures, and the increased stability facilitates analysis of this metabolite from samples. Thus, samples may be stored at, or 4° C, -20° C, or -80° C.

In some embodiments, a sample is treated to remove cells or other biological

particulates. Methods for removing cells from a blood or other sample are well known in the art and may include e.g., centrifugation, sedimentation, ultrafiltration, immune selection, etc.

The subject may be an animal (such as a mammal, such as a human). The subject may be a pediatric, a newborn, a neonate, an infant, a child, an adolescent, a pre-teen, a teenager, an adult, or an elderly patient. The subject may be in critical care, intensive care, neonatal intensive care, pediatric intensive care, coronary care, cardiothoracic care, surgical intensive care, medical intensive care, long-term intensive care, an operating room, an ambulance, a field hospital, or an out-of-hospital field setting.

The sample may be obtained from an individual before or after administration to the subject of a DHODH inhibitor. For example, the sample may be obtained 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more before administration of a DHODH inhibitor, or it may be obtained 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more after administration of a DHODH inhibitor.

Providing DHODH inhibitors and other therapeutic agents

Methods of the invention may include providing a DHODH inhibitor and/or agent that inhibits immunosuppression to subject according to a dosing regimen or dosage determined as described above. Providing the DHODH inhibitor or another therapeutic agent to the subject may include administering it to the subject. A dose may be administered as a single unit or in multiple units. The DHODH inhibitor or other therapeutic agent may be administered by any suitable means. For example and without limitation, the DHODH inhibitor or other therapeutic agent may be administered orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, intravenously, subcutaneously, nasally, pulmonarily, or with or on an implantable medical device (e.g., stent or drug-eluting stent or balloon

equivalents).

In some embodiments, the methods include assessing the level of a metabolite, such as DHO, in a sample from a subject, and determining whether that level is within a threshold range (e.g., above a minimal threshold and/or below a potential toxicity threshold) that warrants dosing, and/or that warrants dosing at a particular level or in a particular amount.

The methods may include administering at least one dose of the DHODH inhibitor to a subject whose plasma DHO level has been determined and is below a pre-determined threshold (e.g., a pre-determined potential toxicity threshold and/or a pre-determined potential efficacy threshold). In some embodiments, the predetermined threshold reflects percent inhibition of DHODH in the subject relative to a baseline determined for the subject. In some embodiments, the baseline is determined by an assay. For example, in some embodiments, in order to maintain inhibition of DHODH at an effective threshold, multiple doses of the DHODH inhibitor may be administered. In some embodiments, dosing of the DHODH inhibitor can occur at different times and in different amounts. The present disclosure encompasses those methods that can maintain inhibition of DHODH at a consistent level at or above the efficacy threshold throughout the course of treatment. In some embodiments, the amount of inhibition of DHODH is measured by the amount of DHO in the plasma of a subject.

In some embodiments, more than one dose of the DHODH inhibitor is administered to the subject. In some embodiments, the method further comprises a step of re-determining the subject’s plasma DHO level after administration of the at least one dose. In some embodiments, the subject’s plasma DHO level is re-determined after each dose. In some embodiments, the method further comprises administering at least one further dose of the DHODH inhibitor after the subject’s plasma DHO level has been determined again (e.g., after administering a first or previous dose), and is below the pre-determined threshold. If the subject’s plasma DHO level is determined to be above a pre-determined threshold, dosing can be discontinued. In some embodiments, therefore, no further dose of the DHODH inhibitor is administered until the subject’s plasma DHO level has been determined to again be below a pre-determined threshold.

The methods may include administering a DHODH inhibitor to a subject at a dosage level at or near a cell-lethal level. Such dosage can be supplemented with a later dose at a reduced level, or by discontinuing of dosing. For example, in some embodiments, the present disclosure provides a method of administering a dihydroorotate dehydrogenase inhibitor to a subject in need thereof, the method comprising: administering a plurality of doses of a DHODH inhibitor, according to a regimen characterized by at least first and second phases, wherein the first phase involves administration of at least one bolus dose of a DHODH inhibitor at a cell- lethal level; and the second phase involves either: administration of at least one dose that is lower than the bolus dose; or absence of administration of a DHODH inhibitor.

In some embodiments, a DHODH inhibitor is not administered during a second phase. In some embodiments, a second phase involves administration of uridine rescue therapy. In some embodiments, a bolus dose is or comprises a cell lethal dose. In some embodiments, a cell lethal dose is an amount of a DHODH inhibitor that is sufficient to cause apoptosis in normal (e.g., non-cancerous) cells in addition to target cells (e.g., cancer cells). In some embodiments, the first phase and the second phase each comprise administering a DHODH inhibitor. In some embodiments, the first phase and the second phase are at different times. In some embodiments, the first phase and the second phase are on different days. In some embodiments, the first phase lasts for a period of time that is less than four days. In some embodiments, the first phase comprises administering a DHODH inhibitor, followed by a period of time in which no DHODH inhibitor is administered. In some embodiments, the period of time in which no DHODH inhibitor is administered is 3 to 7 days after the dose during the first phase. In some embodiments, the first phase comprises administering more than one dose.

In some embodiments, a DHODH inhibitor is administered during a second phase. In some embodiments, a DHODH inhibitor is administered sub-cell-lethal levels during the second phase. In some embodiments, the first phase is repeated after the second phase. In some embodiments, both the first and second phases are repeated.

In some embodiments, the present disclosure provides a method of administering a DHODH inhibitor to a subject in need thereof, according to a multi-phase protocol comprising: a first phase in which at least one dose of the DHODH inhibitor is administered to the subject; and a second phase in which at least one dose of the DHODH inhibitor is administered to the subject, wherein one or more doses administered in the second phase differs in amount and/or timing relative to other doses in its phase as compared with the dose(s) administered in the first phase.

In some embodiments, a DHO level is determined in a sample from the subject between the first and second phases. In some embodiments, the sample is a plasma sample. In some embodiments, the timing or amount of at least one dose administered after the DHO level is determined or differs from that of at least one dose administered before the DHO level was determined.

In some embodiments, the amount of DHODH inhibitor that is administered to the patient is adjusted in view of the DHO level in the subject’s plasma. For example, in some

embodiments, a first dose is administered in the first phase. In some embodiments, DHO level is determined at a period of time after administration of the first dose.

In some embodiments, if the DHO level is below a pre-determined level, the amount of DHODH inhibitor administered in a second or subsequent dose is increased and/or the interval between doses is reduced. For example, in some such embodiments, the amount of DHODH inhibitor administered may be increased, for example, by 100 mg/m . In some embodiments, the amount of DHODH inhibitor administered in a second or subsequent dose is increased by 150 mg/m . In some embodiments, the amount of DHODH inhibitor administered in a second or subsequent dose is increased by 200 mg/m . In some embodiments, the amount of DHODH inhibitor administered may be increased by an adjustment amount determined based on change in DHO levels observed between prior doses of different amounts administered to the subject.

In some embodiments, if the DHO level is above a pre-determined level, the amount of DHODH inhibitor administered in a second or subsequent dose is the same as the amount administered in the first or previous dose and/or the interval between doses is the same.

In some embodiments, if the DHO level is above a pre-determined level, the amount of DHODH inhibitor in a second or subsequent dose is decreased and/or the interval between doses is increased. For example, in some such embodiments, the amount of DHODH inhibitor administered may be decreased, for example, by 50 mg/m . In some embodiments, if the DHO level is above a pre-determined level, the amount of DHODH inhibitor in a second or subsequent dose is decreased by 75 mg/m . In some embodiments, if the DHO level is above a pre determined level, the amount of DHODH inhibitor in a second or subsequent dose is decreased by 100 mg/m . In some embodiments, the amount of DHODH inhibitor administered may be decreased by an adjustment amount determined based on change in DHO levels observed between prior doses of different amounts administered to the subject.

In some embodiments, the present disclosure provides a method of administering a later dose of a DHODH inhibitor to a patient who has previously received an earlier dose of the DHODH inhibitor, wherein the patient has had a level of DHO assessed subsequent to administration of the earlier dose, and wherein the later dose is different than the earlier dose.

The later dose may be different from the earlier dose in amount of DHODH inhibitor included in the dose, time interval relative to an immediately prior or immediately subsequent dose, or combinations thereof. The amount of DHODH inhibitor in the later dose may be less than that in the earlier dose.

The method may include administering multiple dose of the DHODH inhibitor, separated from one another by a time period that is longer than 2 days and shorter than 8 days for example, the time period may be about 3 days.

In some embodiments, the DHO level is determined in a sample from the subject before each dose is administered, and dosing is delayed or skipped if the determined DHO level is above a pre-determined threshold. For example, the DHO level may be determined about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours after administration of a DHODH inhibitor

The method may include administering the DHODH inhibitor according to a regimen approved in a trial in which a level of DHO was measured in patients between doses of the DHODH inhibitor. The regimen may include multiple doses whose amount and timing were determined in the trial to maintain the DHO level within a range determined to indicate a degree of DHODH inhibition below a toxic threshold and above a minimum threshold. The regimen may include determining the DHO level in the subject after administration of one or more doses of the DHODH inhibitor.

In some embodiments, the regimen includes a dosing cycle in which an established pattern of doses is administered over a first period of time. In some embodiments, the regimen comprises a plurality of the dosing cycles. In some embodiments, the regimen includes a rest period during which the DHODH inhibitor is not administered between the cycles.

Incorporation by Reference

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

Equivalents

Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Claims What is claimed is:

1. A method of promoting differentiation of myeloid-derived suppressor cells (MDSC) in a subject, the method comprising providing to a subject an inhibitor of dihydroorotate

dehydrogenase (DHODH) in a therapeutically effective amount to promote differentiation of MDSC in the subject.

2. The method of claim 1, wherein the DHODH inhibitor is selected from the group consisting of brequinar, leflunomide, teriflunomide, and a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation of any of the aforementioned compounds.

3. The method of claim 2, wherein the DHODH inhibitor is brequinar or a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation thereof.

4. The method of claim 1, wherein the subject has cancer.

5. The method of claim 4, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer (e.g., squamous cell carcinoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, and B cell lymphoma), melanoma, Merkel cell carcinoma, myeloma (e.g., multiple myeloma), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

6. The method of claim 1, further comprising providing to the subject an agent that inhibits immunosuppression in the subject.

7. The method of claim 6, wherein the agent inhibits immunosuppression mediated by MDSC.

8. The method of claim 7, wherein the agent is selected from the group consisting of all- trans retinoic acid, axitinib, CFS-1R inhibitor, entinostat, gemcitabine, and phenformin.

9. The method of claim 8, wherein the agent is all-trans retinoic acid.

10. The method of claim 6, wherein the agent that inhibits immunosuppression is an immune checkpoint inhibitor.

11. The method of claim 10, wherein the immune checkpoint inhibitor is selected from the group consisting of a CTLA-4 inhibitor, PD-1 inhibitor, and a PD-L1 inhibitor.

12. A method of treating cancer in a subject, the method comprising providing to a subject having cancer an inhibitor of dihydroorotate dehydrogenase (DHODH) in a therapeutically effective amount to promote differentiation of myeloid-derived suppressor cells (MDSC) in the subject.

13. The method of claim 12, wherein the DHODH inhibitor is selected from the group consisting of brequinar, leflunomide, teriflunomide, and a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation of any of the aforementioned compounds.

14. The method of claim 13, wherein the DHODH inhibitor is brequinar or a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation thereof.

15. The method of claim 12, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer (e.g., squamous cell carcinoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, and B cell lymphoma), melanoma, Merkel cell carcinoma, myeloma (e.g., multiple myeloma), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

16. The method of claim 12, further comprising providing to the subject an agent that inhibits immunosuppression in the subject.

17. The method of claim 16, wherein the agent inhibits immunosuppression mediated by MDSC.

18. The method of claim 17, wherein the agent is selected from the group consisting of all- trans retinoic acid, axitinib, CFS-1R inhibitor, entinostat, gemcitabine, and phenformin.

19. The method of claim 18, wherein the agent is all-trans retinoic acid.

20. The method of claim 16, wherein the agent that inhibits immunosuppression is an immune checkpoint inhibitor.

21. The method of claim 20, wherein the immune checkpoint inhibitor is selected from the group consisting of a CTLA-4 inhibitor, PD-1 inhibitor, and a PD-L1 inhibitor.

22. A method of treating cancer in a subject, the method comprising providing to a subject having cancer:

an inhibitor of dihydroorotate dehydrogenase (DHODH); and

an agent that inhibits immunosuppression in the subject.

23. The method of claim 22, wherein the DHODH inhibitor is selected from the group consisting of brequinar, leflunomide, teriflunomide, and a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation of any of the aforementioned compounds.

24. The method of claim 23, wherein the DHODH inhibitor is brequinar or a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation thereof.

25. The method of claim 22, wherein the agent inhibits immunosuppression mediated by myeloid-derived suppressor cells (MDSC).

26. The method of claim 25, wherein the agent is selected from the group consisting of all- trans retinoic acid, axitinib, CFS-1R inhibitor, entinostat, gemcitabine, and phenformin.

27. The method of claim 26, wherein the agent is all-trans retinoic acid.

28. The method of claim 22, wherein the agent that inhibits immunosuppression is an immune checkpoint inhibitor.

29. The method of claim 28, wherein the immune checkpoint inhibitor is selected from the group consisting of a CTLA-4 inhibitor, PD-1 inhibitor, and a PD-L1 inhibitor.

30. The method of claim 22, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer (e.g., squamous cell carcinoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, and B cell lymphoma), melanoma, Merkel cell carcinoma, myeloma (e.g., multiple myeloma), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.

31. A method of amplifying an effect of an agent that inhibits immunosuppression in a subject, the method comprising providing to a subject that has been given an agent that inhibits immunosuppression in the subject an inhibitor of dihydroorotate dehydrogenase (DHODH).

32. The method of claim 31, wherein the DHODH inhibitor is selected from the group consisting of brequinar, leflunomide, teriflunomide, and a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation of any of the aforementioned compounds.

33. The method of claim 32, wherein the DHODH inhibitor is brequinar or a salt, analog, derivative, prodrug, sustained release formulation, or micellar formulation thereof.

34. The method of claim 31, wherein the agent inhibits immunosuppression mediated by myeloid-derived suppressor cells (MDSC).

35. The method of claim 34, wherein the agent is selected from the group consisting of all- trans retinoic acid, axitinib, CFS-1R inhibitor, entinostat, gemcitabine, and phenformin.

36. The method of claim 35, wherein the agent is all-trans retinoic acid.

37. The method of claim 31, wherein the agent that inhibits immunosuppression is an immune checkpoint inhibitor.

38. The method of claim 37, wherein the immune checkpoint inhibitor is selected from the group consisting of a CTLA-4 inhibitor, PD-1 inhibitor, and a PD-L1 inhibitor.

39. The method of claim 31, wherein the effect is MDSC differentiation.

40. The method of claim 31, wherein the subject has cancer.

41. The method of claim 40, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, head and neck cancer (e.g., squamous cell carcinoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, and B cell lymphoma), melanoma, Merkel cell carcinoma, myeloma (e.g., multiple myeloma), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, and uterine cancer.