Methods and compositions for initiating, regulating, and modulating weight loss, and their therapeutic applications

Abstract

Methods, systems, compounds, and compositions are provided for modulating or regulating body weight in a subject and / or for inducing weight loss and for other diseases and conditions. Various methods of administration are described and provided for modulating or regulating or inducing weight loss and for other diseases and conditions. In addition, methods for weight loss and for the treatment of obesity are provided, for example, by modulating one or more enzymes in the ferrotosis pathway.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 350,218, filed on June 8, 2022, which is hereby incorporated by reference in its entirety.

Summary of the Invention

Means for Solving the Problems

[0002] Overview Methods and compositions for use in inducing weight loss, fat loss, or both in a subject, the method comprising administering to the subject a feroptosis inducer in an amount sufficient to induce weight loss, fat loss, or both, are disclosed herein. In some embodiments, it is a method for use in inducing weight loss. In some embodiments, it is a method for use in inducing fat loss. In some embodiments, it is a method for use in inducing weight loss and fat loss. In some embodiments, the method for use further comprises administering to the subject a priming agent prior to administering the feroptosis inducer to the subject. In some embodiments, the adipose tissue in the subject, after administration of the feroptosis inducer to the subject, has a decrease in weight, volume, or both compared to the weight, volume, or both of the adipose tissue in the subject prior to administration. In some embodiments, the adipose tissue in the subject, after administration of the feroptosis inducer to the subject, as determined by a pinch clamp test, dual energy x-ray absorptiometry (DEXA) scan, decrease in abdominal circumference measurement, computed axial tomography (CAT) scan, or any combination thereof, has a decrease in volume compared to the volume of the adipose tissue prior to administration. In some embodiments, the adipose tissue in the subject, after administration of the feroptosis inducer to the subject, as determined by a pinch clamp test, dual energy x-ray absorptiometry (DEXA) scan, decrease in abdominal circumference measurement, computed axial tomography (CAT) scan, or any combination thereof, has a decrease in volume compared to the volume of the adipose tissue prior to administration. In some embodiments, the adipose tissue in the subject, after administration of the feroptosis inducer to the subject, has a decrease in weight compared to the weight of the adipose tissue prior to administration. In some embodiments, the decrease in weight is determined by the subject having a lower total body weight compared to the total body weight of the subject prior to administration of the feroptosis inducer, after administration of the feroptosis inducer.In some embodiments, a method and composition for use, wherein weight loss is determined by comparing DEXA scan results of a subject obtained before and after administration of a ferroptosis inducer to the subject. In some embodiments, the method and composition for use further comprises that the cell size, cell volume, or both of a plurality of cells in the adipose tissue of the subject is reduced after administration of the ferroptosis inducer to the subject as compared to the cell size, cell volume, or both of a plurality of cells in the adipose tissue of the subject before administration. In some embodiments, the method and composition for use is such that the subject is a human. In some embodiments, the method and composition for use is such that the ferroptosis inducer is administered orally. In some embodiments, the administration is carried out for about 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 10 days, 2 weeks, 15 days, 20 days, 3 weeks, 25 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, for the lifespan, or as needed. In some embodiments, the method and composition for use is such that the ferroptosis inducer is administered in an amount in the range of about 0.1 ng to about 25,000 mg. In some embodiments, the method and composition for use is such that the ferroptosis inducer is administered in an amount of about 1 ng, 10 ng, 100 ng, 1 microgram, 10 micrograms, 100 micrograms, 1 mg, 10 mg, 100 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 15 g, 20 g, or 25 g. In some embodiments, the method and composition for use is such that the ferroptosis inducer is administered in an amount in the range of about 1 ng / kg to 1,000 mg / kg, where mg is the mg of the ferroptosis inducer and kg is the kg of the subject's body weight. In some embodiments, the method and composition for use is such that the ferroptosis inducer is administered orally. In some embodiments, the method and composition for use is such that the ferroptosis inducer is administered by one of the following routes: oral administration, subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, intracranial, intrathecal, transdermal, rectal, or directly into adipose tissue.In some embodiments, methods and compositions for use in treating at least one of obesity, metabolic syndrome, elevated blood glucose, diabetes, type 2 diabetes, type 3 diabetes, insulin resistance, hypertension, cardiovascular disease, coronary artery disease, cerebrovascular disease, stroke, rheumatic heart disease, arteriosclerosis, atherosclerotic arteriosclerosis, liver disease, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or any combination thereof. In some embodiments, methods and compositions for use, wherein the subject is a human male. In some embodiments, methods and compositions for use, wherein the subject is a human female. In some embodiments, methods and compositions for use, wherein the subject ranges from about 1 year to about 18 years of age. In some embodiments, methods and compositions for use, wherein the subject ranges from about 18 years to about 120 years of age. In some embodiments, methods and compositions for use, wherein the subject is a subject in need thereof. In some embodiments, methods and compositions for use, wherein the ferroptosis inducer is included in a pharmaceutical composition comprising a pharmaceutically acceptable dosage form, carrier, or diluent. In some embodiments, methods and compositions for use, wherein the pharmaceutical composition is in unit dosage form. In some embodiments, methods and compositions for use, further comprising administering a further therapeutic agent to the subject. In some embodiments, methods and compositions for use, wherein the further therapeutic agent is administered in combination with the ferroptosis inducer. In some embodiments, methods and compositions for use. In some embodiments, methods and compositions for use, wherein the further therapeutic agent is continuously administered with the ferroptosis inducer. In some embodiments, methods and compositions for use, wherein the ferroptosis inducer is administered directly to adipose tissue. In some embodiments, methods and compositions for use, wherein the administration is continuous.In some embodiments, the administration is for about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, and the method and composition for use. In some embodiments, the adipose tissue is heated to a temperature of about 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 degrees Celsius, and the method and composition for use. In some embodiments, the ferroptosis inducer is administered in the form of an aqueous solution, and the method and composition for use. In some embodiments, the concentration of the ferroptosis inducer in the aqueous solution is from about 0.1 nM to about 500 μM, and the method and composition for use. In some embodiments, the administration results in a reduction in the size or number of adipocytes in the subject, and the reduction is determined by measuring the size or number of adipocytes before and after administration of a therapeutically effective amount of a ferroptosis modulator, optionally using microscopic evaluation of a biopsy. In some embodiments, the method further comprises reducing the epididymal white adipose tissue of the subject, and the method and composition for use. In some embodiments, the subject is overweight or obese, and the method and composition for use. In some embodiments, the subject is obese and has a BMI of about 30.0 or higher, and the method and composition for use. In some embodiments, the subject is overweight and has a BMI of about 25.0 to about 30, and the method and composition for use. In some embodiments, the additional therapeutic agent has glucagon-like peptide-1 (GLP-1) receptor agonist activity, and the method and composition for use. In some embodiments, the additional therapeutic agent comprises semaglutide, dulaglutide, liraglutide, exenatide, tizepatide, or any combination thereof, and the method and composition for use.

[0003] Methods and compositions for use are also disclosed herein. In some embodiments, methods and compositions for use in inducing, regulating, or modulating weight loss, body composition, or fat loss or reduction in a subject are disclosed herein. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an autophagy modulator, a glutamate-cysteine ligase (GCL) modulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby inducing, regulating, or modulating weight loss, body composition, or fat loss or reduction in the subject. In some embodiments, the methods and compositions for use are such that the subject is a human. In some embodiments, the methods and compositions for use are such that the subject is male. In some embodiments, the autophagy modulator, the glutamate-cysteine ligase (GCL) modulator, the agent that binds to GCL, the agent that inhibits GCL, or any combination thereof are orally delivered as a solution having a concentration of at least about 5 mg of the agent per mL of solution. In some embodiments, the administration is discontinuous and the administration is performed daily for about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year. In some embodiments, the autophagy inducer is selected from the group consisting of (1S,3R)-RSL3, ML-162, ML-210, JKE-1674, JKE-1716, elastin, jacareic acid, trigonelline, glutamate, sulfasalazine, auranofin, bursatol, sorafenib, sorafenib-d3, sorafenib tosylate, trigonelline, FIN56, FINO2, CIL56, dihydroisotanshinone I, GPX4-IN-3, sulfoximine, BSO, and salts of any of these.In some embodiments, it is a method and composition for use, and in some embodiments, administration results in a reduction in the size of adipocytes in a subject, and the reduction is determined by measuring adipocyte size before and after administration of a ferroptosis inducer, optionally using microscopic evaluation of a biopsy. In some embodiments, administration results in a reduction in the number of adipocytes in a subject, and the reduction is determined by measuring the number of adipocytes before and after administration of a therapeutic amount of a ferroptosis modulator, optionally using microscopic evaluation of a biopsy. In some embodiments, it is a method and composition for use that includes reducing the epididymal white adipose tissue of a subject. In some embodiments, the subject is overweight or obese. In some embodiments, the subject is obese and has a BMI of about 30.0 or higher. In some embodiments, the subject is overweight and has a BMI of about 25.0 to about 30. In some embodiments, an additional agent or treatment is administered to the subject, either concomitantly or sequentially. In some embodiments, the additional agent or treatment is selected from the group consisting of GLP-1 agonists, gastric inhibitory polypeptide analogs, naltrexone-bupropion or salts thereof, orlistat or salts thereof, phentermine or salts thereof, phentermine-topiramate or salts thereof, selenium, exercise therapy, surgery, dietary modification, and any combination thereof. In some embodiments, the additional agent or treatment includes administering a GLP-1 agonist comprising dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, or salts of any of these. In some embodiments, the additional agent or treatment includes administering a gastric inhibitory polypeptide comprising tildepazide or derivatives thereof.In some embodiments, the method and composition for use comprises an additional agent or treatment, including a surgical procedure comprising a gastric sleeve, gastric bypass, adjustable gastric band, a ball placed in the stomach, or any combination thereof. In some embodiments, the method and composition for use is such that the subject is not diagnosed with cancer. In some embodiments, it is a method and composition for use, and in some embodiments, it is a method and composition for use where the subject is a subject in need thereof. In some embodiments, the method and composition for use is such that the subject has type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome. In some embodiments, the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition is in unit dosage form.

[0004] Methods and compositions for use are also described herein, wherein the composition comprises: i) a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, or an agent that inhibits GCL; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof. In some embodiments, the composition comprises: i) a glutamate-cysteine ligase (GCL) regulator; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof. In some embodiments, the methods and compositions for use are pharmaceutical compositions. In some embodiments, the methods and compositions for use are such that the composition is in unit dosage form. In some embodiments, the methods and compositions are for use in the treatment of a disease or condition. In some embodiments, the methods and compositions are for use in the treatment of a kidney disease or condition. In some embodiments, the methods and compositions are for use in the treatment of symptoms associated with a kidney disease or condition. In some embodiments, the disease or condition is weight loss, weight maintenance, a disease or condition associated with iron metabolism, or a disease or condition associated with high, low, or abnormal cholesterol.

[0005] Methods and compositions for use in modulating iron metabolism in multiple cells in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, a glutamate-cysteine ligase (GCL) modulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby modulating iron metabolism in the subject and thereby inducing ferroptosis in the subject, are also described herein. In some embodiments, the methods and compositions for use are such that immediately following modulation, the majority of the cells of the multiple cells remain viable.

[0006] Methods and compositions for use in modulating iron metabolism in multiple cells in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, thereby modulating iron metabolism in the subject, are also described herein. In some embodiments, the methods and compositions for use are such that immediately following modulation, the majority of the cells of the multiple cells remain viable.

[0007] Methods and compositions for use in treating, inducing, regulating, or modulating iron metabolism or a disease or condition associated with iron metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis modulator, a glutamate-cysteine ligase (GCL) modulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, or modulating iron metabolism or a disease or condition associated with iron metabolism in the subject, are also described herein. In some embodiments, the methods and compositions for use are such that the subject is a human. In some embodiments, the methods and compositions for use are such that the subject is a subject in need thereof. In some embodiments, the methods and compositions for use are such that a therapeutically effective amount of a ferroptosis modulator is added.

[0008] Methods and compositions for use in modulating cholesterol metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of an autophagy regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby modulating cholesterol metabolism in the subject. Methods and compositions are also described herein. In some embodiments, the subject is a human. In some embodiments, the subject is a subject in need thereof. In some embodiments, an autophagy inducer is administered.

[0009] Methods and compositions for use in modulating cholesterol metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of an autophagy inducer, thereby modulating cholesterol metabolism in the subject. Methods and compositions are also described herein. In some embodiments, the subject is a human. In some embodiments, the subject is a subject in need thereof. In some embodiments, an autophagy inducer is administered.

[0010] Methods and compositions for use in treating, inducing, modulating, or decreasing cholesterol metabolism in a subject or a disease associated with cholesterol metabolism, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, modulating, or decreasing cholesterol metabolism or a disease or condition associated with cholesterol metabolism in the subject. Methods and compositions are also described herein. In some embodiments, the subject is a human. In some embodiments, the subject is a subject in need thereof. In some embodiments, the ferroptosis regulator is administered.

[0011] Methods and compositions for use in treating, inducing, modulating, decreasing, or increasing reactive oxygen species or a disease or condition associated with reactive oxygen species in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, modulating, decreasing, or increasing reactive oxygen species or a disease or condition associated with reactive oxygen species in the subject. Methods and compositions are also described herein. In some embodiments, the subject is a human. In some embodiments, the subject is a subject in need thereof. In some embodiments, the ferroptosis regulator is administered.

[0012] Methods and compositions for use in increasing reactive oxygen species in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby increasing reactive oxygen species in the subject, are also described herein. In some embodiments, the subject is a human. In some embodiments, the subject is a subject in need thereof. In some embodiments, the ferroptosis regulator is administered.

[0013] Methods and compositions for use in promoting at least one of treating, surviving from, modulating, or reducing acute stress, chronic stress response, immune hypersensitivity, cachexia, kidney disease, neurodegeneration, cardiotoxicity, or cardiotoxicity caused at least in part by chemotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, reducing, or increasing acute stress, chronic stress response, immune hypersensitivity, cachexia, kidney disease, neurodegeneration, cardiotoxicity, or cardiotoxicity caused at least in part by chemotherapy in the subject, are also described herein. In some embodiments, the subject is a human. In some embodiments, the subject is a subject in need thereof. In some embodiments, the ferroptosis regulator is administered.

[0014] Methods and compositions for use in promoting at least one of treating kidney disease in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis modulator, a glutamate-cysteine ligase (GCL) modulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating kidney disease in the subject, the methods and compositions also being described herein. In some embodiments, the subject is a human, for use. In some embodiments, the subject is a subject in need thereof, for use. In some embodiments, the ferroptosis modulator is administered, for use.

[0015] Methods and compositions for use in promoting at least one of treating kidney disease in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, thereby treating the kidney disease in the subject, methods and compositions are also described herein. In some embodiments, the subject is a human, for use methods and compositions. In some embodiments, the subject is a subject in need thereof, for use methods and compositions. In some embodiments, the ferroptosis inducer is administered, for use methods and compositions. In some embodiments, the method comprises reducing the subject's epididymal white adipose tissue, for use methods and compositions. In some embodiments, the subject is overweight or obese, for use methods and compositions. In some embodiments, the subject is obese and has a BMI of 30.0 or higher, for use methods and compositions. In some embodiments, the subject is overweight and has a BMI of 25.0 - <30, for use methods and compositions. In some embodiments, an additional agent or treatment is administered to the subject concomitantly or continuously, for use methods and compositions. In some embodiments, the additional agent or treatment is selected from the group consisting of GLP-1 receptor agonists, gastric inhibitory polypeptide analogs, naltrexone-bupropion or salts thereof, orlistat or salts thereof, phentermine or salts thereof, phentermine-topiramate or salts thereof, selenium, exercise therapy, surgery, dietary modification, and any combination thereof, for use methods and compositions. In some embodiments, the additional agent or treatment comprises administering a GLP-1 receptor agonist comprising dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, or salts of any of these, for use methods and compositions. In some embodiments, the additional agent or treatment comprises administering a gastric inhibitory polypeptide comprising tildepagliflozin or derivatives thereof, for use methods and compositions.In some embodiments, the method and composition for use include an additional agent or treatment, including a surgical procedure comprising a gastric sleeve, gastric bypass, adjustable gastric band, a ball placed in the stomach, or any combination thereof. In some embodiments, the method and composition for use are such that the subject has not been diagnosed with cancer. In some embodiments, the method and composition for use are such that the subject is a subject in need thereof. In some embodiments, the method and composition for use are such that the subject has type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome. In some embodiments, the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition is in unit dosage form. In some embodiments, the agent is a compound or a salt thereof in Table 1. In some embodiments, the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the pharmaceutical composition is in unit dosage form.

[0016] Also described herein are methods and compositions for use comprising a composition including: i) a ferroptosis-modulating agent, a glutamate-cysteine ligase (GCL)-modulating agent, an agent that binds to GCL, an agent that inhibits GCL; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof. In some embodiments, the method and composition for use comprises administering an additional agent or treatment, a GLP-1 receptor agonist comprising dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, or a salt of any of these. In some embodiments, the method and composition for use comprises administering an additional agent or treatment, a gastric inhibitory polypeptide comprising tildepaglutide or a derivative thereof. In some embodiments, the method and composition for use comprises administering an additional agent or treatment, a surgical procedure comprising a gastric sleeve, gastric bypass, adjustable gastric band, a ball placed in the stomach, or any combination thereof. In some embodiments, the method and composition for use is such that the subject is not diagnosed with cancer. In some embodiments, the method and composition for use is such that the subject is a subject in need thereof. In some embodiments, the method and composition for use is such that the subject has type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome. In some embodiments, the method and composition for use is such that the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the method and composition for use is such that the pharmaceutical composition is in unit dosage form. In some embodiments, the method and composition for use is such that the agent is a compound or a salt thereof in Table 1. In some embodiments, the method and composition for use is such that the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the method and composition for use is such that the pharmaceutical composition is in unit dosage form.

[0017] (i) A ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, or an agent that inhibits GCL, and (ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof. Methods and compositions comprising such a composition are also described herein. In some embodiments, the methods and compositions are for use as a pharmaceutical composition. In some embodiments, the methods and compositions are for use in unit dosage form. In some embodiments, the methods and compositions are for use in the treatment of a disease or condition. In some embodiments, the methods and compositions are for use in the treatment of a kidney disease or condition. In some embodiments, the methods and compositions are for use in the treatment of symptoms associated with a kidney disease or condition. In some embodiments, the disease or condition is weight loss, weight maintenance, a disease or condition associated with iron metabolism, or a disease or condition associated with high, low, or abnormal cholesterol.

[0018] Methods for inducing, regulating, or modulating weight loss in a subject are also disclosed herein. In some embodiments, the method comprises administering to the subject, such as a mammal, such as a mouse, such as a male, a therapeutically effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, an agent that modulates an enzyme in the ferroptosis pathway, or any combination thereof, thereby inducing, modulating, or regulating weight loss.

[0019] Methods for inducing, regulating, or modulating weight loss in a subject are disclosed herein. In some embodiments, the method comprises administering to the subject, e.g., a mammal, e.g., a human, e.g., a male, a therapeutically effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, an agent that modulates an enzyme in the ferroptosis pathway, or any combination thereof, thereby inducing, modulating, or regulating weight loss.

[0020] In some embodiments, the agent can be delivered orally, e.g., as a pill or capsule or liquid, or intravenously. When delivered as a liquid, the liquid can have a concentration of at least about 5 mg of the agent per mL of solution.

[0021] In some embodiments, the administration can be discontinuous. The administration can be performed daily for about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year. In some embodiments, the subject can be a mammal, e.g., a mouse or a human, and can be male.

[0022] A method for treating, inducing, regulating, or modulating iron metabolism or a disease or condition related to iron metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, or modulating iron metabolism or a disease or condition related to iron metabolism in the subject, is also disclosed herein.

[0023] Furthermore, provided herein is a method of treating, inducing, regulating, modulating, or reducing cholesterol metabolism or a disease or condition related to cholesterol metabolism in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, or reducing cholesterol metabolism or a disease or condition related to cholesterol metabolism in the subject.

[0024] In addition, provided herein is a method of treating, inducing, regulating, modulating, reducing, or increasing reactive oxygen species or a disease or condition related to reactive oxygen species in a subject, comprising administering to the subject a therapeutically effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, reducing, or increasing reactive oxygen species or a disease or condition related to reactive oxygen species in the subject.

[0025] Furthermore, a method for promoting survival from, modulating, or reducing, or treating a disease or condition in a subject, including acute stress, chronic stress, or stress response, apoptosis resistance, immune hypersensitivity, cachexia, kidney disease, neurodegeneration, cardiotoxicity, or cardiotoxicity at least partially caused by chemotherapy in the subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby promoting survival from, modulating, or reducing, or treating the disease or condition in the subject, is disclosed herein. In some embodiments, the kidney disease is one or more of the following disease situations or conditions: multicystic dysplastic kidney, Fabry disease, cystinosis, glomerulonephritis, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome (aHUS), nephrotic syndrome, kidney injury, polycystic kidney disease (PKD), renal artery stenosis, tubular acidosis, simple renal cyst, or one or more of a single kidney or a single-functional kidney. In some embodiments, the compositions disclosed herein are for use in the treatment of a kidney disease or condition. In some embodiments, the compositions disclosed herein are for use in the treatment of symptoms associated with a kidney disease or condition.

[0026] In some embodiments, the agent can be a compound or a salt thereof in Table 1.

[0027] In some embodiments, the agent can be selected from the group consisting of (1S,3R)-RSL3, ML-162, ML-210, JKE-1674, JKE-1716, elastin, jacareic acid, trigonelline, glutamate, sulfasalazine, auranofin, bursatol, sorafenib, sorafenib-d3, sorafenib tosylate, trigonelline, FIN56, FINO2, CIL56, dihydroisotanshinone I, GPX4-IN-3, sulfoximine, buthionine sulfoximine (BSO), or any salt or pharmaceutically acceptable salt thereof.

[0028] In some embodiments, administration can result in a reduction in the adipose cell size, cell number, or both, of the subject. In some embodiments, administration can result in a reduction in the adipose cell size of the subject. In some embodiments, administration can result in a reduction in the number of adipose cells in a subject.

[0029] In some embodiments, the reduction can be determined by measuring the adipose cell size before and after administration of a therapeutically effective amount of the ferroptosis modulator. The measurement can be performed, for example, by microscopy of a biopsy derived from the subject.

[0030] In some embodiments, the method can include reducing the epididymal white adipose tissue of the subject. In some embodiments, the subject can be overweight or obese. In some embodiments, the subject can be obese and have a body mass index (BMI) of 30.0 or higher. In some embodiments, the subject can be overweight and have a BMI of from 25.0 to less than 30.

[0031] In some embodiments, a second weight loss treatment may be administered concomitantly or continuously. In some embodiments, the second weight loss treatment may be selected from GLP-1 agonists, gastric inhibitory polypeptide analogs, naltrexone-bupropion or salts thereof, orlistat or salts thereof, phentermine or salts thereof, and phentermine-topiramate or salts thereof, selenium, exercise therapy, surgery, diet modification, and any combination thereof.

[0032] In some embodiments, the second treatment may include administering a GLP-1 agonist, including dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, analogs, derivatives, or salts of any of these.

[0033] In some embodiments, the second treatment may include administering a gastric inhibitory polypeptide, including tirlizepatide or derivatives thereof.

[0034] In some embodiments, the second treatment may include surgery, including gastric sleeve, gastric bypass, adjustable gastric band, a ball placed in the stomach, or any combination thereof.

[0035] In some embodiments, the subject is not diagnosed with cancer.

[0036] In some embodiments, the subject may be a subject in need thereof. In some embodiments, the subject in need thereof has, is suspected of having, or is diagnosed with type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome. In some embodiments, the subject is at risk of developing type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome.

[0037] In some embodiments, the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

[0038] In some embodiments, the pharmaceutical composition is in unit dosage form.

[0039] Also described herein are compositions comprising: (i) an agent, ferroptosis modulator or inducer, glutamate-cysteine ligase (GCL) modulator, agent that binds to GCL, or agent that inhibits GCL; and (ii) a GLP-1 agonist or its salt, gastric inhibitory polypeptide analog, naltrexone-bupropion or its salt, orlistat or its salt, phentermine or its salt, and phentermine-topiramate or its salt, or selenium or its salt.

[0040] In some embodiments, the composition can be a pharmaceutical composition. In some embodiments, the pharmaceutical composition is in unit dosage form.

[0041] In some embodiments, administration of the pharmaceutical composition can be at an effective amount or a therapeutically effective amount, orally or intravenously. In some embodiments, said administration can be once, twice, three times, or four times daily over the course of 1 day, 1 week, 1 month, 6 months, 1 year, or longer, or as needed.

[0042] The novel features of the invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that illustrates exemplary embodiments and in which the principles of the invention are utilized, as well as the appended drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0043]

Figure 1

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Figure 2

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Figure 3

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Figure 4A - B

Figure 4C - D

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Figure 6A - C

Figure 6D - E

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Figure 8

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Figure 10 - 2

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BRIEF DESCRIPTION OF THE DRAWINGS

[0057] DETAILED DESCRIPTION OF THE DISCLOSURE The following description and examples illustrate in detail embodiments of the invention. It should be understood that the invention is not limited to the specific embodiments described herein and can, therefore, vary. Those skilled in the art will recognize that numerous variations and modifications of the invention exist and that they are included within the scope of the invention.

[0058] DEFINITIONS Throughout this disclosure, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an absolute limitation on the scope of any embodiment. Thus, a range description should be considered to specifically disclose all possible sub-ranges as well as the individual numerical values within the range down to one tenth of the unit of the lower limit, unless the context clearly indicates otherwise. For example, a range description such as 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual values within the range, such as 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included within a smaller range and are included within this disclosure subject to any specifically excluded limits within the described range. When the described range includes one or both of the above limits, ranges excluding any or both of these included limits are also included within this disclosure, unless the context clearly indicates otherwise.

[0059] Unless specifically recited or apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance in the art, e.g., within 2 standard deviations of the average. "About" can be understood to be within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. Unless the context is otherwise clear, all numerical values provided herein are modified by the term "about". When a particular value is recited in the present application and claims, the term "about" is assumed to mean within the acceptable error range of that particular value and is meant in that context, unless otherwise recited.

[0060] Unless otherwise noted, the structures shown in this specification also include all isomers of that structure (e.g., enantiomers, diastereomers, geometric (or conformational) forms, e.g., the L and S notations for each chiral center, the R and S configurations for each chiral center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers). Thus, single stereochemical isomers of the compound, as well as mixtures of enantiomers, diastereomers, and geometric (or conformational) forms are within the scope of this disclosure.

[0061] Unless otherwise noted, compounds having one or more chiral centers referred to in this specification include their enantiopure, diastereomeric, and racemic mixtures.

[0062] As used herein, the term "adjacent" and its grammatical equivalents refer to being immediately next to the object being referenced. For example, in the context of cells or tissues, the term adjacent may mean that no other cells or tissues are present in between.

[0063] As used herein, the term "analog" and its grammatical equivalents refer to molecules that have similar but not identical structural characteristics. An analog of a drug or agent is a drug or agent that is related to a reference agent (e.g., the agents provided in Table 1) but may have a different chemical structure. Generally, an analog exhibits activity similar to the reference drug or agent, but the activity may be increased, decreased, or otherwise improved. Generally, an analog form of a compound or drug means that the skeletal core of the structure has been modified or changed compared to the reference drug.

[0064] As used herein, the term "cancer" and its grammatical equivalents refer to the uncontrolled growth, lack of differentiation, local tissue invasion, and metastasis that result from the loss of normal regulation that is characteristic of excessive cell growth. With respect to the methods provided herein, cancer can be any cancer including, but not limited to, any one of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal, anal canal, rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal cancer, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, colorectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and / or bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue, for example, of a malignant or benign type.

[0065] The terms "effective amount" or "therapeutically effective amount" and their grammatical equivalents refer to an amount sufficient to achieve or at least partially achieve the desired effect.

[0066] As used herein, the term "expression" and its grammatical equivalents refer to the biosynthesis of a gene product. For example, in the case of a structural gene, expression includes the transcription of the structural gene into mRNA and the translation of the mRNA into one or more polypeptides.

[0067] The term "ferroptosis" refers to a form of cell death that involves the generation of reactive oxygen species mediated by iron and is characterized in part by lipid peroxidation. The terms "ferroptosis-inducing agent", "ferroptosis activator", "ferroptosis inducer", or "ferroptosis-inducing compound" refer to agents that promote or activate ferroptosis in cells.

[0068] As used herein, the term "hyperproliferative cell" and its grammatical equivalents refer to cells characterized by unwanted cell growth, or abnormally rapid or persistent cell division, that is independent of or uncoordinated with that of surrounding normal tissue.

[0069] As used herein, the term "in vitro" and its grammatical equivalents refer to events that occur in an artificial environment, such as in a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0070] As used herein, the term "in vivo" and its grammatical equivalents refer to events that occur within a multicellular organism, such as a non-human animal.

[0071] As used herein, the term "iron-dependent cell death agent" and its grammatical equivalents refer to agents that induce, promote, or activate iron-mediated cell death. In some instances within the present disclosure, the term "iron-dependent cell death agent" is used interchangeably with ferroptosis inducer.

[0072] As used herein, the term "normal cell" and its grammatical equivalents refer to cells that undergo controlled cell division, cells that undergo controlled activation, or quiescent cells.

[0073] The terms used in this specification are for the purpose of describing particular embodiments only and are not intended to limit any embodiments. As used in this specification, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises" and / or "comprising", when used in this specification, indicate the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used in this specification, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0074] Overview Compositions and methods useful for treating a disease or condition, such as overweight or obesity, are provided herein. Compositions for use in any of the methods described herein are also provided herein. Also provided herein are treatment regimens for the treatment of various diseases or conditions, such as overweight or obesity. The treatment regimen may include administering a drug or a ferroptosis modulator, alone or in combination with a second treatment, each in an effective amount or a therapeutically effective amount, thereby treating the disease or condition. In instances where two or more drugs or active agents are administered, the administration may be continuous or combined, and either separately or in a single dosage form that may be a fixed-dose combination drug.

[0075] Ferroptosis Cell death is a cellular process involved in the development of proliferative diseases, such as cancer, cellular homeostasis, and prevention. Programmed cell death can take various forms, such as apoptosis, mitotic cell death, necrosis, senescence, and autophagy. Although each of these processes ultimately leads to cell death, the pathways and mechanisms are considered to be unique at both the molecular and cellular levels.

[0076] Ferroptosis is a regulated form of non-apoptotic oxidative cell death associated with the accumulation of lipid hydroperoxides and lipid peroxides in the cell plasma membrane. Cells undergoing ferroptosis do not exhibit the characteristics or functions of cells associated with apoptosis, which is the canonical form of cell death. Examples of apoptotic cell characteristics include, for example, mitochondrial cytochrome c release, caspase activation, and chromatin fragmentation. Ferroptosis is also characterized by an increase in intracellular reactive oxygen species (ROS) levels that can be prevented by iron chelation and genetic inhibition of intracellular iron uptake. The addition of iron can enhance ferroptosis signaling in cells, but not with other divalent transition metal ions.

[0077] Cellular components related to and regulating ferroptosis include, among others, the cysteine-glutamate antiporter (system Xc), glutathione peroxidase 4 (GPX4), p53, cargo receptor NCOA4, glutathione synthetase (GSH), and glutamate-cysteine ligase (GCL). Inactivation or inhibition of some of these molecules, such as system Xc, GPX4, or glutathione synthetase, results in iron-dependent cell death or ferroptosis.

[0078] Hyperproliferative cells in a drug-resistant state, such as drug-resistant cancer cells, have been found to exhibit dysregulation in the apoptotic cell pathway. Surprisingly, drug resistance to apoptotic agents by hyperproliferative cells can have an enhanced ability to undergo ferroptosis. Apoptosis-resistant cells can be killed through ferroptosis induction due to their "flammable" ferroptosis-sensitive state.

[0079] In some embodiments, interference with ferroptosis regulation can be used to treat diseases and conditions associated with reactive oxygen species, iron metabolism, lipid metabolism, glutathione biosynthesis, cholesterol metabolism, selenium metabolism, or any combination thereof.

[0080] In some embodiments, interference with ferroptosis regulation can affect lipid droplet accumulation. Interference with ferroptosis regulation can reduce lipid accumulation and triglycerides. In some cases, interference with ferroptosis regulation can reduce obesity and liver fat. Interference with ferroptosis regulation can lead to the prevention of metabolic diseases. In some cases, selenium and / or interference with ferroptosis regulation can increase UCP1 expression. In some cases, selenium and / or interference with ferroptosis regulation can increase thermogenesis and energy consumption, thereby improving the prevention of metabolic diseases.

[0081] Method for characterizing ferroptosis-sensitive cells A method for identifying ferroptosis-sensitive cells in a subject and characterizing them is provided herein. In some embodiments, the characterization is performed prior to treating the subject with a ferroptosis inducer provided herein. Ferroptosis-sensitive cells can be identified by the following characteristics provided herein: (1) a selenium concentration higher than that in the corresponding normal cells, (2) an iron concentration higher than that in the corresponding normal cells, (3) a polyunsaturated fatty acid (PUFA) concentration higher than that in the corresponding normal cells, (4) a peroxidizability index (PI) higher than that in the corresponding normal tissue, and / or (5) among other morphological and histological features, by the expression of one or more markers indicative of a mesenchymal state. Methods for measuring the analyte concentrations of selenium, iron, and PUFA include, for example, mass spectrometry, chromatography, immunoassay, immunosorbent assay, absorbance and colorimetric analysis assays, and microwave plasma atomic emission mass spectrometry. Methods for measuring markers of the mesenchymal cell state include, for example, immunoassay, polymerase chain reaction (PCR) assay, and sequencing assay.

[0082] (1) Selenium (Se) concentration and selenoprotein Selenium (Se) is a micronutrient that promotes the synthesis of selenoproteins in cells. Dietary selenium is found in meats, nuts, grains, mushrooms, and vegetables. The selenium content in the human body is about 13 milligrams (mg) to 20 mg. Selenium is involved in the synthesis of selenoproteins and the cellular process of ferroptosis. Selenoproteins are rare proteins that contain selenocysteine (Sec) residues instead of cysteine. Non-limiting examples of selenoproteins include GPX1, GPX2, GPX3, GPX4, GPX6, TXNRD1, TXNRD2 (TXRD2), TXNRD3, DIO1, DIO2, DIO3, SEPHS2, SEPS1, SEPP1, SEP15, SEPN1 (SELENON), SEPX1, SEPW1 (SELENOW), SEPT1, SELH, SELI, SELK, SELM (SELENOM), SELO, and SELV. Selenoproteins exhibit biochemical activities such as redox, selenocysteine synthesis, and / or selenium transport. GPX4 is a phospholipid hydroperoxidase that catalyzes the reduction of hydrogen peroxide and organic peroxides, thereby protecting cells from membrane lipid peroxidation and oxidative stress. GPX4 is a regulator of the ferroptosis pathway, and inhibition of GPX4 induces ferroptotic cell death.

[0083] Methods for identifying ferroptosis-sensitive cells in mammalian tissues based on selenium concentration are provided herein. In some embodiments, the methods provided herein include measuring the concentration of selenium (Se) in one cell, multiple cells, or a mammalian tissue. In some embodiments, the Se concentration in one or multiple cells of a mammalian tissue is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher than the Se concentration in cells of healthy tissue. In some embodiments, the Se concentration in multiple cells of a mammalian tissue is 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% higher than the Se concentration in cells of healthy tissue. In some embodiments, the methods provided herein include administering to a mammal an effective amount of a ferroptosis inducer, wherein the multiple cells of the mammalian tissue have a higher Se concentration than the Se concentration of cells of normal or healthy tissue, and ferroptosis is induced in the multiple cells.

[0084] (2) Iron concentration Ferroptosis is an iron-dependent cellular process, and ferroptosis-sensitive cells have increased intracellular iron concentration compared to normal cells. Treatment of cells with deferoxamine (DFO), an iron chelator used to treat iron overload and a drug reported to block ferroptosis, can inhibit cell death. Alternatively, iron loading into cells by treatment with ferric ammonium citrate (FAC) is sufficient to induce ferroptosis in cells mimicking particle treatment and in amino acid-starved states. An increase in iron uptake in cells can cause depletion of glutathione, perhaps due to an increase in ROS production that leads to ferroptosis induction.

[0085] Methods are provided herein for identifying ferroptosis-sensitive cells in mammalian tissue by iron concentration. In some embodiments, the methods provided herein include measuring the concentration of iron or iron oxide in one cell, multiple cells, or mammalian tissue. In some embodiments, ferroptosis-sensitive cells have an increased intracellular iron concentration of at least about 7 parts per billion (ppb) or higher, about 8 ppb or higher, about 9 ppb or higher, about 10 ppb or higher, about 20 ppb or higher, about 30 ppb or higher, about 40 ppb or higher, about 50 ppb or higher, about 60 ppb or higher, about 70 ppb or higher, about 80 ppb or higher, about 90 ppb or higher, about 100 ppb or higher, about 110 ppb or higher, about 120 ppb or higher, about 130 ppb or higher, about 140 ppb or higher, about 150 ppb or higher, about 160 ppb or higher, up to 170 ppb. In some embodiments, ferroptosis-sensitive cells have an increased intracellular iron concentration of at least about 2 micromolar (μM) or higher, 2.5 μM or higher, 3.0 μM or higher, 4.0 μM or higher, 5.0 μM or higher, up to 10 μM higher than that of normal cells. In some embodiments, the iron concentration in one or more cells of a mammalian tissue is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher than the iron concentration in cells of healthy tissue. In some embodiments, the iron concentration in multiple cells of a mammalian tissue is 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% higher than the iron concentration in cells of healthy tissue.In some embodiments, the methods provided herein include administering to a mammal an effective amount of a ferroptosis inducer, wherein a plurality of cells of the mammalian tissue have an iron concentration higher than that of cells of normal or healthy tissue, and ferroptosis is induced in the plurality of cells. In some embodiments, the methods provided herein include administering to a mammal an effective amount of a ferroptosis inducer, wherein a plurality of cells of the mammalian tissue have an iron concentration higher than that of cells of normal or healthy tissue, thereby affecting iron metabolism and inducing ferroptosis in the plurality of cells.

[0086] (3) PUFA status Apoptosis-resistant cells gain an advantage by being in a ferroptosis-susceptible state with high polyunsaturated fatty acid (PUFA) levels. Apoptosis-resistant cells can be killed via ferroptosis induction due to their "flammable" high-PUFA state. The flammable state is defined by a high membrane PUFA prevalence (compared to MUFA, monosaturated fatty acid), which is prone to undergo uncontrolled lipid peroxidation, a radical chain reaction of polyunsaturated fatty acids that leads to ferroptotic cell death.

[0087] PUFAs are classified as omega-3 (n-3) and omega-6 (n-6) according to the position of the last double bond with reference to the methyl end of the molecule. Non-limiting examples of PUFAs include hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA, timnodonic acid), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA, clupanodonic acid), docosahexaenoic acid (DHA, cervonic acid), tetracosahexaenoic acid (nisinic acid), tetracosapentaenoic acid, linoleic acid (LA), gamma-linolenic acid (GLA), eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA), docosadienoic acid, adrenic acid (AdA), docosapentaenoic acid (osbond acid), tetracosatetraenoic acid, and tetracosapentaenoic acid. Humans can synthesize all fatty acids utilized by the body, except for linoleic acid (LA, C18:2n-6) and alpha-linolenic acid (ALA, C18:3n-3).

[0088] Methods are provided herein for identifying ferroptosis-sensitive cells in mammalian tissues by the concentration of polyunsaturated fatty acids (PUFAs). In some embodiments, the methods provided herein include administering to a mammal an effective amount of a ferroptosis inducer, wherein a plurality of cells of the mammalian tissue have a PUFA concentration that is higher than the polyunsaturated fatty acid (PUFA) concentration of cells of normal or healthy tissue, and ferroptosis is induced in the plurality of cells. In some embodiments, the PUFA concentration in a plurality of cells of the mammalian tissue is higher than the PUFA concentration in cells of healthy or non-malignant tissue of the mammal. In some embodiments, the PUFA concentration in a plurality of cells of the mammalian tissue is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher than the PUFA concentration in cells of healthy tissue. In some embodiments, the PUFA concentration in a plurality of cells of the mammalian tissue is 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% higher than the PUFA concentration in cells of healthy tissue. In some embodiments, the PUFA concentration in a plurality of cells of the mammalian tissue is higher than a predetermined PUFA concentration. In some embodiments, the predetermined PUFA concentration is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mole percent of total lipid. In some embodiments, the predetermined PUFA concentration is about 10 - 20, 20 - 30, 30 - 40, 40 - 50, 50 - 60, 60 - 70, 70 - 80, or 80 - 90 mole percent of total lipid. In some embodiments, the predetermined PUFA concentration is about 20 mole percent of total lipid.

[0089] (4) PI index The cell membrane composition must contain a threshold amount of polyunsaturated fatty acid acyl chains sufficient to assist in enzymatic and / or non-enzymatic lipid peroxidation. The peroxidizability of polyunsaturated fatty acids (PUFAs) depends linearly on the number of allylic double bonds present in the molecule. The sensitivity of the cell membrane to lipid peroxidation can be estimated using the peroxidizability index (PI), which is calculated as follows from the measured fatty acid composition (% w / w): PI = (dienoic acid % × 1) + (trienoic acid % × 2) + (tetraenoic acid % × 3) + (pentaenoic acid % × 4) + (hexaenoic acid % × 5). Alternatively, PI can be calculated as: PI = (monoenoic acid % × 0.025) + (dienoic acid % × 1) + (trienoic acid % × 2) + (tetraenoic acid % × 4) + (pentaenoic acid × 6) + (hexaenoic acid × 8). Lipidomic measurements of the cell membrane composition are used to determine the peroxidizability index. Cell lines with low PI values (less than 50) are less sensitive to ferroptosis induction (e.g., GPX4 inhibition, GSH depletion, addition of pro-oxidant compounds). Cells are more prone to ferroptosis as the membrane PI value increases.

[0090] Cells grown in vitro have a different fatty acid profile and lower PI levels than those of cells in vivo. Vertebrate cells cannot synthesize PUFAs de novo and rely on dietary sources for such molecules. Typical cell culture methods use media supplemented with serum (typically 10% v / v), which is the sole source of exogenous lipids and contains 1% of the PUFAs available to cells in the body. As a result, cells grown in culture have half the PUFA levels and twice the amount of monounsaturated fatty acids (MUFAs) of cells in vivo.

[0091] The ferroptosis susceptibility of cell lines can be modulated by including fatty acids in the culture medium. Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and deuterated PUFAs protect cells from undergoing ferroptosis, while on the other hand, the addition of PUFAs increases the cellular susceptibility to ferroptosis induction interference. By supplementing the cell culture medium with exogenous PUFAs, the in vivo PUFA concentration can be stimulated, and a membrane composition with a higher PI value can be induced. A modulatory profiling assay using fatty acid supplementation and a ferroptosis inducer enables the experimental determination of the specific membrane PUFA content and PI value sufficient for ferroptosis for a given cell line. For example, the peroxidation index (PI) of sarcoma and other cancer cells is higher than that of non-malignant tissues due to the preferential uptake of PUFAs. A number of sarcomas preferentially take up PUFAs, incorporate polyunsaturated fatty acid acyl chains into membrane lipids, and result in a higher membrane peroxidation index value (PI > 100) compared to non-malignant tissues (average PI = 91). The difference in membrane peroxidation provides a therapeutic window for ferroptosis induction that selectively targets sarcoma cells relative to non-malignant tissues. The more peroxidative membrane state is consistent with the observation that lipid peroxidation stress is at a higher level in primary bone and soft tissue sarcomas. The addition of exogenous PUFAs can increase oxidative stress in osteosarcoma cells and exhibit a selective cytotoxic effect.

[0092] Methods are provided herein for identifying ferroptosis-sensitive cells in mammalian tissue by a peroxidation index (PI). In some embodiments, the methods provided herein include administering to a mammal an effective amount of a ferroptosis inducer, and a plurality of cells of the mammalian tissue have a PI that is higher than the PI of cells of normal or healthy tissue, and ferroptosis is induced in the plurality of cells. In some embodiments, the PI in a plurality of cells of a mammalian tissue is higher than a predetermined PI. In some embodiments, the predetermined PI is about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150. In some embodiments, the predetermined PI is about 90. In some embodiments, the PI in a plurality of cells of a mammalian tissue is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% higher than the PI in cells of healthy or non-malignant tissue.

[0093] (5) Mesenchymal cell state Therapeutic resistant cells have three highly mesenchymal cells and a patient-derived signature. The first cell signature is the expression of mesenchymal cell markers. Ferroptosis-sensitive cells exhibit one or more markers of the mesenchymal cell state. Mesenchymal cell state markers that can be used to identify ferroptosis-sensitive cells include, but are not limited to, ZEB1, ACSL4, FADS2, PPARγ, Fsp1, SLC7A11, SLC3A2, and LPCAT3. The second cell signature of ferroptosis-sensitive cells is the reduced expression of endothelial cell markers compared to normal cells. Non-limiting examples of endothelial cell markers include vimentin, E-cadherin, and beta (β)-actin. The third cell signature of ferroptosis-sensitive cells is sensitivity to GPX4 knockdown that results in cell death. GPX4 dependency is more prominent in cancer cells with a treatment-resistant mesenchymal state compared to normal mesenchymal cell lines. Methods for reducing or silencing GPX4 expression can be achieved, for example, by CRISPR / Cas9, siRNA, or shRNA, among others.

[0094] Methods are provided herein for identifying ferroptosis-sensitive cells in mammalian tissues by the expression of one or more mesenchymal cell state markers. In some embodiments, the methods provided herein include administering to a mammal an effective amount of a ferroptosis inducer, wherein a plurality of cells of the mammalian tissue express one or more markers of the mesenchymal cell state and ferroptosis is induced in the plurality of cells. In some embodiments, the expression of mesenchymal cell markers in a plurality of cells of a mammalian tissue is about 1% - 10%, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% higher than the expression of mesenchymal cell markers in cells of healthy or non-malignant tissue.

[0095] (6) Additional morphological features of ferroptosis Cells undergoing ferroptosis are morphologically characterized by the presence of mitochondria that are smaller than normal with condensed mitochondrial membrane density, reduction or disappearance of mitochondrial cristae, and rupture of the outer mitochondrial membrane. Histological methods and immunoassays can be used to determine whether a tissue is cancerous, shows hyperplasia or fibrosis, and to identify ferroptosis-sensitive cells within mammalian tissues. The cell membrane of cells in a ferroptotic state lacks the rupture and bleb formation of the plasma membrane that are normally associated with apoptosis. The nuclear size of ferroptotic cells is normal and lacks chromatin condensation.

[0096] In some embodiments, the methods provided herein include obtaining a biological sample (e.g., a blood sample and a tissue biopsy) from a subject. In some embodiments, the methods provided herein further include fixing, processing, embedding, sectioning, and staining the biological sample for histological analysis. In some embodiments, the tissue includes histological abnormalities. In some embodiments, the histological abnormalities are determined by tissue biopsy before or during the targeted and continuous administration of a ferroptosis inducer to the tissue. In some embodiments, the histological abnormalities are hyperplasia, angiogenesis / neovascularization, or fibrosis. Hyperplasia is identified by an increase in the number of cells in the tissue compared to normal healthy tissue. Angiogenesis and neovascularization are identified in tissue samples by immunoassays for vascular markers such as vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2). Fibrosis is characterized by abnormal collagen deposition between cells, identified in tissue samples, for example, by Masson's trichrome, Sirius red, or collagen staining.

[0097] Cell Death and Ferroptosis Inducers A method of inducing ferroptosis in a tissue in a subject, comprising: (a) continuously administering a therapeutically effective amount of a ferroptosis inducer; (b) contacting the tissue in vivo with an effective amount of an iron-dependent cell death agent for a period of time; and / or (c) contacting a mammalian tissue with a priming agent and then contacting the mammalian tissue in vivo with an effective amount of a ferroptosis inducer for a period of time, is provided herein. Exemplary targets in the ferroptosis pathway are provided in Figure 1.

[0098] The methods provided herein include administering an agent that modulates cell death to a cell, tissue, or subject. In some embodiments, administering induces cell death. In some embodiments, administering inhibits cell death or rescues cells from cell death. In some embodiments, administering modulates ferroptosis. In some embodiments, administering induces ferroptosis in vivo. In some embodiments, administering inhibits ferroptosis in vivo. In some embodiments, the agent is a ferroptosis inducer. In some embodiments, the agent is an iron-dependent cell death inducer. Agents useful for inducing ferroptosis in vivo and treating a disease or disorder are discussed in further detail below.

[0099] (1) Ferroptosis regulators Agents that modulate ferroptosis in a subject are provided herein. In some embodiments, the agent is a small molecule, peptide, or nucleic acid. In some embodiments, the ferroptosis regulator is an inhibitor of glutathione peroxidase 4 (GPX4), glutathione synthetase, glutamate-cysteine ligase, phosphoserine-TRNA kinase (PSTK), eukaryotic elongation factor selenocysteine-TRNA specific (EEFSEC), selenophosphate synthetase 2 (SEPHS2), Sep(O-phosphoserine)TRNA:Sec (selenocysteine)TRNA synthetase (SEPSECS), or SECIS binding protein 2 (SECISBP2).

[0100] In some embodiments, the agent is an inhibitor of glutathione peroxidase 4 (GPX4). Glutathione peroxidase 4 (GPX4), also known as MCSP, SMDS, GPx-4, PHGPx, snGPx, GSHPx-4, snPHGPx, belongs to the glutathione peroxidase family, and its members catalyze the reduction of hydrogen peroxide, organic hydroperoxides, and lipid hydroperoxides, thereby protecting cells from oxidative damage. GPX4 activation directly reduces the level of phospholipid hydroperoxides in the cell membrane. Several isozymes of this gene family are present in vertebrates, and their intracellular locations and substrate specificities are diverse. GPX4 has a high preference for lipid hydroperoxides and protects cells against membrane lipid peroxidation and cell death. This isozyme is also a selenoprotein that contains the rare amino acid selenocysteine (Sec) in its active site. Representative human GPX4 cDNA and human GPX4 protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). Human glutathione peroxidase 4 peroxidase isoform B precursor (NM_001039847.3 and NP_001034936.1), isoform C (NM_001039848.4 and NP_001034937.1), isoform D (NM_001367832.1 and NP_001354761.1), isoform A precursor (NM_002085.5 and NP_002076.2).

[0101] Depletion of GPX4 induces lipid peroxidation-dependent cell death. Cancer cells in a drug-induced treatment-resistant state enhance their dependence on the lipid peroxidase activity of GPX4 to prevent undergoing ferroptotic cell death. Lipophilic antioxidants, such as ferrostatin, can rescue cells from ferroptosis induced by GPX4 inhibition. For example, mesenchymal GPX4 knockout cells can survive in the presence of ferrostatin, but when the supply of ferrostatin is stopped, these cells undergo ferroptosis. GPX4 inhibition can be rescued by blocking other components of the ferroptosis pathway, such as lipid ROS scavengers (ferrostatin, liproxstatin), lipoxygenase inhibitors, iron chelators, and caspase inhibitors, which cannot be rescued by apoptosis inhibitors. Therefore, GPX4 inhibitors may be useful for inducing ferroptotic cell death.

[0102] In some embodiments, the agent is an inhibitor of glutathione synthetase (GSS). Glutathione synthetase (GSS), also known as GSHS, HEL-S-64p, HEL-S-88n, is a homodimer that catalyzes the ATP-dependent conversion of gamma-L-glutamyl-L-cysteine, the second step of glutathione biosynthesis, to glutathione. Representative human GSS cDNA and human GSS protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). Human glutathione synthetase (NM_000178.4 and NP_000169.1, NM_001322494.1 and NP_001309423.1, NM_001322495.1 and NP_001309424.1).

[0103] In some embodiments, the agent is an inhibitor of glutamate-cysteine ligase (GCL). Glutamate-cysteine ligase (GCL), a central node of the ferroptosis pathway, has been overlooked as a target. Ablation of GCL activity induces ferroptosis in sensitive cells and kills only the most ferroptosis-sensitive cells. Representative human GCL cDNA and human GCL protein sequences are publicly available from the National Center for Biotechnology Information (NCBI). Human glutamate-cysteine ligase catalytic subunit isoform b (NM_001197115.2 and NP_001184044.1, which lacks an in-frame exon in the 5' coding region compared to variant 1. This results in a shorter protein (isoform b) compared to isoform a) and glutamate-cysteine ligase catalytic subunit isoform a (NM_001498.4 and NP_001489.1, which represents a longer transcript and encodes a longer isoform (a)).

[0104] In some embodiments, the agent is an inhibitor of phosphoseryl - tRNA kinase (PSTK). PSTK is an enzyme that mobilizes selenocysteine, which is encoded by UGA. Sec is formed in the tRNA - dependent conversion of serine that is bound to tRNA Sec by seryl - tRNA synthetase. PSTK phosphorylates Ser - tRNA Sec to Sep - tRNA Sec, which is then converted to Sec - tRNA Sec by Sep - tRNA:Sec - tRNA synthetase (SepSecS). Representative human PSTK cDNA and human PSTK protein sequences are well - known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human L - seryl - tRNA(Sec) kinase isoform 1 (NM_001363531.2 and NP_001350460.1) and L - seryl - tRNA(Sec) kinase isoform 2 (NM_153336.3 and NP_699167.2).

[0105] In some embodiments, the inhibitor is an inhibitor of eukaryotic elongation factor selenocysteine - tRNA specific (EEFSEC). EEFSEC is also known as the selenoprotein translation factor selb. Representative human EEFSEC cDNA and human EEFSEC protein sequences are publicly available from the National Center for Biotechnology Information (NCBI) as follows: selenocysteine - specific elongation factor (NM_021937.5 and NP_068756.2), selenocysteine - specific elongation factor isoform X4 (XM_024453695.1 and XP_024309463.1), selenocysteine - specific elongation factor isoform X3 (XM_024453694.1 and XP_024309462.1), selenocysteine - specific elongation factor isoform X1 (XM_024453692.1 and XP_024309460.1), selenocysteine - specific elongation factor isoform X2 (XM_024453693.1 and XP_024309461.1), selenocysteine - specific elongation factor isoform X5 (XM_005247696.3 and XP_005247753.1), selenocysteine - specific elongation factor isoform X7 (XM_011513066.2 and XP_011511368.1), and selenocysteine - specific elongation factor isoform X6 (XM_024453696.1 and XP_024309464.1). EEFSEC is a specialized translation elongation factor responsible for co - translationally incorporating selenocysteine into proteins by recoding of the UGA stop codon in the presence of a downstream mRNA hairpin loop.

[0106] In some embodiments, the agent is an inhibitor of selenophosphate synthetase 2 (SEPHS2). Selenophosphate synthetase 2 (SEPHS2) catalyzes the production of monoselenophosphate (MSP) from selenide and ATP. MSP is a selenium donor required for the synthesis of selenocysteine (Sec), which is usually co-translationally incorporated into selenoproteins at the in-frame UGA codon that normally signals translational termination. This protein is itself a selenoprotein that contains a Sec residue in its active site, suggesting the presence of a self-regulatory mechanism. SEPHS2 is preferentially expressed in tissues and sites of blood cell development involved in selenoprotein synthesis. Furthermore, genome-scale cancer dependency profiling has identified selenoprotein synthase as a target for ferroptosis induction. The loss of selenoprotein synthase induces ferroptosis in sensitive cells. Furthermore, the loss of selenophosphate synthetase 2 (SEPHS2) exhibits a novel two-sided ferroptosis mechanism of action. SEPHS2 loss induced ferroptosis much more rapidly than the loss of other selenoprotein biosynthetic enzymes. An SEPHS2 inhibitor can induce ferroptosis in certain diseases. For example, aggressive liver cancer can be selectively targeted by SEPHS2 inhibition. Representative human SEPHS2 cDNA and human SEPHS2 protein sequences are well known in the art and publicly available from the National Center for Biotechnology Information (NCBI). Selenide, water dikinase 2 (NM_012248.4 and NP_036380.2).

[0107] In some embodiments, the agent is an inhibitor of Sep(O-phosphoserine)tRNA:Sec(selenocysteine)tRNA synthase (SEPSECS). Sep(O-phosphoserine)tRNA:Sec(selenocysteine)tRNA synthase (SEPSECS) catalyzes the conversion of O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec), which is the third step in the process of selenocysteine synthesis. Representative human SEPSECS cDNA and human SEPSECS protein sequences are publicly available from the National Center for Biotechnology Information (NCBI) as follows: O-phosphoseryl-tRNA(Sec) selenotransferase (NM_016955.4 and NP_058651.3), O-phosphoseryl-tRNA(Sec) selenotransferase isoform X1 (XM_017008277.1 and XP_016863766.1), O-phosphoseryl-tRNA(Sec) selenotransferase isoform X5 (XM_017008278.1 and XP_016863767.1), O-phosphoseryl-tRNA(Sec) selenotransferase isoform X4 (XM_011513848.1 and XP_011512150.1), O-phosphoseryl-tRNA(Sec) selenotransferase isoform X2 (XM_011513846.2 and XP_011512148.1), and O-phosphoseryl-tRNA(Sec) selenotransferase isoform X3 (XM_011513847.2 and XP_011512149.1).

[0108] In some embodiments, the agent is an inhibitor of selenocysteine insertion sequence binding protein 2 (SECISBP2). SECISBP2 is one of the polypeptide components of the mechanism involved in the co-translational insertion of selenocysteine (Sec) into selenoproteins. Sec is encoded by the UGA codon, which normally signals translation termination. Recoding of UGA as a Sec codon requires a Sec insertion sequence (SECIS) element present in the 3' untranslated region of eukaryotic selenoprotein mRNAs. This protein specifically binds to the SECIS element, which is stimulated by Sec-specific translation elongation factors. Representative human SECISBP2 cDNA and human SECISBP2 protein sequences are publicly available from the National Center for Biotechnology Information (NCBI) as follows: selenocysteine insertion sequence binding protein 2 isoform 2 (NM_001282688.2 and NP_001269617.1), selenocysteine insertion sequence binding protein 2 isoform 3 (NM_001282689.2 and NP_001269618.1), selenocysteine insertion sequence binding protein 2 isoform 4 (NM_001282690.1 and NP_001269619.1), selenocysteine insertion sequence binding protein 2 isoform 5 (NM_001354696.2 and NP_001341625.1), selenocysteine insertion sequence binding protein 2 isoform 6 (NM_001354697.2 and NP_001341626.1), selenocysteine insertion sequence binding protein 2 isoform 7 (NM_001354698.2 and NP_001341627.1), selenocysteine insertion sequence binding protein 2 isoform 8 (NM_001354702.2 and NP_001341631.1), selenocysteine insertion sequence binding protein 2 isoform 1 (NM_024077.5 and NP_076982.3), selenocysteine insertion sequence binding protein 2 isoform X9 (XM_024447669.1 and XP_024303437.1), selenocysteine insertion sequence binding protein 2 isoform X8 (XM_024447667.1 and XP_024303435.1), selenocysteine insertion sequence binding protein 2 isoform X5 (XM_017015122.2 and XP_016870611.1), selenocysteine insertion sequence binding protein 2 isoform X6 (XM_024447666.1 and XP_024303434.1), selenocysteine insertion sequence binding protein 2 isoform X9 (XM_024447668.1 and XP_024303436.1), selenocysteine insertion sequence binding protein 2 isoform X1 (XM_011519000.2 and XP_011517302.1), selenocysteine insertion sequence binding protein 2 isoform X2 (XM_011519001.1 and XP_011517303.1), selenocysteine insertion sequence binding protein 2 isoform X3 (XM_011519002.1 and XP_011517304.1), selenocysteine insertion sequence binding protein 2 isoform X4 (XM_011519003.1 and XP_011517305.1), and selenocysteine insertion sequence binding protein 2 isoform X7 (XM_006717282.2 and XP_006717345.1).

[0109] In some embodiments, the agent is an inhibitor of nuclear factor-erythroid 2-related factor 2 (NRF2). NRF2 is a member of the cap "n" collar (CNC) subfamily of basic region leucine zipper (bZip) transcription factors. NRF2 mediates the induction of a set of drug-metabolizing enzymes, such as glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), by antioxidants and electrophiles. NRF2 also regulates GPX4 protein content, intracellular labile iron content, and mitochondrial function, thereby modulating ferroptosis. The NRF2 protein sequence is publicly available from the National Center for Biotechnology Information (NCBI) as follows: Nrf2 [Homo sapiens], GenBank: AAB32188.1, transcription factor Nrf2 - human, PIR: I59340.

[0110] In some embodiments, the agent is an inhibitor of the cystine transporter SLC7A11 (also referred to as xCT). SLC7A11 (widely known as xCT) functions to transport cystine for glutathione biosynthesis and antioxidant defense and is overexpressed in multiple human cancers. The SLC7A11 (xCT) protein sequences are publicly available from the National Center for Biotechnology Information (NCBI) as follows: cystine / glutamate transporter [Homo sapiens] NP_055146.1 and cystine / glutamate transporter isoform X1 [Homo sapiens] XP_011530104.1.

[0111] In some embodiments, the agent is an inhibitor of system Xc. System Xc-, also referred to as the cystine / glutamate antiporter, is an intracellular antioxidant element composed of the light chain SLC7A11 (xCT) and the heavy chain SLC3A2 (4F2hc) and functions as a raw material for the synthesis of glutathione (GSH). The system Xc protein sequences are publicly available from the National Center for Biotechnology Information (NCBI) as follows: cystine / glutamate transporter [Homo sapiens] NP_055146.1, cystine / glutamate transporter isoform X1 [Homo sapiens] XP_011530104.1, 4F2 cell surface antigen heavy chain isoform f [Homo sapiens] NP_001013269.1, 4F2 cell surface antigen heavy chain isoform c [Homo sapiens] NP_002385.3, 4F2 cell surface antigen heavy chain isoform b [Homo sapiens] NP_001012680.1, and 4F2 cell surface antigen heavy chain isoform e [Homo sapiens] NP_001012682.1.

[0112] In some embodiments, the agent is an inhibitor of thioredoxin reductase (TXNRD). TXNRD is involved in the reversible S-nitrosylation of cysteine in certain proteins. The TXNRD protein sequences are publicly available from the National Center for Biotechnology Information (NCBI) as follows: Thioredoxin reductase [Homo sapiens] AAB35418.1, Thioredoxin reductase [Homo sapiens] AAF15900.1 GI: 6538774, Thioredoxin reductase [Homo sapiens] AAD25167.1, Thioredoxin reductase [Homo sapiens] AAD19597.1, and Thioredoxin reductase [Homo sapiens] CAA04503.1.

[0113] In some embodiments, the ferroptosis inducer is a class of molecules, including at least one of salts of said molecules, pharmaceutically acceptable salts, solvates, hydrates, enantiomers, diastereomers, racemates, crystalline forms, or any combination thereof (for said molecules within said class), which is a molecule.

[0114] In some embodiments, the priming agent is a class of molecules, including at least one of salts of said molecules, pharmaceutically acceptable salts, solvates, hydrates, enantiomers, diastereomers, racemates, crystalline forms, or any combination thereof (for said molecules within said class), which is a molecule.

[0115] In some embodiments, the agent is a statin. Exemplary statins include, but are not limited to, atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[0116] In some embodiments, the agent that induces or modulates ferroptosis in tissues or systemically is selected from Table 1. Exemplary ferroptosis modulating agents are provided in Table 1 along with their formulas, chemical identifiers, and respective targets and / or mechanisms of action.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

[0117] In some embodiments, the ferroptosis regulator is selected from the group consisting of (1S,3R)-RSL3, ML-162, ML-210, JKE-1674, JKE-1716, elastin, jacaric acid, butionine sulfoximine (BSO), trigonelline, glutamate, sulfasalazine, auranofin, bursatol, sorafenib, sorafenib-d3, sorafenib tosylate, trigonelline, FIN56, FINO2, CIL56, dihydroisotanshinone I, GPX4-IN-3, analogs, salts, or derivatives thereof. In some embodiments, the agents in Table 1 are in a pharmaceutically acceptable salt form of a small molecule.

[0118] (2) Priming agent A method of inducing targeted cell death in mammalian tissue in vivo, comprising: (a) contacting the mammalian tissue with a priming agent; and (b) a plurality of cells in the mammalian tissue being (i) a plurality of cells containing a selenium concentration higher than the selenium concentration in the mammalian tissue before contacting with the priming agent, (ii) a plurality of cells containing an iron concentration higher than the iron concentration in the mammalian tissue before contacting with the priming agent, (iii) a plurality of cells containing a PUFA concentration higher than the PUFA concentration in the mammalian tissue before contacting with the priming agent, (iv) a plurality of cells expressing one or more markers indicating a mesenchymal state, (v) a plurality of cells containing a peroxidation index (PI) higher than the PI in the mammalian tissue before contacting with the priming agent, and / or (vi) responsive to the priming agent when determined by detecting excessive cell proliferation in the mammalian tissue, contacting the mammalian tissue in vivo with an effective amount of a ferroptosis inducer for a period of at least 4 hours, wherein the ferroptosis inducer induces targeted cell death in the mammalian tissue in vivo, is provided herein. In some embodiments, the priming agent is administered prior to the administration of the ferroptosis inducer provided herein. In some embodiments, the priming agent is administered in vivo, in vitro, or ex vivo. The priming agent is an agent that prepares the subject or tissue for the administration of a therapeutically effective dose of the ferroptosis inducer or ferroptosis modulator provided herein. In some embodiments, the priming agent is a ferroptosis inhibitor. In some embodiments, the priming agent makes the cells in the tissue sensitive to ferroptosis. In some embodiments, the priming agent is a lipophilic antioxidant or a radical trap. In some embodiments, the priming agent is a polyunsaturated fatty acid. In some embodiments, the priming agent is an iron chelator. In some embodiments, the priming agent is a lipid peroxidation inhibitor. In some embodiments, the priming agent modulates blood oxygen levels. In some embodiments, the priming agent is a hydroperoxide.In some embodiments, the priming agent is selected from the group consisting of reproxastatin-1, ferrostatin-1, deferoxamine (DFO), iron, selenium, vitamin E, erythropoietin, polyunsaturated fatty acids, N-acetylcysteine, pifithrin-alpha-HBr, and methylnaphthalene-4-propionate endoperoxide (MNPE). In some embodiments, the polyunsaturated fatty acids are selected from the group consisting of hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA, timnodonic acid), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA, clupanodonic acid), docosahexaenoic acid (DHA, cervonic acid), tetracosahexaenoic acid (nisinic acid), tetracosapentaenoic acid, linoleic acid (LA), gamma-linolenic acid (GLA), eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA), docosadienoic acid, adrenic acid (AdA), docosapentaenoic acid (osbond acid), tetracosatetraenoic acid, and tetracosapentaenoic acid. Non-limiting examples of the priming agent are provided in Table 2.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0119] In some embodiments, the methods provided herein include administering any one of the agents listed in Table 2. Further provided herein are pharmaceutical compositions comprising a ferroptosis inducer or modulator and a priming agent. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent.

[0120] In some embodiments, a composition for use in a method of reducing the number of adipocytes in a subject in need thereof. In some embodiments, a composition for use in regulating, modulating, or inducing weight loss or fat loss. In some embodiments, a composition for use in regulating or modulating iron metabolism. In some embodiments, a composition for use in the treatment of obesity. In some embodiments, a composition for use in the treatment of kidney disease. In some embodiments, a composition for use in the treatment of kidney disease. In some embodiments, a composition for use in the treatment of a disease or condition associated with iron metabolism.

[0121] In some embodiments, a composition comprising an agent in Table 1 for use in the manufacture of a medicament for the treatment of a disease or condition described herein, such as a kidney disease or condition. In some embodiments, a composition comprising an agent in Table 1 for use in the manufacture of a medicament for the treatment of elevated cholesterol. In some embodiments, a composition comprising an agent in Table 1 for use in the manufacture of a medicament for inducing ferroptosis in a subject. In some embodiments, a composition comprising a ferroptosis inducer for use in the manufacture of a medicament for the treatment of a kidney disease, kidney condition, a disease or condition associated with lipid metabolism, or a disease or condition associated with elevated cholesterol.

[0122] (3) Additional Treatments In some embodiments, the methods provided herein include administering to a subject at least one additional treatment (e.g., a second weight loss treatment). In some cases, the second weight treatment is selected from GLP-1 agonists, gastric inhibitory polypeptide analogs, naltrexone-bupropion or salts thereof, orlistat or salts thereof, phentermine or salts thereof, and phentermine-topiramate or salts thereof, selenium, exercise therapy, surgery, dietary modification, and any combination thereof. In some cases, the GLP-1 agonist includes dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, or salts of any of these. In some cases, the gastric inhibitory polypeptide includes tizepatide or derivatives thereof. In some cases, the surgery is a gastric sleeve, gastric bypass, adjustable gastric band, or any combination thereof. In some cases, selenium can include inorganic selenium (selenate and selenite), organic selenium (selenomethionine and selenocysteine), or any combination thereof.

[0123] In some embodiments, the methods provided herein include performing at least one additional treatment on a subject. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is a dietary supplement. Non-limiting examples of dietary supplements include probiotics, selenium, iron, vitamins (e.g., vitamin A, vitamin C, vitamin E), curcumin, fish oil, beta-carotene, hydrogen sulfide, fatty acids, methionine, cysteine, homocysteine, taurine, cystine, or di-cystine. In some embodiments, the dietary supplement is a high-selenium dietary supplement.

[0124] In some embodiments, the additional treatment is an additional therapeutic agent. In some embodiments, the methods provided herein include administering an additional agent in combination with a ferroptosis inducer, an iron-dependent cell death inducer, and / or a priming agent provided herein. In some embodiments, the additional agent is a cell death inducer. In some embodiments, the additional agent is an anti-cancer agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. The chemotherapeutic agent or compound is any agent or compound useful in the treatment of cancer. Cancer chemotherapeutic agents that can be used in combination with a ferroptosis inducer or an iron-dependent cell death agent provided herein include, but are not limited to, mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, vindesine, and Navelbine™ (vinorelbine, 5'-noranhydroblastine). In still other cases, cancer chemotherapeutic agents include topoisomerase I inhibitors, such as camptothecin compounds. As used herein, “camptothecin compounds” include Camptosar™ (irinotecan HCL), Hycamtin™ (topotecan HCL), and other compounds derived from camptothecin and its analogs. Another category of cancer chemotherapeutic agents that can be used in the methods and compositions disclosed herein are podophyllotoxin derivatives, such as etoposide, teniposide, and mitopodoside. The present disclosure further encompasses other cancer chemotherapeutic agents known as alkylating agents that alkylate genetic material in tumor cells. These include, without limitation, cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, bendamustine, uracil mustard, chromafazine, and dacarbazine. The present disclosure encompasses anti-metabolites as chemotherapeutic agents. Examples of these types of agents include cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprime, and procarbazine.Additional categories of cancer chemotherapeutic agents that can be used in the methods and compositions disclosed herein include antibiotics. Examples include, but are not limited to, doxorubicin, bleomycin, dactinomycin, daunorubicin, mitomycin, mitomycin C, and daunomycin. There are numerous commercially available liposomal formulations of these compounds. The present disclosure further encompasses other cancer chemotherapeutic agents including, but not limited to, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, ifosfamide, and mitoxantrone.

[0125] The disclosed agents provided herein can be administered in combination with other antitumor agents, including cytotoxic / antineoplastic agents and antiangiogenic agents. Cytotoxic / antineoplastic agents can be defined as agents that attack and kill cancer cells. Some cytotoxic / antineoplastic agents can be alkylating agents that alkylate genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, bendamustine, uracil mustard, chromafazine, and dacarbazine. Other cytotoxic / antineoplastic agents can be antimetabolites against tumor cells, such as cytarabine, fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine. Other cytotoxic / antineoplastic agents can be antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mitomycin, mitomycin C, and daunomycin. There are numerous commercially available liposomal formulations of these compounds. Still other cytotoxic / antineoplastic agents can be mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / antineoplastic agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0126] Angiogenesis inhibitors can also be used. Suitable angiogenesis inhibitors for use in the disclosed methods and compositions include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other inhibitors of angiogenesis include angiostatin, endostatin, interferon, interleukin-1 (including α and β), interleukin-12, retinoic acid, and tissue inhibitors of metalloproteinase-1 and -2 (TIMP-1 and -2). Small molecules including topoisomerase, for example, razoxane, a topoisomerase II inhibitor having anti-angiogenic activity, can also be used.

[0127] Other anticancer agents that can be used in combination with the ferroptosis inducer provided in this specification include acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldosterone, altretamine, amphomycin, ametantrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, avastin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bevacizumab, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate, brequinar sodium, broxuridine, busulfan, calicheamicin, caracemide, carbetimer, carboplatin, carmustine, carboquone hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diacron, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, drostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enoplatin, empromate, epipropidine, epirubicin hydrochloride, erbuzole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, folic acid, fosquidone, fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, interleukin II (including recombinant interleukin II or rIL2), interferon alpha-2a, interferon alpha-2b,Interferon alpha-n1, Interferon alpha-n3, Interferon beta-I a, Interferon gamma-I b, Iproplatin, Irinotecan hydrochloride, Lanreotide acetate, Letrozole, Leuprolide acetate, Lialozole hydrochloride, Lometrexol sodium, Lomustine, Losoxantrone hydrochloride, Masoprocol, Mitamycin, Mechlorethamine hydrochloride, Megestrol acetate, Melenegestrol acetate, Melphalan, Menogaril, Mercaptopurine, Methotrexate, Methotrexate sodium, Methopterin, Metsulredepa, Mitindomide, Mitocarcin, Mitocromin, Mitogirin, Mitomalcin, Mitomycin, Mitosper, Mitotan, Mitoxantrone hydrochloride, Mycophenolic acid, Nocodazole, Nogalamycin, Ormaplatin, Oxisuran, Paclitaxel, Pegaspargase, Peliomycin, Pentamustine, Pepromycin sulfate, Perfosfamide, Pipobroman, Piposulfan, Pyroxantrone hydrochloride, Plicamycin, Promestane, Porfimer sodium, Porfiromycin, Prednimustine, Procarbazine hydrochloride, Puromycin, Puromycin hydrochloride, Pyrazofurin, Riboprine, Logretimide, Safingol, Safingol hydrochloride, Semustine, Simtrazene, Sparfosate sodium, Sparsomycin, Spirogermanium hydrochloride, Spiro-mustine, Spiroplatin, Streptozocin, Streptonigrin, Streptozocin, Thio-TEPA, Thiamiprine, Thioguanine, Thiotepa, Thiazofurin, Tirabazamine, Toremifene citrate, Trestolone acetate, Trisilibine phosphate, Trimethoprim, Trimethoprim glucuronide, Triptorelin, Tubulozole hydrochloride, Uracil mustard, Uredepa, Bapreotide, Verteporfin, Vinblastine sulfate, Vincristine sulfate, Vindesine, Vindesine sulfate, Vinetepidine sulfate,Examples include, but are not limited to, vincristine sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, borazole, zinoplatin, dinostatin, zorubicin hydrochloride. Other anticancer agents include 20-epi-1,25-dihydroxyvitamin D3, 5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecipenol, adozelesin, aldosterone, ALL-TK agonist or antagonist, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitor, antagonist D, antagonist G, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogen drug, prostate cancer, antiestrogen drug, antineoplaston, antisense oligonucleotide, aphidicolin glycinate, apoptosis gene modulator, apoptosis regulator, apurinic acid, ara-CDP-DL-PTBA, arginine deiminase, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azaserone, azatoxin, azatyrosine, baccatin III derivative, baranol, batimastat, BCR / ABL antagonist or agonist, benzchlorin, benzoyl staurosporine, beta-lactam derivative, beta-aretin, beta-clarithromycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinyl spermine, bisnafide, bistratene A, bizelesin, breflate, broxuridine, budotitane, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivative, canaripox IL-2, capecitabine, carboxamide-amino-triazole, carboxamide triazole, CaRest M3, CARN 700, cartilage-derived inhibitor, carzelesin, casein kinase inhibitor (ICOS), castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost,Cis-porphyrin, cladribine, clomiphene analog, clotrimazole, corismycin A, corismycin B, combretastatin A4, combretastatin analog, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivative, classin A, cyclopentanthraquinone, cycloplatin, sipeamycin, cytarabine ocfosfate, cytolytic factor, cytostatic, daclizumab, decitabine, dehydrodidemnin B, deslorelin, dexamethasone, dexifosfamide, dexrazoxane, dexverapamil, diadicone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, 9-, dioxamycin, diphenylspiromustin, docetaxel, docosanol, drotrecogin, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemen, emitefur, epirubicin, epristeride, estramustine analog, estrogen agonist, estrogen antagonist, etanidazole, etoposide phosphate, exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorubicin hydrochloride, formestane, fosfomycin, fotemustine, gadolinium texaphyrin, gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitor, gemcitabine, glutathione inhibitor, hepsulfam, heregulin, hexamethylenebisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imiquimod, immunostimulatory peptide, insulin-like growth factor-1 receptor inhibitor, interferon agonist, interferon, interleukin, yobengamine, iododoxorubicin, ipomeanol, 4-(ipomeanol, 4-)Iroplact, ilsogladine, isobengazole, isohomohalicondrin B, itacetron, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, reinosamycin, lenograstim, lentinan sulfate, leptostatin, retrozole, leukemia inhibitory factor, leukocyte alpha interferon, leuprorelin + estrogen + progesterone, leuprorelin, levamisole, liarozole, linear polyamine analog, lipophilic disaccharide peptide, lipophilic platinum compound, lissoclinamide 7, lobaplatin, lombricin, lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine, larotrectin, lutetium texaphyrin, lysophilin, soluble peptide, maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysin inhibitor, matrix metalloproteinase inhibitor, menogaril, melvalone, metelethrin, methioninase, metoclopramide, MIF inhibitor, mifepristone, miltefosine, milimostim, mismatched double-stranded RNA, mitoguazone, mitolactol, mitomycin analog, mitonafide, mitotoxin fibroblast growth factor-saporin, mitoxantrone, mofarotene, molgramostim, monoclonal antibody, human chorionic gonadotropin, monophosphoryl lipid A + mycobacterium cell wall sk, mopidamol, multidrug resistance gene inhibitor, treatment based on multiple tumor suppressor 1, mustard anticancer agent, mycaperoxide B, mycobacterium cell wall extract, myriaporon, N-acetyl dinarine, N-substituted benzamide, naphthalene, nagrestip, naloxone + pentazocine, napabine, naphterpin, naltrindole, nedaplatin, nemorubicin, nerydroic acid, neutral endopeptidase, nilutamide, nisamycin, nitric oxide modulator, nitrooxide antioxidant, nitrullyn, O6-benzylguanine, octreotide, oxenone, oligonucleotide, onapristone, ondansetron,Ondansetron, oracin, oral cytokine inducer, ormaplatin, osaterone, oxaliplatin, oxaunomycin, paclitaxel, paclitaxel analog, paclitaxel derivative, paraumine, palmitoyl lysophosphatidic acid, pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, perdicane, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perfosfamide, perillyl alcohol, phenazinomycin, phenyl acetate, phosphatase inhibitor, picibanil, pilocarpine hydrochloride, pirarubicin, pirimtrexate, placetin A, placetin B, plasminogen activator inhibitor, platinum complex, platinum compound, platinum-triamine complex, porfimer sodium, porphy, Romamycin, prednisone, propylbis-acridone, prostaglandin J2, proteasome inhibitor, protein A-based immunomodulator, protein kinase C inhibitor, protein kinase C inhibitor, microalgae, protein tyrosine phosphatase inhibitor, purine nucleoside phosphorylase inhibitor, purpurin, pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonist, raltitrexed, lamotrigine, ras farnesyl protein transferase inhibitor, ras inhibitor, ras-GAP inhibitor, demethylated leptin, rhenium Re186 etidronate, lysokin, ribozyme, RII retinamide, logretimide, rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol, sintopine, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetic, semustine, senescence-derived inhibitor 1, sense oligonucleotide, signal transduction inhibitor, signal transduction regulator, single-chain antigen-binding protein, schizophyllan, sobuzoxane, sodium borocapitate, sodium phenylacetate, solverol, somatomedin-binding protein, sonermin, sparfosic acid, spicamycin D, spiromustin, sprionpentine, spongistatin 1, squalamine, stem cell inhibitor, stem cell division inhibitor, spithiamide, stromelysin inhibitor, sulfinosine, superactive vasoactive intestinal peptide antagonist, suradista, suramine, swine sonin, synthetic glycosaminoglycan, talimustine, tamoxifen methiodide, tauromustine, tazarotene, sodium tecogalansodium), tegafur, terapyllium, telomerase inhibitor, temoporfin, temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine, taliblastine, thiocholin, thrombopoietin, thrombopoietin mimetic, timalphasin, thymopoietin receptor agonist, thymotrinan, thyroid stimulating hormone, ethyl etiopurpurin tin, tirapazamine, titanocene bichloride, topsecentin, tamoxifen, pluripotent stem cell factor, translation inhibitor, tretinoin, triacetyluridine, triciribine, trimethoprate, tryptoreline, tropisetron, turossteride, tyrosine kinase inhibitor, tilostatin, UBC inhibitor, ubenimex, non-urogenital sinus-derived growth inhibitor, urokinase receptor agonist or antagonist, vapreotide, valiorin B, erythrocyte gene therapy, veraresol, veramine, bergenin, verteporfin, vinorelbine, vincasar, vitaxin, borozole, zanoterone, zinoplatin, dillascolb, and dinostatin stimalamer, among others, but not limited thereto. Any of the aforementioned chemotherapeutic agents can be administered at a clinically effective dose. The chemotherapeutic agent can also be administered about 14 days before, about 13 days before, about 12 days before, about 11 days before, about 10 days before, about 9 days before, about 8 days before, about 7 days before, about 6 days before, about 5 days before, about 4 days before, about 3 days before, about 2 days before, about 1 day before, on about day 0, about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, about day 7, about day 8, about day 9, about day 10, about day 11, about day 12, about day 13, or up to about 14 days after the administration of the agent provided herein. In some cases, the subject may have refractory cancer that is not responsive to the chemotherapeutic agent.

[0128] Pharmaceutical composition A pharmaceutical composition is provided herein that comprises an agent selected from Table 1 or a combination of agents selected from Table 1 and / or Table 2, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an apoptosis inducer. In some embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent. In some embodiments, the pharmaceutical compositions provided herein are in suspension, optionally a homogeneous suspension. In some embodiments, the pharmaceutical compositions provided herein are in emulsion form. In some embodiments, the pharmaceutical compositions provided herein comprise a salt form of any one of the agents provided herein. In some embodiments, the salt is a methanesulfonate.

[0129] Also provided herein are pharmaceutical compositions comprising a ferroptosis inducer or an iron-dependent cell death agent provided herein. Pharmaceutical compositions comprising a ferroptosis modulator are also provided herein. In some cases, the pharmaceutical composition can be in unit dosage form. In some embodiments, the agents provided herein are combined with pharmaceutically acceptable salts, excipients, and / or carriers to form a pharmaceutical composition. The pharmaceutical salts, excipients, and carriers can be selected based on the route of administration, the location of the target tissue, and the time course of drug delivery. Pharmaceutically acceptable carriers or excipients can include solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents compatible with pharmaceutical administration.

[0130] In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid, or gas (aerosol). Injectable preparations, for example, sterile aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be utilized are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, a sterile fixed oil is used as a solvent or suspending medium. For this purpose, any bland fixed oil including synthetic monoglycerides or diglycerides can be utilized. In addition, fatty acids such as oleic acid are used in injectable preparations. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0131] Exemplary carriers and excipients include dextrose, sodium chloride, sucrose, lactose, cellulose, xylitol, sorbitol, mannitol, gelatin, polymers, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and any combination thereof. In some embodiments, an excipient such as dextrose or sodium chloride can be present in a percent of about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, or up to about 15%.

[0132] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the encapsulated or non-encapsulated conjugate is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or (a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, etc., (c) humectants, such as glycerol, (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents, such as paraffin, (f) absorption promoters, such as quaternary ammonium compounds, (g) wetting agents, such as cetyl alcohol and glycerol monostearate, etc., (h) absorbents, such as kaolin and bentonite clay, and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include buffering agents.

[0133] The tablets may be film-coated or enteric-coated according to methods known in the art. Liquid preparations for oral administration may, for example, be in the form of solutions, syrups, or suspensions, or they may be presented as dry products for reconstitution with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable carriers and additives such as suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats, emulsifying agents, for example, lecithin or acacia, non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils, and preservatives, for example, methyl or propyl - p - hydroxybenzoates, or sorbic acid. The preparations may also, if appropriate, contain buffer salts, flavoring agents, coloring agents, and / or sweetening agents. If desired, the preparations for oral administration may preferably be formulated to provide controlled release of the active compound.

[0134] Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth, as well as pastilles containing the compound in an inert base, for example, gelatin and glycerin or sucrose and acacia.

[0135] The ferrotosis inducer or ferrotosis regulator provided herein may be formulated as a rectal composition, for example, a suppository or a retention enema, containing, for example, conventional suppository bases such as cocoa butter or other glycerides, or gel-forming agents such as carbomers.

[0136] The pharmaceutical composition can also be administered by controlled release formulations and / or delivery devices (see, for example, U.S. Patent No. 5,733,566).

[0137] A variety of delivery vehicles are known, including but not limited to encapsulation into liposomes, microparticles, microcapsules, nanoparticles, vectors, and recombinant cells, and can be used to administer the ferrotosis inducer or ferrotosis modulator provided herein. Liposomes and / or nanoparticles can also be utilized in the administration of the compositions herein. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also referred to as multilamellar vesicles (MLV)). MLV generally have a diameter ranging from 25 nm to 4 μm. Sonication of MLV results in the formation of small unilamellar vesicles (SUV) having a diameter in the range of 200 - 500 angstroms and containing an aqueous solution in the core.

[0138] Phospholipids can form various structures other than liposomes depending on the molar ratio of lipid to water when dispersed in water. At low ratios, liposomes are formed. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes can exhibit low permeability to ionic and polar substances, but at high temperatures, they undergo a phase transition and their permeability changes significantly. The phase transition involves a change from a tightly packed ordered structure known as the gel state to a loosely packed less ordered structure known as the fluid state. This occurs at a characteristic phase transition temperature and results in an increase in permeability to ions, sugars, and drugs.

[0139] Liposomes interact with cells by different mechanisms, including endocytosis by phagocytic cells of the reticuloendothelial system, such as macrophages and neutrophils, adsorption to the cell surface either by non-specific weak hydrophobic or electrostatic forces or by specific interactions with cell surface components, fusion with the plasma membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, which is accompanied by the simultaneous release of the liposome contents into the cytoplasm, and transfer of the liposome lipids into or from the cell membrane or submembrane without any association of the liposome contents. By changing the composition of the liposome, the mechanism that operates can be altered, although more than one may operate simultaneously. Nanocapsules can generally take up compounds in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, such ultrafine particles (of the order of 0.1 μm size) should be designed using polymers that can degrade in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles can also be used as delivery vehicles.

[0140] The tissue-specific targeting nanoparticle carriers provided herein can also be used as pharmaceutically acceptable carriers. In some embodiments, the nanoparticles are gold nanoparticles, platinum nanoparticles, iron oxide nanoparticles, lipid nanoparticles, selenium nanoparticles, tumor-targeting glycol chitosan nanoparticles (CNP), cathepsin B-sensitive nanoparticles, hyaluronic acid nanoparticles, paramagnetic nanoparticles, or polymer nanoparticles.

[0141] A pharmaceutical formulation of a ferroptosis inducer or a ferroptosis modulator suitable for transdermal application contains an effective amount of the agent together with a carrier. The carrier includes an absorbable pharmacologically acceptable solvent that aids in passing through the skin of the subject. For example, a transdermal device is in the form of a bandage or a patch and includes a backing member, a reservoir containing the compound, optionally together with a carrier, a rate control barrier for delivering the compound to the skin of the host over a long period of time at a controlled or predetermined rate, and means for securing the device to the skin. Matrix transdermal formulations can also be used. For example, formulations suitable for topical application to the skin and eyes are preferably aqueous solutions, ointments, creams, or gels well known in the art. The formulation may contain solubilizers, stabilizers, isotonicity enhancers, buffers, and preservatives.

[0142] In certain embodiments, the ferroptosis inducer or ferroptosis modulator provided herein is formulated as a depot composition. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. The ferroptosis inducer or ferroptosis modulator can be formulated using a suitable polymeric material or a hydrophobic material (e.g., as an acceptable oil-in-emulsion), an ion exchange resin, a biodegradable polymer, or a poorly soluble derivative, e.g., a poorly soluble salt.

[0143] In some embodiments, one or more of the agents provided herein are formulated as a pharmaceutical food composition (also referred to as a medical food). The food composition can be for consumption by a mammal, e.g., a human or non-human mammal. The agents provided herein can be formulated as dietary supplements or medical foods. In some embodiments, the agents provided herein are administered with a food component. The food component is any product, composition, or constituent of a food that is known to have a nutritional effect or is disclosed to have such. Foods include various meats (e.g., beef, pork, chicken, fish, etc.), dairy products (e.g., milk, cheese, eggs), fruits, vegetables, grains, breads, etc., as well as their constituents. The food can be fresh food or preserved food, e.g., canned, dried, frozen, or smoked. The food can be provided in an unprocessed, untreated, and / or natural state, or in a cooked, processed, and / or combined state. In some embodiments, the food component is selected from the group consisting of lipids, carbohydrates, proteins, fibers, nutritional balancers, and mixtures thereof. In some embodiments, the pharmaceutical food composition provided herein further comprises one or more of proteins or amino acids. In some embodiments of any of these aspects, the pharmaceutical food composition further comprises adenine, one or more vitamins (e.g., vitamin E), potassium, fatty acids, and / or calcium carbonate.

[0144] Methods of Administering the Agent A treatment regimen and methods for administering to a subject having a disease or disorder (e.g., cancer, autoimmune disease, obesity, or fibrosis) can be provided herein. In some embodiments, the administration is a continuous administration of a therapeutically effective amount of a ferroptosis inducer or ferroptosis modulator. In some embodiments, the continuous administration of the ferroptosis inducer is to administer the ferroptosis inducer at least about 10 ng / mm in the tissue 2Provided to the tissue in an amount sufficient to achieve the distribution for at least a 4-hour period, thereby inducing ferroptosis in the tissue. In some embodiments, the sustained administration further forms a gradient of a sub-therapeutic amount of a ferroptosis inducer adjacent to the site of administration within the tissue. In some embodiments, the sustained administration of a ferroptosis inducer or a ferroptosis modulator comprises an additional administration step. In some embodiments, the ferroptosis inducer or the ferroptosis modulator is administered multiple times. In some embodiments, the administration is by the system provided herein. In some embodiments, the administration is local administration within the tissue. In some embodiments, the tissue is contacted in vivo with an effective amount of an iron-dependent cell death agent for at least a 4-hour period. In some embodiments, the administration comprises contacting mammalian tissue with a priming agent and contacting mammalian tissue with an effective amount of a ferroptosis inducer provided herein, wherein the ferroptosis inducer induces targeted cell death in mammalian tissue in vivo. In some embodiments, the administration is local or systemic administration. In some embodiments, the step of administering or contacting is by intratumoral injection, oral administration, transdermal injection, inhalation, nasal administration, topical administration, vaginal administration, ophthalmic administration, intracerebral administration, rectal administration.

[0145] In some cases, the agents or combinations of agents provided herein are administered as a unit dosage form. A number of agents can be administered orally as a liquid, capsule, tablet, or chewable tablet. The oral route is the most convenient and is most often used because it is usually the safest and least expensive. However, it has limitations because the drug typically moves through the digestive tract. For drugs administered orally, absorption can begin in the mouth and stomach. However, most drugs are usually absorbed from the small intestine. The drug passes through the intestinal wall and travels to the liver, after which it is transported to its target site through the bloodstream. The intestinal wall and liver chemically alter (metabolize) many drugs and reduce the amount of drug reaching the bloodstream. As a result, these drugs are often provided at lower doses than would be obtained by intravenous injection to achieve the same effect.

[0146] In some embodiments, the agents provided herein are formulated for oral administration. In some embodiments, the agents provided herein are formulated for administration / use in administration by subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, intracranial, intrathecal, intratumoral, or oral routes. For the subcutaneous route, the needle is inserted into the adipose tissue just under the skin. After the drug is injected, it then moves into small blood vessels (capillaries) and is carried far away by the blood stream. Alternatively, the drug reaches the blood stream through lymphatic vessels. The intramuscular route is preferred over the subcutaneous route when larger amounts of drug product are required. Since muscle is present under the skin and adipose tissue, longer needles are used. The agent is usually injected into the muscles of the upper arm, thigh, or buttocks. The rate at which the drug is absorbed into the blood stream depends in part on the blood supply to the muscle, and the less the blood supply, the longer it takes for the drug to be absorbed. For the intravenous route, the needle is inserted directly into a vein. The solution containing the drug can be provided as a single bolus or by continuous infusion. For infusion, the solution moves through thin flexible tubing, usually by gravity (from a collapsible plastic bag) or more commonly by an infusion pump, to a tube (catheter) inserted into a vein in the forearm. In some cases, the agent or treatment regimen is administered as an infusion. The infusion can be carried out over a period of time. For example, the infusion can be the administration of the agent or treatment regimen over a period of about 5 minutes to about 5 hours. The infusion can be carried out over a period of about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or up to about 5 hours.

[0147] In some embodiments, intravenous administration is used to deliver an accurate dose quickly and in a well-controlled manner throughout the body. It is also used for irritating solutions that would cause pain and damage tissue if provided by subcutaneous or intramuscular injection. Intravenous injection can be more difficult to administer than subcutaneous or intramuscular injection because inserting a needle or catheter into a vein can be difficult, especially if the person is obese. When provided intravenously, the drug is delivered directly into the bloodstream and tends to act more quickly than when provided by any other route. As a result, healthcare providers monitor a person receiving an intravenous injection more closely for signs that the drug is acting or causing unwanted side effects. Also, the effects of drugs provided by this route tend to be of short duration. Thus, some drugs must be provided by continuous infusion to maintain their effect. For the intrathecal route, the needle is inserted into the space around the spinal cord between two vertebrae in the lower spine. The drug is then injected into the spinal canal. A small amount of local anesthetic is often used to numb the injection site. This route is used when the drug needs to have a rapid or local effect on the brain, spinal cord, or the layers of tissue (meninges) that cover them, for example, to treat an infection of these structures.

[0148] For administration by inhalation, the ferrostosis inducer or ferrostosis regulator provided herein can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer by the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, for use in an inhaler or insufflator can be formulated to contain the compound and a suitable powder base, such as a powder mix of lactose or starch. Drugs administered by inhalation through the mouth can be sprayed into smaller droplets than those administered by the nasal route so that the drug can pass through the trachea (windpipe) into the lungs. How deep the drug enters the lungs depends on the size of the droplets. The smaller the droplets, the deeper they enter and the greater the amount of drug absorbed. Inside the lungs, they are absorbed into the bloodstream.

[0149] Drugs applied to the skin are usually used for their local action and are thus most commonly used to treat superficial skin disorders such as psoriasis, eczema, skin infections (viral, bacterial, and fungal), itching, and dry skin. The drug is mixed with an inert substance. Depending on the consistency of the inert substance, the formulation can be an ointment, cream, lotion, solution, powder, or gel.

[0150] In some cases, the treatment regimen can be administered according to the weight of the subject. In subjects determined to be obese (BMI > 35), it may be necessary to use the actual weight. The BMI is calculated as follows: BMI = weight (kg) / [height (m)] 2 。

[0151] In some cases, the treatment regimen may be administered with a carrier or excipient. The ferroptosis inducer or ferroptosis modulator provided herein may be administered sequentially or in combination with one or more of the second agents by either the same route of administration or different routes of administration. When administered sequentially, the time between administrations is selected, inter alia, so as to obtain a benefit in the therapeutic efficacy and / or safety of the combination treatment. In certain embodiments, the agents provided herein may administer the second agent after the first agent, or alternatively, the second agent may be administered first, followed by the agent of the present disclosure (e.g., the ferroptosis inducer / modulator of Table 1). By way of example and not limitation, the time between administrations is about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 16 hours, or about 20 hours. In certain embodiments, the time between administrations is an additional number of days of about 1, about 2, about 3, about 4, about 5, about 6, or about 7. In some embodiments, the time between administrations is about 1 week, 2 weeks, 3 weeks, or 4 weeks, or longer. In some embodiments, the time between administrations is about 1 month or 2 months or longer.

[0152] In some embodiments, the ferrotosis inducer provided herein contacts mammalian tissue for at least about 4 hours, at least about 6 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 36 hours, at least about 48 hours, up to 72 hours. In some embodiments, the ferrotosis inducer provided herein contacts mammalian tissue for about 4 hours. In some embodiments, the ferrotosis inducer provided herein contacts mammalian tissue for about 6 hours. In some embodiments, the ferrotosis inducer provided herein contacts mammalian tissue for about 10 hours. In some embodiments, the ferrotosis inducer provided herein contacts mammalian tissue for about 12 hours. In some embodiments, the ferrotosis inducer provided herein contacts mammalian tissue for about 24 hours. In some embodiments, the ferrotosis inducer provided herein contacts mammalian tissue for about 48 hours. In some embodiments, the ferrotosis inducer provided herein contacts mammalian tissue for about 72 hours.

[0153] When administered in combination, the agents may be administered separately by the same or different routes at the same time as the second agent, or may be administered in a single pharmaceutical composition by the same route. In certain embodiments, standard dosages and standard dosing frequencies used for a particular compound may be used for the dosage and frequency of administration of the second agent. Administration and Tissue Distribution

[0154] The methods provided herein involve administering to a subject an agent or pharmaceutical composition provided herein in an amount effective to induce ferroptosis in tissues in vivo. The agents and pharmaceutical compositions for administration to a subject in need thereof can be formulated in unit dosage form for ease of administration and uniformity of dosage. A unit dosage form is a physically distinct unit of the composition provided herein appropriate for the subject to be treated. However, it is understood that the total usage of the composition provided herein will be determined by the attending physician within the scope of sound medical judgment. For any composition provided herein, a therapeutically effective dosage can first be estimated in cell culture assays or animal models, such as mice, rabbits, dogs, pigs, or non-human primates. Animal models can also be used to achieve desired concentration ranges and routes of administration. Such information can then be used to determine dosages and routes useful for administration in humans. The therapeutic efficacy and toxicity of the compositions provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (This dosage is therapeutically effective in 50% of the population) and LD 50 (This dosage is lethal to 50% of the population). The dosage ratio of toxic effect to therapeutic effect is the therapeutic index and can be expressed as the ratio of LD 50 / ED 50 . Pharmaceutical compositions exhibiting a large therapeutic index may be useful in some embodiments. Data obtained from cell culture assays and animal studies can be used to form the range of dosage for use in humans.

[0155] Typical human dosages of the agents (e.g., ferroptosis inducers / modulators) provided herein can range from about 10 μg / kg body weight / day to 10,000 mg / kg / day. In some embodiments, the dosage of the agents provided herein is from about 0.1 mg / kg to about 1000 mg / kg, 1 mg / kg to 1000 mg / kg, 1 mg / kg to 800 mg / kg, from about 1 mg / kg to about 700 mg / kg, from about 2 mg / kg to about 500 mg / kg, from about 3 mg / kg to about 400 mg / kg, 4 mg / kg to about 300 mg / kg, or from about 5 mg / kg to about 200 mg / kg. In certain embodiments, suitable dosages of the agent can be about 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, 2,000 mg / kg, 3,000 mg / kg, 4,000 mg / kg, 5,000 mg / kg, 6,000 mg / kg, 7,000 / mg / kg, 8,000 mg / kg, 9,000 mg / kg, up to 9,600 mg / kg. In some embodiments, the dosage of the agents provided herein is about 100 mg / kg / day to about 6,400 mg / kg / day, four times a day. In some embodiments, the dosage of the agents provided herein is from about 50 mg / kg / day to about 25 mg / kg / day. In some embodiments, the dosage of the agents provided herein is from about 400 mg / kg / day to about 800 mg / kg / day. In certain embodiments, the dosage of the agent may be administered once a day or divided into fractional dosages and administered multiple times, e.g., two, three, or four times a day.

[0156] In some embodiments, the agent provided herein is administered in an amount of at least about 10 nanograms (ng) or more, about 20 ng or more, about 30 ng or more, about 40 ng or more, about 50 ng or more, about 60 ng or more, about 70 ng or more, about 80 ng or more, about 90 ng or more, up to 100 ng. In some embodiments, the agent is administered in an amount of at least about 1 microgram (μg) or more, about 5 μg or more, about 10 μg or more, about 20 μg or more, about 30 μg or more, about 40 μg or more, about 50 μg or more, about 60 μg or more, about 70 μg or more, about 80 μg or more, about 90 μg or more, up to 100 μg.

[0157] In some embodiments, the agents provided herein are administered at a concentration of at least about 0.1 micromolar (μM) or higher, about 1 μM or higher, about 2 μM or higher, about 3 μM or higher, about 4 μM or higher, about 5 μM or higher, about 6 μM or higher, about 7 μM or higher, about 8 μM or higher, about 9 μM or higher, about 10 μM or higher, about 15 μM or higher, about 20 μM or higher, about 25 μM or higher, about 30 μM or higher, about 35 μM or higher, about 40 μM or higher, about 45 μM or higher, about 50 μM or higher, about 55 μM or higher, about 60 μM or higher, about 65 μM or higher, about 70 μM or higher, about 75 μM or higher, about 80 μM or higher, about 85 μM or higher, about 90 μM or higher, about 95 μM or higher, about 100 μM or higher, about 110 μM or higher, about 120 μM or higher, about 130 μM or higher, about 140 μM or higher, about 150 μM or higher, about 160 μM or higher, about 170 μM or higher, about 180 μM or higher, about 190 μM or higher, about 200 μM or higher, about 300 μM or higher, about 400 μM or higher, about 500 μM or higher, up to a concentration of 1 mM. In some embodiments, the agents provided herein are administered at a concentration of at least about 0.1 μM and up to about 500 μM. In some embodiments, the agents provided herein are administered at a concentration of at least about 1 μM and up to 500 μM. In some embodiments, the agents provided herein are administered at a concentration of at least about 0.1 μM and up to 10 μM. In some embodiments, the agents provided herein are administered at a concentration of at least about 1 μM and up to 10 μM.

[0158] In some embodiments, the ferroptosis inducer or ferroptosis regulator provided herein is administered intravenously. In some embodiments, the ferroptosis inducer or ferroptosis regulator provided herein is administered intravenously at a concentration of at least about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, about 1000 mg / kg, about 1100 mg / kg, about 1200 mg / kg, about 1300 mg / kg, about 1400 mg / kg, about 1500 mg / kg, about 2000 mg / kg, about 2200 mg / kg, about 2400 mg / kg, up to about 2500 mg / kg.

[0159] In some embodiments, the ferroptosis inducer or ferroptosis regulator provided herein is administered orally. In some embodiments, the ferroptosis inducer or ferroptosis regulator provided herein is orally administered at a concentration of at least about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 200 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, about 1000 mg / kg, about 1100 mg / kg, about 1200 mg / kg, about 1300 mg / kg, about 1400 mg / kg, about 1500 mg / kg, about 2000 mg / kg, about 2200 mg / kg, about 2400 mg / kg, up to about 2500 mg / kg. In some embodiments, the ferroptosis inducer or ferroptosis regulator provided herein is orally administered once daily at a concentration of about 25 mg / kg. In some embodiments, the ferroptosis inducer or ferroptosis regulator provided herein is orally administered twice daily at a concentration of about 25 mg / kg. In some embodiments, the ferroptosis inducer or ferroptosis regulator provided herein is orally administered at a concentration of about 1300 mg / kg / day. In some embodiments, the ferroptosis inducer or ferroptosis regulator provided herein is orally administered at a concentration of about 2400 mg / kg / day.

[0160] The methods provided herein can be characterized by, or further include, measuring the distribution of the agent in the target tissue. The distribution of the agent provided herein can be determined by the amount or concentration of the agent in 1 square millimeter (mm 2 ) or 1 cubic millimeter (mm 3 ) of tissue. For example, with respect to local administration of the agent to a tumor, the tissue is about 6 - 7 mm in diameter, 36 - 42 mm 2 , or 216 - 294 mm 3This may be the case. Data obtained from animal studies may be used when formulating the extent of drug distribution in mammalian tissues. Examples of methods for determining the tissue distribution of a drug or agent include, for example, mass spectrometry, chromatography, imaging techniques, and immunoassays. The distribution of the agents provided herein can be determined using the systems provided herein.

[0161] In some embodiments, the tissue is administered a therapeutically effective amount of a ferrotosis inducer, and the administration comprises providing to the tissue a ferrotosis inducer in an amount sufficient to achieve the desired drug distribution. In some embodiments, the agents provided herein are at least about 1 ng / mm 2 or more, about 5 ng / mm 2 or more, about 10 ng / mm 2 or more, about 15 ng / mm 2 or more, about 20 ng / mm 2 or more, about 25 ng / mm 2 or more, about 30 ng / mm 2 or more, about 35 ng / mm 2 or more, about 40 ng / mm 2 or more, about 45 ng / mm 2 or more, about 50 ng / mm 2 or more, about 55 ng / mm 2 or more, about 60 ng / mm 2 or more, about 65 ng / mm 2 or more, about 70 ng / mm 2 or more, about 75 ng / mm 2 or more, about 80 ng / mm 2 or more, about 85 ng / mm 2 or more, about 90 ng / mm 2 or more, about 95 ng / mm 2 or more, about 100 ng / mm 2Or more than that, about 110 ng / mm 2 Or more than that, about 120 ng / mm 2 Or more than that, about 130 ng / mm 2 Or more than that, about 140 ng / mm 2 Or more than that, about 150 ng / mm 2 Or more than that, about 160 ng / mm 2 Or more than that, about 170 ng / mm 2 Or more than that, about 180 ng / mm 2 Or more than that, about 190 ng / mm 2 Or more than that, about 200 ng / mm 2 Or more than that, about 300 ng / mm 2 Or more than that, about 400 ng / mm 2 Or more than that, up to 500 ng / mm 2 to achieve tissue distribution. In some embodiments, the agents provided herein are at least about 1 ng / mm 3 Or more than that, about 5 ng / mm 3 Or more than that, about 10 ng / mm 3 Or more than that, about 15 ng / mm 3 Or more than that, about 20 ng / mm 3 Or more than that, about 25 ng / mm 3 Or more than that, about 30 ng / mm 3 Or more than that, about 35 ng / mm 3 Or more than that, about 40 ng / mm 3 Or more than that, about 45 ng / mm 3 Or more than that, about 50 ng / mm 3 Or more than that, about 55 ng / mm 3 Or more than that, about 60 ng / mm 3 Or more than that, about 65 ng / mm 3 Or more than that, about 70 ng / mm 3 Or more than that, about 75 ng / mm 3 Or more than that, about 80 ng / mm 3Or more than that, about 85 ng / mm 3 Or more than that, about 90 ng / mm 3 Or more than that, about 95 ng / mm 3 Or more than that, about 100 ng / mm 3 Or more than that, about 110 ng / mm 3 Or more than that, about 120 ng / mm 3 Or more than that, about 130 ng / mm 3 Or more than that, about 140 ng / mm 3 Or more than that, about 150 ng / mm 3 Or more than that, about 160 ng / mm 3 Or more than that, about 170 ng / mm 3 Or more than that, about 180 ng / mm 3 Or more than that, about 190 ng / mm 3 Or more than that, about 200 ng / mm 3 Or more than that, about 300 ng / mm 3 Or more than that, about 400 ng / mm 3 Or more than that, up to 500 ng / mm 3 to achieve the tissue distribution of.

[0162] In some embodiments, the ferroptosis inducer or ferroptosis modulator provided herein is administered at least once a day, twice a day, three times a day, four times a day, or five times a day. In some embodiments of any of these aspects, the ferroptosis inducer or ferroptosis modulator is administered at least once every about week, at least once every about two weeks, or at least once every about three weeks. The amount of drug administered depends on the size of the tissue, the type of disease being treated, and the type of administration (e.g., local administration to tissue in vivo using the system provided herein). The effective dose varies depending on the type of disease being treated, the route of administration, the amount of excipient used, and the potential for co-use with other therapeutic treatments. Efficacy

[0163] The therapeutic efficacy of the agents and / or pharmaceutical compositions provided herein can be determined by evaluating and comparing the symptoms and quality of life of the patient before and after administration. Such methods are applicable regardless of the mode of administration. In some embodiments, before administration refers to evaluating the symptoms and quality of life of the patient before the start of treatment, and after administration refers to evaluating the symptoms and quality of life of the patient at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks after the start of treatment. In certain embodiments, the evaluation after administration is performed about 2 - 8 weeks, 2 - 6 weeks, 4 - 6 weeks, or 4 weeks after the start of treatment. In certain embodiments, the symptoms (e.g., symptoms associated with cancer, fibrosis, obesity, or autoimmune diseases) and quality of life of the patient before and after administration are evaluated by clinical evaluation and questionnaire evaluation.

[0164] The agents and methods provided herein can be used to reduce the growth or survival of cancer cells in vivo or in vitro. Methods for evaluating tumor progression or cell growth are known in the art. In some embodiments, the overall response is evaluated as follows from a time - point response assessment (based on tumor volume): · Complete response (CR): Disappearance of all target lesions. Any pathologic lymph nodes (regardless of being target or non - target) must have a short axis reduction to less than 10 mm. · Partial response (PR): At least a 30% decrease in the sum of the diameters of the target lesions based on the baseline sum diameter. · Progressive disease (PD): An increase of at least 20% in the sum of the diameters of target lesions, based on the minimum sum (including the minimum sum at baseline if it is the minimum during the study). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also considered progression). · Stable disease (SD): Absence of shrinkage sufficient to qualify for PR or increase sufficient to qualify for PD, based on the minimum sum diameter during the study.

[0165] In some embodiments, in vitro cell proliferation assays are used to evaluate the effectiveness of one or more of the ferrotosis-inducing agents provided herein. The compositions and methods provided herein result in a reduction in the proliferation or survival of multiple cells. For example, after treatment with one or more of the agents provided herein, cell proliferation or survival is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to cell proliferation or survival prior to treatment.

[0166] In some embodiments, an animal model is used to evaluate the effectiveness of one or more of the ferroptosis inducers and ferroptosis modulators provided herein in vivo. The ferroptosis inducers and methods provided herein can result in a reduction in the size or volume of hyperproliferative tissue (e.g., a tumor). For example, after treatment, the tissue size is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to its size prior to treatment. The size of the tissue (e.g., a tumor) can be measured by any reproducible means of measurement. The size of the tissue can be measured as the diameter of the tumor or by any reproducible means of measurement. A ferroptosis inhibitor (e.g., the agents in Table 2) can be used to determine the effectiveness of a particular test agent (also referred to herein as an activator) for inducing ferroptosis in a tissue. For example, a combination of a ferroptosis inducer paired with a ferroptosis inhibitor (e.g., liproxstatin-1) can be used to determine whether the test agent targets a protein or nucleic acid involved in the ferroptosis pathway (see FIG. 1). Further provided herein is a method of rescuing one or more cells from cell death and / or ferroptosis in vivo, comprising administering a ferroptosis inhibitor to a subject. In some embodiments, the method further comprises administering a ferroptosis inducer. Further provided herein is a method of screening a plurality of cells in a tissue for ferroptosis susceptibility, comprising contacting the tissue with a ferroptosis inducer and a ferroptosis inhibitor and measuring one or more parameters indicative of ferroptosis. In some embodiments, the ferroptosis inducer is an agent or test agent in Table 1. In some embodiments, the ferroptosis inhibitor is any of the agents listed in Table 2. In some embodiments, the ferroptosis inhibitor is liproxstatin-1.In some embodiments, one or more parameters indicative of ferroptosis are the PUFA concentration, the PI index, modulation of mesenchymal cell state marker expression, or modulation of iron or selenium concentration. The screening methods provided herein can be readily scaled for high-throughput analysis to enable the evaluation or prediction of the ferroptosis-inducing activity of test agents. Similarly, the screening methods can be performed in animal models in the presence and absence of a ferroptosis inhibitor, as discussed above.

[0167] Treating a disease or disorder (e.g., cancer, obesity, or fibrotic disease) can further result in a decrease in the number of hyperproliferative tissues and / or adipose tissues (e.g., tumors or fat). For example, after treatment, the number of hyperproliferative tissues or tumors is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to the number before treatment. The number of tumors, cells, or tissues provided herein can be measured by any reproducible means of measurement. The number of tumors, cells, or tissues can be measured by counting tumors, cells, or tissues visible to the naked eye or at a specified magnification (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or 50-fold). In some embodiments, the methods and ferroptosis inducers provided herein decrease the number of tumors in a subject.

[0168] In some embodiments, when administered to a subject in need thereof, the ferrotosis inducer or ferrotosis modulator provided herein decreases the number of adipocytes in the subject. In some cases, after treatment, the number of adipose cells (fat cells) in the subject may be reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or greater compared to the amount prior to treatment. In some cases, the amount of adipocytes may be reduced by about 5% to 99%, 25 to 75%, or 40 to 80% after treatment compared to before treatment. In some cases, after treatment, the amount of epididymal white adipose tissue in the subject may be reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or greater compared to before treatment. In some cases, the amount of epididymal white adipose tissue may be reduced by about 5% to 99%, 25 to 75%, or 40 to 80% after treatment compared to the amount prior to treatment. In some cases, the reduction can be determined by measuring the amount of epididymal white adipose tissue before and after administration of a therapeutic amount of the ferrotosis modulator.

[0169] In some cases, after treatment, there may be a decrease in the size of adipocytes. In some cases, the size of adipocytes may be reduced by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or greater compared to the size prior to treatment. In some cases, the size of adipocytes may be reduced by about 5% to 99%, 25 to 75%, or 40 to 80% after treatment compared to the size before treatment. In some cases, the reduction can be determined by measuring adipocyte size before and after administration of a therapeutic amount of the ferrotosis modulator.

[0170] In some embodiments, the methods and ferroptosis inducers provided herein increase the number or activity of leukocytes in the tumor microenvironment. In some embodiments, the leukocytes specifically target cancer cells that have a high PUFA concentration compared to normal cells.

[0171] Treating cancer can result in a decrease in the number of metastatic nodules in other tissues or organs distal to the primary tumor site. For example, after treatment, the number of metastatic nodules is reduced by 5% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) compared to the number before treatment. The number of metastatic nodules can be measured by any reproducible means of measurement. The number of metastatic nodules can be measured by counting the metastatic nodules visible to the naked eye or at a specified magnification (e.g., 2-fold, 10-fold, or 50-fold).

[0172] Treating a disease or disorder (e.g., cancer) can result in an increase in the average survival time of a population of subjects treated according to the present invention compared to an untreated population of subjects. For example, the average survival time is extended to more than 30 days (more than 60 days, more than 90 days, more than 120 days, or more). The increase in the average survival time of the population can be measured by any reproducible means. The increase in the average survival time of the population can be measured, for example, by calculating the average length of the survival time after the start of treatment with the compound of the present invention for the population. The increase in the average survival time of the population can also be measured, for example, by calculating the average length of the survival time after the completion of the first treatment with the compound of the present invention for the population.

[0173] Treating a disease or disorder (e.g., cancer) can also result in a decrease in the mortality rate of the treated subject population as compared to an untreated population. For example, the mortality rate decreases by more than 2% (e.g., more than 5%, more than 10%, more than 25%, or greater). The decrease in the mortality rate of the treated subject population can be measured by any reproducible means, e.g., by calculating the average number of disease-related deaths per unit time after initiation of treatment with a compound of the invention for the population. The decrease in the mortality rate of the population can also be measured, e.g., by calculating the average number of disease-related deaths per unit time after completion of the first treatment with a ferroptosis inducer for the population.

[0174] In addition, treating a disease or disorder can also result in a decrease in at least one symptom associated with the disease, disorder, or condition. In some embodiments, the methods provided herein reduce at least one symptom of a disease or disorder by at least 10%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, or more as compared to the number prior to treatment. In some embodiments, cell death can be detected upon or subsequent to contact of mammalian tissue with a ferroptosis inducer or administration of a ferroptosis inducer. In some embodiments, the methods provided herein increase cell death by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to the number prior to treatment.

[0175] Therapeutic applications Methods for treating a disease or disorder in a subject are provided herein. Compositions for use in using any of the methods described herein are also provided herein. In some embodiments, the subject has, is suspected of having, or is at risk of developing a proliferative disease or condition. In some embodiments, the methods provided herein further comprise obtaining a tissue biopsy for histological analysis. In some embodiments, the tissue comprises histological abnormalities, which are hyperplasia or fibrosis.

[0176] Methods for treating a disease, such as obesity or overweight, are provided herein. In some cases, the methods provided herein induce weight loss in a subject by administering a therapeutically effective amount of a ferroptosis modulator. In some cases, the method may include reducing the adipocyte size of the subject. In some cases, the reduction is determined by measuring the adipocyte size before and after administration of a therapeutically effective amount of a ferroptosis modulator. In some cases, the method includes reducing the epididymal white adipose tissue of the subject. In some cases, the reduction is determined by measuring the amount of epididymal white adipose tissue before and after administration of a therapeutically effective amount of a ferroptosis modulator.

[0177] In some embodiments, the subject has, is suspected of having, or is at risk of developing a metabolic disease. In some embodiments, the metabolic disease is weight gain or obesity. In some embodiments, the subject has, is suspected of having, or is at risk of developing weight gain. In some embodiments, the subject is obese. In some cases, the subject is not diagnosed with cancer. In some embodiments, the subject is overweight. In some embodiments, the subject has, is suspected of having, or is at risk of developing a disease or condition associated with obesity. In some cases, the subject has a BMI of from 25.0 to less than 30, which is overweight. In some cases, the subject has a BMI of 30.0 or higher, which is obese. In some cases, the subject can be class 1 obesity (BMI from 30 to less than 35), class 2 obesity (BMI from 35 to less than 40), or class 3 obesity (BMI 40 or higher).

[0178] In some embodiments, the subject may have, be suspected of having, be at risk of developing, or be diagnosed with type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome.

[0179] In some cases, the agent or therapeutic agent or molecule can be a glucagon-like peptide 1 (GLP-1) agonist.

[0180] In some embodiments, the subject has, is suspected of having, or is at risk of developing a disease or condition associated with abnormal angiogenesis or vasculogenesis. Diseases or conditions associated with abnormal angiogenesis or vasculogenesis include, but are not limited to, ocular neovascularization, macular degeneration, retinopathy, sarcoma, polycystic kidney disease, benign hyperplasia, leiomyoma, adenoma, lipoma, hemangioma, fibroma, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions, intraepithelial carcinoma, and cancer. In some embodiments, the subject has, is suspected of having, or is at risk of developing an autoimmune disease. Non-limiting examples of related autoimmune diseases include rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, oral hairy leukoplakia, and psoriasis. In some embodiments, the subject has, is suspected of having, or is at risk of developing fibrosis. Non-limiting examples of diseases and conditions associated with fibrosis include keloid scar, hypertrophic scar, systemic sclerosis, pulmonary arterial hypertension, cardiac fibrosis, hypertrophic cardiomyopathy valvular disease, myelofibrosis, myelodysplastic syndrome, chronic myeloid leukemia, portal hypertension, hepatocellular carcinoma, retroperitoneal fibrosis, intestinal fibrosis, intestinal disease, subretinal fibrosis, epiretinal fibrosis, cystic fibrosis, emphysema, pancreatic fibrosis, chronic pancreatitis, ductal obstruction, joint fibrosis, renal fibrosis, nephrogenic systemic fibrosis, renal anemia, chronic kidney disease, Dupuytren's disease, Ledderhose's disease (plantar fibromatosis), primary biliary cholangitis (PBC), non-alcoholic steatohepatitis (NASH), scleroderma, diabetic neuropathy, hypertensive nephrosclerosis, allograft nephropathy, cirrhosis, and pulmonary fibrosis. In some embodiments, the subject has, is suspected of having, or is at risk of developing a disease or condition associated with polycystic dysplastic kidney, Fabry disease, cystinosis, glomerulonephritis, IgA nephropathy, lupus nephritis, atypical hemolytic uremic syndrome (aHUS), nephrotic syndrome, kidney injury, polycystic kidney disease (PKD), renal artery stenosis, tubular acidosis, simple renal cyst, or a disease or condition associated with a single kidney or a single-functional kidney.

[0181] In some embodiments, the subject has cancer, is suspected of having cancer, or is at risk of developing cancer. In some embodiments, the subject has a benign tumor. In some embodiments, the subject has a pre-cancerous lesion. In some embodiments, the subject has basal cell carcinoma (BCC) or squamous cell carcinoma (SCC). In some embodiments, the subject has a metastatic tumor. In some embodiments, the cancer is a solid cancer or a blood cancer. In some embodiments, the blood cancer is leukemia or lymphoma. In some embodiments, the subject has a solid tumor. In some embodiments, the solid tumor is cancer, melanoma, or sarcoma. In some embodiments, the melanoma is a dedifferentiated melanoma or a melanin-deficient melanoma. In some embodiments, the subject has a melanoma having a B-Raf proto-oncogene serine / threonine kinase (BRAF) mutation. In some embodiments, the subject has a sarcoma having a Kirsten rat sarcoma (KRAS) mutation. In some embodiments, the sarcoma is a soft tissue sarcoma. In some embodiments, the sarcoma is a leiomyosarcoma. In some embodiments, the cancer is colorectal adenocarcinoma.

[0182] Non-limiting examples of cancers that can be treated with the agents provided herein include acoustic neuroma, adenocarcinoma, adrenal cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, angiosarcoma), appendiceal cancer, benign monoclonal gammaglobulinemia, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, mammary cancer, breast medullary carcinoma), brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma), bronchial cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, connective tissue cancer, epithelial cancer, epithelioma, endothelial sarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familial eosinophilia, gall bladder cancer, gastric cancer (e.g., gastric adenocarcinoma), gastrointestinal stromal tumor (GIST), germ cell cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematologic cancer (e.g., leukemia, e.g., acute lymphoblastic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myeloid leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myeloid leukemia (CML) (e.g., B-cell CIVIL, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)), lymphoma, e.g., Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) as well as non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL, e.g., diffuse large B-cell lymphoma (DLCL) (e.g., diffuse large B-cell type lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (e.g.,Waldenström macroglobulinemia, hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, and primary central nervous system (CNS) lymphoma, and T cell NHL, such as precursor T lymphoblastic lymphoma / leukemia, peripheral T cell lymphoma (PTCL) (e.g., cutaneous T cell lymphoma (CTCL) (e.g., mycosis fungoides, Sézary syndrome), angioimmunoblastic T cell lymphoma, extranodal natural killer T cell lymphoma, enteropathy-type T cell lymphoma, cutaneous panniculitis-like T cell lymphoma, and anaplastic large cell lymphoma), a mixture of one or more of the above leukemias / lymphomas, and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, hypopharyngeal cancer, inflammatory myofibroblastic tumor, immunoglobulin amyloidosis, kidney cancer (e.g., nephroblastoma, also known as Wilms tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), muscle cancer, myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocythemia (ET), angioectatic myeloid metaplasia (AMM), also known as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelogenous leukemia (CIVIL), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or 2, schwannoma), neuroendocrine cancer (e.g., gastrointestinal pancreatic neuroendocrine tumor (GEP NET), carcinoid tumor), osteosarcoma (e.g., bone cancer), ovarian cancer (e.g., cystadenocarcinoma, ovarian fetal cancer, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), islet cell tumor), penile cancer (e.g., penile and scrotal Paget's disease), pineal tumor, primitive neuroectodermal tumor (PNT), plasmacytic neoplasia, paraneoplastic syndrome, intraepithelial neoplasia, prostate cancer (e.g., prostate adenocarcinoma), colorectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC))Small bowel cancer (e.g., appendiceal cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), adenocarcinoma, small intestine cancer, sweat gland cancer, synovial sarcoma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid cancer (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer, and vulvar cancer (e.g., Paget's disease of the vulva).

[0183] Methods of administering a ferroptosis inducer to a tissue, wherein the tissue comprises different cell types, are provided herein. In some embodiments, the tissue comprises a heterogeneous cell population, and the heterogeneous cell population comprises at least one of a pre-cancerous cell and a non-cancerous cell. In some embodiments, the tissue comprises a heterogeneous cell population, and the heterogeneous cell population comprises an immune cell population.

[0184] Methods of inducing recruitment of immune cells to a tumor, comprising administering to a subject a ferroptosis inducer provided herein by any of the methods provided herein, are provided herein. In some embodiments, the administration is a continuous administration for at least about 10 hours, thereby recruiting immune cells to the tumor site. In some embodiments, the immune cells are white blood cells. In some embodiments, recruitment of immune cells can be detected at or after the time of contact of the mammalian tissue with the ferroptosis inducer, at the time of or subsequent to contact of the mammalian tissue with the ferroptosis inducer. In some embodiments, the administration reduces the size of the tumor and / or increases the number of white blood cells within the tumor.

[0185] Systems and kits Systems for the delivery of ferroptosis inducers or iron-dependent cell death inducers provided herein are provided herein. Systems for the delivery of ferroptosis modulators provided herein are provided herein. Further provided herein is a system for inducing ferroptosis in vivo, comprising: (a) a cylindrical support structure having at least one microwell formed on or within the surface of the support structure; (b) a microdose of a ferroptosis inducer within at least one microwell; and (c) a compound release mechanism for sustained administration for controlling the release of the ferroptosis inducer from the microwell, the system comprising an implantable microdevice configured for local administration to tissue, wherein the microdose of the ferroptosis inducer forms a gradient of a sub-therapeutic dose of the ferroptosis inducer at the site of administration within the tissue for a duration of at least 4 hours, the microdevice being configured to permit implantation into tissue using a catheter, cannula, or biopsy needle, and the microdevice being further configured to release the ferroptosis inducer from at least one microwell to the site of administration within apoptosis-resistant tissue adjacent to at least one microwell.

[0186] Further provided herein is a system for identifying ferroptosis induction in an animal model, comprising: (a) an animal model comprising a target tissue of interest; (b) a microdevice configured to permit implantation into the tissue of the animal model using a catheter, cannula, or biopsy needle, the microdevice comprising: (i) at least one microwell comprising one or more active agents; (ii) a microdose of the one or more active agents within the at least one microwell; and (iii) a compound release mechanism comprising a polymer matrix for controlling the release of the one or more active agents from the microwell to the tissue, the system measuring the results of ferroptosis induction in the animal model after administration of the one or more active agents to the tissue compared to baseline tissue without administration of the one or more active agents, and identifying the one or more active agents that induce ferroptosis in the tissue.

[0187] A system for screening for ferroptosis-induced cell death in vivo, comprising: (a) an animal model containing a target tissue of interest; (b) configured to allow implantation into the tissue of the animal model using a catheter, cannula, or biopsy needle, and including: (i) at least one microwell containing one or more activators; (ii) at least one microwell containing one or more ferroptosis inhibitors; (ii) a microdose of one or more activators and / or one or more ferroptosis inhibitors within at least one microwell; (iii) a compound release mechanism including a polymer matrix for controlling the release of one or more activators from the microwell to the tissue, the system including a microdevice, measuring the results of ferroptosis induction in the animal model after administration of one or more activators to the tissue compared to baseline tissue without administration of one or more activators, measuring the results of ferroptosis induction in the animal model after administration of one or more activators to the tissue compared to the administration of one or more activators and one or more ferroptosis inhibitors, and identifying one or more activators that induce ferroptosis in the tissue, is provided herein.

[0188] The systems provided herein generally include a plurality of microwells disposed on or within a support structure. The microwells contain one or more active agents, alone or in combination, in one or more dosages and / or release pharmacokinetics. Preferably, the device is configured to deliver microdoses so as to substantially eliminate overlap of the active agents released from different microwells in tissue. In some embodiments, the device is configured to facilitate implantation and removal in a target tissue. In an exemplary embodiment, the device has a cylindrical shape with symmetric wells on the outside of the device, each well containing one or more drugs at one or more concentrations. The device is sized to allow placement using a catheter, cannula, or stylet. In a preferred embodiment, the device has a guidewire to assist with placement and removal. The device may also include features that assist in maintaining the spatial stability of the tissue excised by the device, such as fins or stabilizers that can expand from the device before or at the time of removal. Optionally, the device has optical fibers, sensors, and / or interactive features, such as remote accessibility (e.g., Wi-Fi), to provide for in situ information retrieval and modification of the release characteristics of the device. In the most preferred embodiments, the optical fibers and / or sensors are individually accessible to separate wells.

[0189] In some embodiments, the systems provided herein are formed from biocompatible silicon, metal, ceramic, or polymer. They may include materials such as radiopaque materials or materials that can be imaged using ultrasound or MRI. They can be manufactured using techniques such as deep reactive ion etching, nanoimprint lithography, micromachining, laser etching, three-dimensional printing, or stereolithography. Drugs can be loaded by injection of a solution or suspension into the wells, followed by removal of the solvent by drying, evaporation, or lyophilization, or placement of the drug in the wells in tablet or particulate form. In a preferred embodiment, the drug is loaded on top of a hydrogel pad within the microwell. The hydrogel pad expands during implantation to deliver the drug to the surrounding tissue. The pharmacokinetics of drug release are a function of the solubility of the drug, excipients, well dimensions, and the tissue into which the device is implanted (more highly vascularized tissues will release at a higher rate than less vascularized tissues).

[0190] In some embodiments, the systems provided herein are implanted directly into a solid tumor or the tissue to be biopsied. After implantation, the systems provided herein locally release an array of active agents at microdoses. Analysis of the tumor response to the subsequent array of active agents can be used to identify specific drugs, drug combinations, and / or dosages that are effective in treating the solid tumor in a patient. By locally delivering an array of microdose drugs, the microassay device can be used to test a patient for responses to a wide range of regimens under rapid and actual physiological conditions without inducing systemic toxicity. These data are used in combination with genomic data as needed to accurately predict the systemic drug response.

[0191] Without limitation, the systems provided herein can administer the agents provided herein by any of the methods provided herein. For example, the systems provided herein can be used to deliver a microdose of an agent to a tissue in vivo. The systems described herein can provide a sustained administration of a therapeutic amount of a ferroptosis inducer to a tissue, wherein the sustained administration of the therapeutic amount comprises providing the ferroptosis inducer to the tissue in an amount sufficient to achieve a distribution of at least about 10 ng / mm 2 in the tissue for a period of at least 4 hours, thereby inducing ferroptosis in the tissue. In some embodiments, the sustained administration further forms a gradient of a sub-therapeutic amount of a ferroptosis inducer adjacent to the site of administration within the tissue. In some embodiments, the sustained administration of a therapeutic amount of a ferroptosis inducer is at least 10 hours. In some embodiments, the therapeutic amount of a ferroptosis inducer is a concentration of at least about 1 μM and up to 10 μM. In some embodiments, the systems provided herein are implanted into a tumor. In some embodiments, the system delivers one or more ferroptosis inducers to the tumor.

[0192] Kits are provided herein that include a ferroptosis modulating agent or a ferroptosis inducer. In some instances, the kits herein may be contained in a container. In some instances, the container can include glass, plastic, metal, or combinations thereof.

[0193] Exemplary embodiments Some compositions and methods are disclosed herein. Specific exemplary embodiments of these compositions and methods are disclosed below. The following embodiments enumerate non-limiting permutations of combinations of the characteristics disclosed herein. Other permutations of combinations of characteristics are also contemplated. Specifically, each of these numbered embodiments is intended to be dependent on or related to any preceding or subsequent numbered embodiment, independent of the order in which they are recited. Embodiment 1. A method for inducing weight loss or fat loss in a subject, comprising administering to the subject a ferroptosis inducer in an amount sufficient to reduce the size or number of adipocytes in the subject. Embodiment 2. The method according to Embodiment 1, wherein the subject is a human. Embodiment 3. The method according to Embodiment 1, wherein the ferroptosis inducer is administered orally. Embodiment 4. The administering is discontinuous, and the administering is performed daily for about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year, the method according to Embodiment 1. Embodiment 5. The administering results in a reduction in the size or number of adipocytes of the subject, and the reduction is determined by measuring the size or number of adipocytes before and after the administration of a therapeutically effective amount of a ferroptosis regulator using microscopic evaluation of a biopsy, if necessary, the method according to Embodiment 1. Embodiment 6. The method according to Embodiment 1, further comprising reducing the epididymal white adipose tissue of the subject. Embodiment 7. The method according to Embodiment 6, wherein the subject is overweight or obese. Embodiment 8. The method according to Embodiment 6, wherein the subject is obese and has a BMI of 30.0 or higher. Embodiment 9. The method according to Embodiment 6, wherein the subject is overweight and has a BMI of 25.0 to <30. Embodiment 10. A method for inducing, regulating, or modulating weight loss, body composition, or fat loss or reduction in a subject, comprising administering to the subject a therapeutically effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby inducing, regulating, or modulating the weight loss, body composition, or fat loss or reduction in the subject. Embodiment 11. The method according to embodiment 10, wherein the subject is a human. Embodiment 12. The method according to embodiment 11, wherein the subject is male. Embodiment 13. The method according to any of the preceding embodiments, wherein the agent is orally delivered as a solution having a concentration of at least about 5 mg of the agent per mL of the solution. Embodiment 14. The method according to embodiment 1, wherein the administration is discontinuous, and the administration is performed daily for about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year. Embodiment 15. The method according to embodiment 1, wherein the agent is selected from the group consisting of (1S,3R)-RSL3, ML-162, ML-210, JKE-1674, JKE-1716, elastin, jacaric acid, trigonelline, glutamate, sulfasalazine, auranofin, bursatol, sorafenib, sorafenib-d3, sorafenib tosylate, trigonelline, FIN56, FINO2, CIL56, dihydroisotanshinone I, GPX4-IN-3, sulfoximine, BSO, and salts of any of these. Embodiment 16. The method according to embodiment 1, wherein the administration results in a reduction in the size of the adipocytes of the subject, and the reduction is determined by measuring the adipocyte size before and after administration of a therapeutic amount of the ferroptosis regulator, optionally using microscopic evaluation of a biopsy. Embodiment 17. The method according to embodiment 1, wherein the administration results in a reduction in the number of adipocytes of the subject, and the reduction is determined by measuring the number of adipocytes before and after administration of a therapeutic amount of the ferroptosis regulator, optionally using microscopic evaluation of a biopsy. Embodiment 18. The method according to embodiment 16 or embodiment 17, wherein the method comprises reducing the epididymal white adipose tissue of the subject. Embodiment 19. The method according to embodiment 18, wherein the subject is overweight or obese. Embodiment 20. The method according to embodiment 19, wherein the subject is obese and has a BMI of 30.0 or higher. Embodiment 21. The method according to embodiment 19, wherein the subject is overweight and has a BMI of 25.0 - 30. Embodiment 22. The method according to any one of the preceding embodiments, wherein an additional agent or treatment is administered to the subject either concomitantly or sequentially. Embodiment 23. The method according to embodiment 22, wherein the additional agent or treatment is selected from the group consisting of GLP-1 agonists, gastric inhibitory polypeptide analogs, naltrexone-bupropion or its salts, orlistat or its salts, phentermine or its salts, phentermine-topiramate or its salts, selenium, exercise therapy, surgery, dietary modification, and any combination thereof. Embodiment 24. The method according to embodiment 23, wherein the additional agent or treatment comprises administering the GLP-1 agonist comprising dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, or a salt of any of these. Embodiment 25. The method according to embodiment 23, wherein the additional agent or treatment comprises administering the gastric inhibitory polypeptide comprising tildesatide or a derivative thereof. Embodiment 26. The method according to embodiment 23, wherein the additional agent or treatment comprises surgery including gastric sleeve, gastric bypass, adjustable gastric band, a ball placed in the stomach, or any combination thereof. Embodiment 27. The method according to any one of the preceding embodiments, wherein the subject is not diagnosed with cancer. Embodiment 28. The method according to any one of the preceding embodiments, wherein the subject is a subject in need thereof. Embodiment 29. The method according to any one of the preceding embodiments, wherein the subject has type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome. Embodiment 30. The method according to any one of the preceding embodiments, wherein the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. The method according to embodiment 30, wherein the pharmaceutical composition is in a unit dosage form. Embodiment 32. A composition comprising: i) a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, or an agent that inhibits GCL; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof. Embodiment 33. A composition comprising: i) a glutamate-cysteine ligase (GCL) regulator; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof. Embodiment 34. The composition according to embodiment 32 or 33, which is a pharmaceutical composition. Embodiment 35. The pharmaceutical composition according to embodiment 34, which is in a unit dosage form. Embodiment 36. The composition according to any one of embodiments 32 to 35, for use in the treatment of a disease or condition. Embodiment 37. The composition according to any one of embodiments 32 to 35, for use in the treatment of a kidney disease or condition. Embodiment 38. The composition according to any one of embodiments 32 to 35, for use in the treatment of a symptom associated with a kidney disease or condition. Embodiment 39. The composition for use according to embodiment 36, wherein the disease or condition is weight loss, weight maintenance, a disease or condition associated with iron metabolism, or a disease or condition associated with high, low, or abnormal cholesterol. Embodiment 40. A method of modulating iron metabolism in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby modulating the iron metabolism in the subject and thereby inducing ferroptosis in the subject. Embodiment 41. A method of modulating iron metabolism in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, thereby modulating the iron metabolism in the subject. Embodiment 42. A method of treating, inducing, regulating, or modulating iron metabolism or a disease or condition associated with iron metabolism in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, or modulating the iron metabolism or the disease or condition associated with the iron metabolism in the subject. Embodiment 43. A method of modulating cholesterol metabolism in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby modulating the cholesterol metabolism in the subject. Embodiment 44. A method of modulating cholesterol metabolism in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, thereby modulating the cholesterol metabolism in the subject. Embodiment 45. A method for treating, inducing, regulating, modulating, or reducing cholesterol metabolism or a disease associated with cholesterol metabolism in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, or reducing the cholesterol metabolism or the disease or condition associated with the cholesterol metabolism in the subject. Embodiment 46. A method for treating, inducing, regulating, modulating, reducing, or increasing reactive oxygen species or a disease or condition associated with reactive oxygen species in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, reducing, or increasing the reactive oxygen species or the disease or condition associated with the reactive oxygen species in the subject. Embodiment 47. A method for increasing reactive oxygen species in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby increasing the reactive oxygen species in the subject. Embodiment 48. A method for promoting at least one of treating, surviving from, modulating, or reducing acute stress, chronic stress response, immune hypersensitivity, cachexia, kidney disease, neurodegeneration, cardiotoxicity, or cardiotoxicity at least partially caused by chemotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, reducing, or increasing the acute stress, chronic stress response, immune hypersensitivity, cachexia, kidney disease, neurodegeneration, cardiotoxicity, or cardiotoxicity at least partially caused by chemotherapy in the subject. Embodiment 49. A method for promoting at least one of treating kidney disease in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating the kidney disease in the subject. Embodiment 50. A method for promoting at least one of treating kidney disease in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, thereby treating the kidney disease in the subject. Embodiment 51. The method according to any one of Embodiments 40 to 50, wherein the method comprises reducing the epididymal white adipose tissue of the subject. Embodiment 52. The method according to Embodiment 51, wherein the subject is overweight or obese. Embodiment 53. The method according to Embodiment 52, wherein the subject is obese and has a BMI of 30.0 or higher. Embodiment 54. The method according to Embodiment 52, wherein the subject is overweight and has a BMI of 25.0 to 30. Embodiment 55. The method according to any one of Embodiments 40 to 54, wherein an additional agent or treatment is administered to the subject, either concomitantly or sequentially. Embodiment 56. The method according to Embodiment 55, wherein the additional agent or treatment is selected from the group consisting of a GLP-1 agonist, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, phentermine-topiramate or a salt thereof, selenium, exercise therapy, surgery, dietary modification, and any combination thereof. Embodiment 57. The method according to Embodiment 56, wherein the additional agent or treatment comprises administering the GLP-1 agonist comprising dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, or a salt of any of these. Embodiment 58. The method according to Embodiment 56, wherein the additional agent or treatment comprises administering the gastric inhibitory polypeptide comprising tildepazide or a derivative thereof. Embodiment 59. The method according to Embodiment 56, wherein the additional agent or treatment comprises surgery comprising a gastric sleeve, gastric bypass, adjustable gastric band, a ball placed in the stomach, or any combination thereof. Embodiment 60. The method according to any one of Embodiments 40 to 59, wherein the subject is not diagnosed with cancer. Embodiment 61. The method according to any one of Embodiments 40 to 59, wherein the subject is a subject in need thereof. Embodiment 62. The method according to any one of Embodiments 40 to 59, wherein the subject has type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome. Embodiment 63. The method according to any one of Embodiments 40 to 59, wherein the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. Embodiment 64. The method according to Embodiment 63, wherein the pharmaceutical composition is in unit dosage form. Embodiment 65. The method according to any of the preceding embodiments, wherein the agent is a compound or a salt thereof in Table 1. The method according to any one of the preceding embodiments, wherein the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent. The method according to embodiment 46, wherein the pharmaceutical composition is in unit dosage form. Embodiment 68. A composition comprising: i) a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, or an agent that inhibits GCL; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof. The composition according to embodiment 68, which is a pharmaceutical composition. The pharmaceutical composition according to embodiment 69, which is in unit dosage form. The composition according to any one of embodiments 68 to 70, for use in the treatment of a disease or condition. The composition according to any one of embodiments 68 to 70, for use in the treatment of a kidney disease or condition. The composition according to any one of embodiments 68 to 70, for use in the treatment of a symptom associated with a kidney disease or condition. The composition for use according to embodiment 71, wherein the disease or condition is weight loss, weight maintenance, a disease or condition associated with iron metabolism, or a disease or condition associated with high, low, or abnormal cholesterol. A composition for use for inducing weight loss, fat loss, or both in a subject, the inducer being present in an amount sufficient to induce weight loss, fat loss, or both. The composition for use according to embodiment 75, for inducing weight loss. The composition for use according to embodiment 75, for inducing fat loss. Embodiment 78. A composition for use according to Embodiment 75 for inducing weight loss and fat loss. Embodiment 79. A composition for use according to Embodiment 75, further comprising administering a priming agent to the subject prior to administering the ferroptosis inducer to the subject. Embodiment 80. The adipose tissue in the subject, after administering the ferroptosis inducer to the subject, has a reduced weight, volume, or both compared to the weight, volume, or both of the adipose tissue in the subject before the administering. A composition for use according to Embodiment 75. Embodiment 81. The adipose tissue in the subject, after administering the ferroptosis inducer to the subject, has a reduced volume compared to the volume of the adipose tissue before the administering when determined by a pinch clamp test, dual-energy x-ray absorptiometry (DEXA) scan, reduction in abdominal circumference measurement, computed axial tomography (CAT) test, or any combination thereof. A composition for use according to Embodiment 80. Embodiment 82. The adipose tissue in the subject, after administering the ferroptosis inducer to the subject, has a reduced weight compared to the weight of the adipose tissue before the administering. A composition for use according to Embodiment 80. Embodiment 83. The reduction in weight is determined by the fact that the total body weight of the subject is less compared to the total body weight of the subject before administering the ferroptosis inducer after administering the ferroptosis inducer. A composition for use according to Embodiment 82. Embodiment 84. The reduction in weight is determined by comparing the DEXA scan results of the subject obtained before and after administering the ferroptosis inducer to the subject. A composition for use according to Embodiment 82. Embodiment 85. A composition for use according to Embodiment 75, further comprising that the cell size, cell volume, or both of a plurality of cells in the adipose tissue of the subject decrease after the administration of the ferroptosis inducer to the subject as compared to the cell size, cell volume, or both of a plurality of cells in the adipose tissue of the subject before the administration. Embodiment 86. A composition for use according to Embodiment 75, wherein the subject is a human. Embodiment 87. A composition for use according to Embodiment 75, wherein the ferroptosis inducer is administered orally. Embodiment 88. A composition for use according to Embodiment 75, wherein the administration is carried out for about 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 10 days, 2 weeks, 15 days, 20 days, 3 weeks, 25 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, for the lifespan, or as needed. Embodiment 89. A composition for use according to Embodiment 87, wherein the ferroptosis inducer is administered in an amount in the range of about 0.1 ng to about 25,000 mg. Embodiment 90. A composition for use according to Embodiment 87, wherein the ferroptosis inducer is administered in an amount of about 1 ng, 10 ng, 100 ng, 1 microgram, 10 micrograms, 100 micrograms, 1 mg, 10 mg, 100 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 15 g, 20 g, or 25 g. Embodiment 91. A composition for use according to Embodiment 87, wherein the ferroptosis inducer is administered in an amount in the range of about 1 ng / kg to 1,000 mg / kg, where mg is the mg of the ferroptosis inducer and kg is the kg of the subject's body weight. Embodiment 92. A composition for use according to Embodiment 75, wherein the ferroptosis inducer is administered orally. Embodiment 93. The composition for use according to Embodiment 75, wherein the ferroptosis inducer is administered by one of the following routes: oral administration, subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, intracranial, intrathecal, transdermal, rectal, or directly into adipose tissue. Embodiment 94. The composition for use according to Embodiment 75, which treats at least one of obesity, metabolic syndrome, elevated blood glucose, diabetes, type 2 diabetes, type 3 diabetes, insulin resistance, hypertension, cardiovascular disease, coronary artery disease, cerebrovascular disease, stroke, rheumatic heart disease, arteriosclerosis, atherosclerotic arteriosclerosis, liver disease, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or any combination thereof. Embodiment 95. The composition for use according to Embodiment 75, wherein the subject is a human male. Embodiment 96. The composition for use according to Embodiment 75, wherein the subject is a human female. Embodiment 97. The composition for use according to Embodiment 75, wherein the subject is in the range of about 1 year to about 18 years old. Embodiment 98. The composition for use according to Embodiment 75, wherein the subject is in the range of about 18 years to about 120 years old. Embodiment 99. The composition for use according to Embodiment 75, wherein the subject is a subject in need thereof. Embodiment 100. The composition for use according to Embodiment 75, wherein the ferroptosis inducer is included in a pharmaceutical composition containing a pharmaceutically acceptable form, carrier, or diluent. Embodiment 101. The composition for use according to Embodiment 100, wherein the pharmaceutical composition is in unit dosage form. Embodiment 102. The composition for use according to Embodiment 75, further comprising administering a further therapeutic agent to the subject. Embodiment 103. The composition for use according to Embodiment 102, wherein the further therapeutic agent is administered in combination with the ferroptosis inducer. Embodiment 104. A composition for use according to Embodiment 102, wherein the further therapeutic agent is continuously administered together with the ferroptosis inducer. Embodiment 105. A composition for use according to Embodiment 75, wherein the ferroptosis inducer is directly administered to adipose tissue. Embodiment 106. A composition for use according to Embodiment 105, wherein the administration is continuous. Embodiment 107. A composition for use according to Embodiment 105, wherein the administration is for about half an hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week. Embodiment 108. A composition for use according to Embodiment 105, wherein the adipose tissue is heated to a temperature of about 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 degrees Celsius. Embodiment 109. A composition for use according to Embodiment 105, wherein the ferroptosis inducer is administered in the form of an aqueous solution. Embodiment 110. A composition for use according to Embodiment 105, wherein the concentration of the ferroptosis inducer in the aqueous solution is from about 0.1 nM to about 500 μM. Embodiment 111. A composition for use according to Embodiment 75, wherein the administration results in a reduction in the adipose cell size or the number of adipose cells of the subject, and the reduction is determined by measuring the adipose cell size or the number of adipose cells before and after the administration of a therapeutic amount of a ferroptosis modulator, using microscopic evaluation of a biopsy if necessary. Embodiment 112. A composition for use according to Embodiment 75, wherein the method further comprises reducing the epididymal white adipose tissue of the subject. Embodiment 113. A composition for use according to Embodiment 75, wherein the subject is overweight or obese. Composition for use according to embodiment 75, wherein the subject is obese and has a BMI of about 30.0 or higher. Composition for use according to embodiment 75, wherein the subject is overweight and has a BMI of about 25.0 to about 30. Composition for use according to embodiment 102, wherein the further therapeutic agent has glucagon-like peptide-1 (GLP-1) receptor agonist activity. Composition for use according to embodiment 116, wherein the further therapeutic agent comprises semaglutide, dulaglutide, liraglutide, exenatide, tirzepatide, or any combination thereof.

Examples

[0194] The following examples are described to more clearly illustrate to those skilled in the art the principles and practices of the embodiments disclosed herein and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise indicated, all parts and percentages are by weight.

[0195] (Example 1) Cell Lines and Culture Conditions Human cancer cell lines are cultured in Ham's F12 medium supplemented with 10% (volume / volume) fetal bovine serum (FBS), penicillin (100 U / mL), and streptomycin (100 μg / mL). Human cancer cells are cultured in RPMI medium supplemented with 10% FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL). The cells are grown in a humidified incubator at 37 °C with 5% carbon dioxide and split every 3 - 4 days using trypsin / EDTA solution.

[0196] Method for adding exogenous PUFA or MUFA: The exogenous fatty acid was dissolved in DMSO and added to the cell culture medium 24 hours after seeding the cells.

[0197] (Example 2) Determination of PUFA status Perform lipidomics using either gas chromatography-mass spectrometry (GC-MS) or direct injection mass spectrometry.

[0198] For GC-MS evaluation of the PUFA status of cells, membrane lipids are transesterified with 500 μL of methanolic HCl, 250 μL of n-hexane, and 500 μL of internal standard (0.8 mg of di-C17-phosphatidylcholine in 1 mL of methanol with 0.2% butylated hydroxytoluene as antioxidant). After cooling, 500 μL of n-hexane and 1 mL of distilled water are added. The upper hexane phase is evaporated with nitrogen. Fatty acid methyl esters (FAME) are taken up in 60 μL of n-hexane. A 1 μL aliquot is injected on-column into a Varian CP 3800 gas chromatograph (Varian, Darmstadt, Germany) equipped with an Omegawax™ 320 column (inner diameter 0.32 mm, length 30 m) (Supelco, Bellefonte, USA). The column temperature was 200 °C.

[0199] For direct infusion MS analysis, lipids are extracted using a two-step chloroform / methanol procedure. The sample is spiked with an internal lipid standard mixture containing cardiolipin 16:1 / 15:0 / 15:0 / 15:0 (CL), ceramide 18:1;2 / 17:0 (Cer), diacylglycerol 17:0 / 17:0 (DAG), hexosylceramide 18:1;2 / 12:0 (HexCer), lysophosphatidate 17:0 (LPA), lysophosphatidylcholine 12:0 (LPC), lysophosphatidylethanolamine 17:1 (LPE), lysophosphatidylglycerol 17:1 (LPG), lysophosphatidylinositol 17:1 (LPI), lysophosphatidylserine 17:1 (LPS), phosphatidate 17:0 / 17:0 (PA), phosphatidylcholine 17:0 / 17:0 (PC), phosphatidylethanolamine 17:0 / 17:0 (PE), phosphatidylglycerol 17:0 / 17:0 (PG), phosphatidylinositol 16:0 / 16:0 (PI), phosphatidylserine 17:0 / 17:0 (PS), cholesteryl ester 20:0 (CE), sphingomyelin 18:1;2 / 12:0;0 (SM), triacylglycerol 17:0 / 17:0 / 17:0 (TAG). After extraction, the organic phase is transferred to an injection plate and dried using a speed vacuum concentrator. The dried extract is resuspended in 7.5 mM ammonium acetate in chloroform / methanol / propanol (1:2:4, volume:volume:volume), and the second-step dried extract is resuspended in a 33% ethanol solution of methylamine in chloroform / methanol (0.003:5:1, volume:volume:volume). The sample is analyzed by direct infusion on a QExactive mass spectrometer (ThermoFisher Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). The sample is analyzed in a single acquisition in both positive and negative ion modes with a resolution of Rm / z = 200 = 280000 for MS and Rm / z = 200 = 17500 for tandem mass spectrometry (MS-MS) assays.MS-MS is triggered by an inclusion list encompassing the corresponding MS mass range scanned in 1 Da increments.

[0200] (Example 3) Cell line profiling using ferroptosis inducers with and without a rescue agent Cell viability assays are performed by seeding 1,000 cells per well (30 μL volume) in opaque white 384-well plates (Corning). After allowing the cells to adhere for 24 hours, they are exposed to the compounds for 72 hours. DMSO stock solutions of the compounds are added to the cells using a CyBio Well Vario liquid dispenser (Analytik Jena AG). The ATP levels of the cells are measured using CellTiter-Glo (Promega) as a surrogate measure of viability. Rescue assays are performed using rescue agents designated as anti-ferroptosis rescue agent (N) (N, 1.5 μM), anti-ferroptosis rescue agent (M) (M, 1 μM), anti-ferroptosis rescue agent (P) (P, 50 μM), selected from Table 2, and other ferroptosis inhibitors added to the cells upon addition to the assay plate.

[0201] Target knockdown using genetic reagents ± anti-ferroptosis rescue agent (N) For lentiviral shRNA production, 293-T cells are seeded in 6-well dishes in antibiotic-free medium (280,000 cells / well). The next day, the cells are transfected using FuGENE with the appropriate shRNA coding plasmid (450 ng), viral packaging plasmid (p-Delta8.9, 400 ng), and viral envelope plasmid (p-VSV-G, 45 ng). After 24 hours, the medium is removed and replaced with fresh medium. Collection of the viral supernatant three times per shRNA is performed over 36 hours and pooled. The combined supernatant is centrifuged, aliquoted, and stored at -80 °C until viral infection.

[0202] Lentiviral infection is performed by seeding cells for 12 hours and replacing the medium with medium supplemented with polybrene (8 μg / mL) and an aliquot of virus supernatant. The plates are incubated for 48 hours, the medium is replaced with medium containing 1.5 μg / mL puromycin, and incubated at 37 °C for 48 hours. Knockdown is evaluated by immunoblotting and RT-qPCR.

[0203] Target knockout using gene reagents ± anti-ferroptosis rescue agent (N) For the generation of a cell line with gene knockout, lentivirus is produced in Lenti-X 293T cells by overnight polyethyleneimine transfection with the target lentiviral plasmid as well as the packaging plasmids pCMV-dR8.2 dvpr and pCMV-VSV-G in DMEM supplemented with 10% FBS. The next day, the medium is replaced with fresh DMEM with 10% FBS. After 24 hours and 48 hours, the virus-containing medium is collected, filtered through a 0.45 μm polyethersulfone filter, combined, and stored at -80 °C until virus infection.

[0204] Cells are transduced with pLenti-CRISPR-V2 encoding the appropriate sgRNA for the target gene using 2 μg / mL polybrene, followed by puromycin selection (1 μg / mL) for 4 days in the presence of ferrostatin-1 (1 μM). Protein knockout is verified by immunoblotting.

[0205] (Example 4) Use of C11-BODIPY to show lipid peroxidation as an indicator of ferroptosis Imaging assay: Human cancer cells are seeded in a CellCarrier Ultra 96-well plate (Perkin-Elmer) at 5,000 cells per well in 150 μl of RPMI medium with 10% FBS. The cells are incubated at 37 °C for 24 hours and then treated with the indicated compound or DMSO (90 minutes, 37 °C). During the last 30 minutes of incubation, 60 nM of DRAQ7 (Abcam), 1 μg ml -1 of Hoechst 33342 (ThermoFisher), and 1 μM of BODIPY 581 / 591 C11 (ThermoFisher) dyes are added. The cells are imaged using an Opera Phenix High-Content Screening System (Perkin-Elmer) equipped with lasers at 405, 488, 560, and 647 nm. Image analysis is performed using Harmony High-Content Imaging and Analysis software (Perkin-Elmer).

[0206] Use of C11-BODIPY to indicate lipid peroxidation (flow cytometry assay) Human cancer cells are seeded in a 96-well plate at 15,000 cells per well in RPMI medium with 10% FBS. After 48 hours, the culture medium is replaced with 200 μl of medium containing either DMSO or the indicated inhibitor (10 μM) and 1 μM of an anti-ferroptosis rescue agent (if indicated). The cultures are incubated at 37 °C for 2 hours. Thirty minutes before the end of the incubation period, 10 μM of BODIPY 581 / 591 C11 (Molecular Probes number C10445) is added to the cells. The cells are collected in 200 μl of PBS + 0.1% BSA and subjected to flow cytometry analysis (BD FACSCanto II).

[0207] (Example 5) Microadministration of a ferroptosis inducer to tumors For allogeneic transplantation studies, tumor cells derived from cancer cell lines were injected into the flanks of male C57BL6 / J mice. Assays were initiated when the tumor diameter reached approximately 6 - 7 mm.

[0208] Microdose drug delivery was performed for the assays described herein. The compounds of Table 3 were loaded into the reservoir of the device using a tapered metal needle. For the initial release of anti-ferroptosis rescue agent (M) (if included), followed by the release of the ferroptosis inducer with a 4 - 6 hour delay, it was loaded into the reservoir. The device was prepared for dosing the mouse tumor. The ferroptosis inducer was delivered to the tissue by the device for 24 - 72 hours. The tumor was then excised and the tissue was snap frozen in liquid nitrogen. The tissue was sectioned using a standard cryotome and 20 μm thick tissue sections were collected from each reservoir for analysis by immunoassay, transcriptomics, and metabolomics assays.

Table 3 - 1

Table 3 - 2

[0209] (Example 6) System for in vivo ferroptosis inducer delivery To establish the pharmacokinetics of the drug, the ferroptosis inducer and / or priming agent is administered systemically to the mammal by injection. Representative drugs to be tested include ferroptosis inducer (A), ferroptosis inducer (C), ferroptosis inducer (B), and anti-ferroptosis rescue agent (M). Representative animal models that can be used include, for example, those having tumors in a flammable membrane state.

[0210] Load a drug delivery system having microwells with approximately 1.5 micrograms of a ferroptosis inducer (crystalline powder) per microwell. Load the same drug into the system based on the results of a systemic test. Each drug is loaded separately and at concentrations greater than one, as well as in combination. After 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours, remove the device and perform a histological examination of the tissue to determine the effect of the ferroptosis inducer on the tumor cells in the immediate vicinity of each well. Evaluate the effect of the compound eluted from the microwells by different techniques. Assay the tissue excised with the device by standard histopathological techniques including immunohistochemistry and immunofluorescence. Visualize the ingrowth of tissue in the range of 20 to approximately 300 microns by staining tissue / device sections by standard immunohistochemical (IHC) techniques including hematoxylin and eosin (H&E) staining or any nuclear cell staining, e.g., DAPI. Use mass spectrometry to measure local biomarkers (e.g., mesenchymal state markers or PUFA concentration) indicative of the effect of the ferroptosis inducer. Perform analyses related to apoptosis, necrosis, mitotic cell death, and proliferation. Determine the local microdose response and use this to define an appropriate treatment regimen for cancer.

[0211] Develop several methods for controlling the release / diffusion of the ferroptosis inducer into the tissue, including the exact spatial arrangement of the microwells along the device mantle, the geometry and size of the microwells, and the formulation of the drug released. The microwells of the device from which the ferroptosis inducer diffuses are engineered to expose only the area of the tissue directly adjacent to the opening of the microwell to the drug being released. This creates distinct local regions in the tissue, and the effect of the compound is evaluated without interference from other compounds released from different microwells. The creation of distinct drug regions is useful for evaluating the effectiveness of different drugs or combinations thereof, and / or dosages and / or release times (continuous, pulsed, delayed, continuous following a bolus, etc.).

[0212] The agent is released upward and diffused into a larger area, or released downward into a relatively small area of the target tissue. By accurately controlling the transport time as a function of the distance from the microwell, the local concentration of the first agent can be obtained as a function of the distance from the microwell at multiple time points after in vivo implantation.

[0213] The concentration gradient region is defined as the distance from the microwell, and as it increases, the concentration of the administered agent decreases. As a function of the distance from the microwell, the percentage area of cleaved caspase 3 positive cells as 3,3'-diaminobenzidine (DAB) staining is one example of a functional readout from the implantable drug delivery system. The drug concentration gradient is formed at approximately 100 - 250 μm from the microwell, with the highest tissue concentration in the region closest to the microwell.

[0214] The system is used to deliver a microdose of a ferroptosis inducer to tissue in vivo. The system is also used to deliver a priming agent (e.g., an anti-ferroptosis rescue agent (M)), followed by a ferroptosis inducer (e.g., a ferroptosis inducer (C)) to tissue in vivo to induce targeted cell death in the tissue. The system is also capable of achieving a drug of at least about 10 ng / mm 2 directly implanted into tumors with a diameter of approximately 6 millimeters (mm) to about 7 mm for at least 4 hours at the site of the microwell.

[0215] (Example 7) Interference with ferroptosis regulation affects lipid accumulation Specific ferroptosis-modulating knockouts that result in ferroptosis were performed in mice. As shown in Figure 2, there was a significant decrease in fat accumulation in the mutant mice compared to wild-type mice. Also, there was a significant decrease in the amount of the epididymal / gonadal white adipose tissue in wild-type mice compared to knockout mice. This decrease in adipose tissue can also be confirmed by pharmacological modulation. The perturbation is safe and tolerable. The partial loss of function results in a lean phenotype when mice are fed a high-fat diet (HFD). Constitutive pathway activation is known to cause fatty liver and hepatomegaly in mice.

[0216] (Example 8) Ferroptosis agent treatment reproduces the weight loss phenotype Male C57Bl / 6 mice (5 - 6 weeks old) were treated with a ferroptosis agent (at 5 mg / mL) to demonstrate weight loss with a glutamate-cysteine ligase inhibitor. Glutathione (GSH) is synthesized by the sequential action of rate-limiting glutamate-cysteine ligase (GCL) and glutathione synthetase. Figures 4A - 4C show that mice treated with the ferroptosis agent had a decrease in weight change (percentage), as well as a decrease in epididymal white adipose tissue (eWAT) weight (g) and eWAT weight (g / g) relative to body weight. Additionally, Figure 4B shows the water intake between the control and the ferroptosis agent-treated group, and Figure 4D shows the decrease in liver GSH levels in mice treated with the ferroptosis agent compared to the control group that had water only. This data shows that inhibition of glutamate-cysteine ligase can result in weight loss.

[0217] Similarly, Figure 5 shows histological samples of male C57Bl / 6 mice (4 - 5 weeks old) treated with 5 mg / mL of the ferroptosis agent for 7 days and control mice treated with water. In the treated samples, a decrease in fat accumulation was observed in the histological samples.

[0218] (Example 9) Ferroptosis agent treatment in high-fat diet (HFD) mice Male C57Bl / 6 mice and diet-induced obesity (DIO) mice were treated with a 5 mg / mL ferroptosis agent to demonstrate weight loss on a high-fat diet. The mice were 8-week-old male mice. The mice were acclimated to the facility for 1 week. Ferroptosis agent treatment was started 2 days prior to HFD diet supplementation. Figures 6A - 6C show that mice treated with the ferroptosis agent (both C57Bl / 6 and DIO mice) had a decrease in weight change (percentage) and a decrease in epididymal white adipose tissue (eWAT) weight (g). Additionally, Figures 6D - 6E show that triglyceride levels decreased in DIO-treated mice and LDL cholesterol levels increased in C57Bl / 6 mice treated with the ferroptosis agent. This data indicates that there was no significant weight gain on the HFD diet in mice when treated with the ferroptosis agent.

Claims

1. A method for inducing weight loss, fat loss, or both in a subject, the method comprising administering to the subject a ferroptosis inducer in an amount sufficient to induce the weight loss, the fat loss, or both.

2. The method according to claim 1, which is a method for inducing weight loss.

3. The method according to claim 1, which is a method for inducing fat loss.

4. The method according to claim 1, which is a method for inducing weight loss and fat loss.

5. The method according to claim 1, further comprising administering to the subject a priming agent prior to said administering of the ferroptosis inducer to the subject.

6. The method according to claim 1, wherein the adipose tissue in the subject has a decrease in weight, volume, or both, compared to the weight, volume, or both of the adipose tissue in the subject prior to said administering, after said administering of the ferroptosis inducer to the subject.

7. The method according to claim 6, wherein the adipose tissue in the subject has a decrease in volume, compared to the volume of the adipose tissue prior to said administering, when determined by a pinch clamp test, a dual-energy x-ray absorptiometry (DXA) scan, a decrease in abdominal circumference measurement, a computed axial tomography (CAT) test, or any combination thereof, after said administering of the ferroptosis inducer to the subject.

8. The method according to claim 6, wherein the adipose tissue in the subject has a decrease in weight, compared to the weight of the adipose tissue prior to said administering, after said administering of the ferroptosis inducer to the subject.

9. The method according to claim 8, wherein the decrease in weight is determined by the subject having a lower total body weight compared to the total body weight of the subject prior to said administering of the ferroptosis inducer, after said administering of the ferroptosis inducer.

10. The method according to claim 8, wherein the decrease in weight is determined by comparing DXA scan results of the subject obtained before and after said administering of the ferroptosis inducer to the subject.

11. The method according to claim 1, further comprising that the cell size, cell volume, or both of a plurality of cells in the adipose tissue of the subject decrease after the administration of the ferroptosis inducer to the subject, as compared to the cell size, cell volume, or both of a plurality of cells in the adipose tissue of the subject before the administration.

12. The method according to claim 1, wherein the subject is a human.

13. The method according to claim 1, wherein the ferroptosis inducer is administered orally.

14. The method according to claim 1, wherein the administration is carried out for about 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 10 days, 2 weeks, 15 days, 20 days, 3 weeks, 25 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, for the lifespan, or as needed.

15. The method according to claim 14, wherein the ferroptosis inducer is administered in an amount in the range of about 0.1 ng to about 25,000 mg.

16. The method according to claim 14, wherein the ferroptosis inducer is administered in an amount of about 1 ng, 10 ng, 100 ng, 1 microgram, 10 micrograms, 100 micrograms, 1 mg, 10 mg, 100 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 15 g, 20 g, or 25 g.

17. The method according to claim 14, wherein the ferroptosis inducer is administered in an amount in the range of about 1 ng / kg to 1,000 mg / kg, where mg is the mg of the ferroptosis inducer and kg is the kg of the subject's body weight.

18. The method according to claim 1, wherein the ferroptosis inducer is administered orally.

19. The method according to claim 1, wherein the ferroptosis inducer is administered by one of the following routes: oral administration, subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, intracranial, intrathecal, transdermal, rectal, or directly into adipose tissue.

20. The method according to claim 1 for treating at least one of obesity, metabolic syndrome, elevated blood glucose, diabetes, type 2 diabetes, type 3 diabetes, insulin resistance, hypertension, cardiovascular disease, coronary artery disease, cerebrovascular disease, stroke, rheumatic heart disease, arteriosclerosis, atherosclerotic arteriosclerosis, liver disease, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or any combination thereof.

21. The method according to claim 1, wherein the subject is a human male.

22. The method according to claim 1, wherein the subject is a human female.

23. The method according to claim 1, wherein the subject is in the range of about 1 year to about 18 years old.

24. The method according to claim 1, wherein the subject is in the range of about 18 years to about 120 years old.

25. The method according to claim 1, wherein the subject is a subject in need thereof.

26. The method according to claim 1, wherein the ferroptosis inducer is included in a pharmaceutical composition comprising a pharmaceutically acceptable form, carrier, or diluent.

27. The method according to claim 26, wherein the pharmaceutical composition is in unit dosage form.

28. The method according to claim 1, further comprising administering a further therapeutic agent to the subject.

29. The method according to claim 28, wherein the further therapeutic agent is administered in combination with the ferroptosis inducer.

30. The method according to claim 28, wherein the further therapeutic agent is continuously administered together with the ferroptosis inducer.

31. The method according to claim 1, wherein the ferroptosis inducer is administered directly to adipose tissue.

32. The method according to claim 31, wherein the administering is continuous.

33. The method according to claim 32, wherein the administering is for about one-half hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week.

34. The method according to claim 31, wherein the adipose tissue is heated to a temperature of about 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 degrees Celsius.

35. The method according to claim 31, wherein the ferroptosis inducer is administered in the form of an aqueous solution.

36. The method according to claim 35, wherein the concentration of the ferroptosis inducer in the aqueous solution is from about 0.1 nM to about 500 μM.

37. The method according to claim 1, wherein the administration results in a reduction in the size or number of adipocytes of the subject, and the reduction is determined by measuring the size or number of adipocytes before and after administration of a therapeutically effective amount of the ferroptosis modulating agent, optionally using microscopic evaluation of a biopsy.

38. The method according to claim 1, further comprising reducing the epididymal white adipose tissue of the subject.

39. The method according to claim 1, wherein the subject is overweight or obese.

40. The method according to claim 1, wherein the subject is obese and has a BMI of about 30.0 or higher.

41. The method according to claim 1, wherein the subject is overweight and has a BMI of about 25.0 to about 30.

42. The method according to claim 28, wherein the further therapeutic agent has glucagon-like peptide-1 (GLP-1) receptor agonist activity.

43. The method according to claim 42, wherein the further therapeutic agent comprises semaglutide, dulaglutide, liraglutide, exenatide, tizepatide, or any combination thereof.

44. A method of inducing, regulating, or modulating weight loss, body composition, or fat loss or reduction in a subject, comprising administering to the subject a therapeutically effective amount of a ferroptosis modulating agent, a glutamate-cysteine ligase (GCL) modulating agent, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby inducing, regulating, or modulating the weight loss, body composition, or fat loss or reduction in the subject.

45. The method according to claim 44, wherein the subject is human.

46. The method according to claim 45, wherein the subject is male.

47. The method according to claim 44, wherein the ferroptosis regulator, the glutamate-cysteine ligase (GCL) regulator, the agent that binds to GCL, the agent that inhibits GCL, or any combination thereof is orally delivered as a solution having a concentration of at least about 5 mg of the agent per mL of solution.

48. The method according to claim 44, wherein the administration is discontinuous and the administration is performed daily for about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year.

49. The ferroptosis inducer is selected from the group consisting of (1S,3R)-RSL3, ML-162, ML-210, JKE-1674, JKE-1716, elastin, jacareic acid, trigonelline, glutamate, sulfasalazine, auranofin, bursatol, sorafenib, sorafenib-d3, sorafenib tosylate, trigonelline, FIN56, FINO ２ , CIL56, dihydroisotanshinone I, GPX4-IN-3, sulfoximine, BSO, and salts of any of these, the method according to claim 1.

50. The method according to claim 1, wherein the administration results in a reduction in the size of the adipocytes of the subject, and the reduction is determined by measuring the size of the adipocytes before and after the administration of the ferroptosis inducer, optionally using microscopic evaluation of a biopsy.

51. The method according to claim 1, wherein the administration results in a reduction in the number of adipocytes of the subject, and the reduction is determined by measuring the number of adipocytes before and after the administration of a therapeutic amount of the ferroptosis regulator, optionally using microscopic evaluation of a biopsy.

52. The method according to claim 50 or claim 51, comprising reducing the epididymal white adipose tissue of the subject.

53. The method according to claim 52, wherein the subject is overweight or obese.

54. The method according to claim 53, wherein the subject is obese and has a BMI of about 30.0 or higher.

55. The method according to claim 53, wherein the subject is overweight and has a BMI of about 25.0 to about 30.

56. The method according to any of the preceding claims, wherein an additional agent or treatment is administered to the subject, either concomitantly or sequentially.

57. The method according to claim 56, wherein the additional agent or treatment is selected from the group consisting of GLP-1 agonists, gastric inhibitory polypeptide analogs, naltrexone-bupropion or salts thereof, orlistat or salts thereof, phentermine or salts thereof, phentermine-topiramate or salts thereof, selenium, exercise therapy, surgery, dietary modification, and any combination thereof.

58. The method according to claim 57, wherein the additional agent or treatment comprises administering a GLP-1 agonist comprising dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, or a salt of any of these.

59. The method according to claim 57, wherein the additional agent or treatment comprises administering the gastric inhibitory polypeptide comprising tildatide or a derivative thereof.

60. The method according to claim 57, wherein the additional agent or treatment comprises a surgical procedure comprising a gastric sleeve, gastric bypass, adjustable gastric band, a ball placed in the stomach, or any combination thereof.

61. The method according to any of the preceding claims, wherein the subject is not diagnosed with cancer.

62. The method according to any of the preceding claims, wherein the subject is a subject in need thereof.

63. The method according to any of the preceding claims, wherein the subject has type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome.

64. The method according to claim 44, wherein the ferroptosis regulator, the glutamate-cysteine ligase (GCL) regulator, the agent that binds to GCL, the agent that inhibits GCL, or any combination thereof is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

65. The method according to claim 64, wherein the pharmaceutical composition is in unit dosage form.

66. A composition comprising: i) a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, or an agent that inhibits GCL; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof.

67. A composition comprising: i) a glutamate-cysteine ligase (GCL) regulator; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof.

68. The composition according to claim 66 or claim 67, which is a pharmaceutical composition.

69. The pharmaceutical composition according to claim 68, which is in unit dosage form.

70. The composition according to any one of claims 66 to 69, for use in the treatment of a disease or condition.

71. The composition according to any one of claims 66 to 69, for use in the treatment of a kidney disease or condition.

72. The composition according to any one of claims 66 to 69, for use in the treatment of symptoms associated with a kidney disease or condition.

73. The composition for use according to claim 70, wherein the disease or condition is weight loss, weight maintenance, a disease or condition associated with iron metabolism, or a disease or condition associated with high, low, or abnormal cholesterol.

74. A method of modulating iron metabolism in a plurality of cells in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, a glutamate-cysteine ligase (GCL) modulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby modulating the iron metabolism in the subject and thereby inducing ferroptosis in the subject.

75. The method according to claim 74, wherein immediately after said modulating, the majority of the cells of the plurality of cells remain viable.

76. A method of modulating iron metabolism in a plurality of cells in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, thereby modulating the iron metabolism in the subject.

77. The method according to claim 76, wherein immediately after said modulating, the majority of the cells of the plurality of cells remain viable.

78. A method of treating, inducing, regulating, or modulating iron metabolism or a disease or condition associated with iron metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, or modulating the iron metabolism or the disease or condition associated with the iron metabolism in the subject.

79. The method according to claim 78, wherein the subject is a human.

80. The method according to claim 78, wherein the subject is a subject in need thereof.

81. The method according to claim 78, wherein a therapeutically effective amount of the ferroptosis regulator is added.

82. A method of modulating cholesterol metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby modulating cholesterol metabolism in the subject.

83. The method according to claim 82, wherein the subject is a human.

84. The method according to claim 82, wherein the subject is a subject in need thereof.

85. The method according to claim 84, wherein a ferroptosis inducer is administered.

86. A method of modulating cholesterol metabolism in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, thereby modulating the cholesterol metabolism in the subject.

87. The method according to claim 86, wherein the subject is a human.

88. The method according to claim 86, wherein the subject is a subject in need thereof.

89. The method according to claim 86, wherein a ferroptosis inducer is administered.

90. A method for treating, inducing, regulating, modulating, or reducing cholesterol metabolism or a disease or condition associated with cholesterol metabolism in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of an autophagy regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, or reducing the cholesterol metabolism or the disease or condition associated with the cholesterol metabolism in the subject.

91. The method according to claim 90, wherein the subject is a human.

92. The method according to claim 90, wherein the subject is a subject in need thereof.

93. The method according to claim 90, wherein the autophagy regulator is administered.

94. A method for treating, inducing, regulating, modulating, reducing, or increasing reactive oxygen species or a disease or condition associated with reactive oxygen species in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of an autophagy regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, reducing, or increasing the reactive oxygen species or the disease or condition associated with the reactive oxygen species in the subject.

95. The method according to claim 94, wherein the subject is a human.

96. The method according to claim 94, wherein the subject is a subject in need thereof.

97. The method according to claim 94, wherein the autophagy regulator is administered.

98. A method for increasing reactive oxygen species in a subject, comprising administering to the subject a therapeutically effective amount or an effective amount of an autophagy regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby increasing the reactive oxygen species in the subject.

99. The method according to claim 98, wherein the subject is a human.

100. The method according to claim 98, wherein the subject is a subject in need thereof.

101. The method according to claim 98, wherein the ferroptosis regulator is administered.

102. A method of promoting at least one of treating, surviving from, modulating, or reducing acute stress, chronic stress response, immune hypersensitivity, cachexia, kidney disease, neurodegeneration, cardiotoxicity, or cardiotoxicity caused at least in part by chemotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating, inducing, regulating, modulating, reducing, or increasing the acute stress, chronic stress response, immune hypersensitivity, cachexia, kidney disease, neurodegeneration, cardiotoxicity, or cardiotoxicity caused at least in part by chemotherapy in the subject.

103. The method according to claim 102, wherein the subject is a human.

104. The method according to claim 102, wherein the subject is a subject in need thereof.

105. The method according to claim 102, wherein the ferroptosis regulator is administered.

106. A method of promoting at least one of treating kidney disease in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, an agent that inhibits GCL, or any combination thereof, thereby treating the kidney disease in the subject.

107. The method according to claim 106, wherein the subject is a human.

108. The method according to claim 106, wherein the subject is a subject in need thereof.

109. The method according to claim 106, wherein the ferroptosis regulator is administered.

110. A method of promoting at least one of treating kidney disease in a subject, the method comprising administering to the subject a therapeutically effective amount or an effective amount of a ferroptosis inducer, thereby treating the kidney disease in the subject.

111. The method according to claim 110, wherein the subject is a human.

112. The method according to claim 110, wherein the subject is a subject in need thereof.

113. The method according to claim 110, wherein the ferroptosis inducer is administered.

114. The method according to any one of claims 74 to 110, comprising reducing the epididymal white adipose tissue of the subject.

115. The method according to claim 114, wherein the subject is overweight or obese.

116. The method according to claim 115, wherein the subject is obese and has a BMI of 30.0 or higher.

117. The method according to claim 115, wherein the subject is overweight and has a BMI of 25.0 to <30.

118. The method according to any one of claims 74 to 117, wherein an additional agent or treatment is administered to the subject either concomitantly or sequentially.

119. The method according to claim 118, wherein the additional agent or treatment is selected from the group consisting of GLP-1 receptor agonists, gastric inhibitory polypeptide analogs, naltrexone-bupropion or salts thereof, orlistat or salts thereof, phentermine or salts thereof, phentermine-topiramate or salts thereof, selenium, exercise therapy, surgery, dietary modification, and any combination thereof.

120. The method according to claim 119, wherein the additional agent or treatment comprises administering the GLP-1 receptor agonist comprising dulaglutide, exenatide, semaglutide, liraglutide, lixisenatide, or salts of any of these.

121. The method according to claim 119, wherein the additional agent or treatment comprises administering the gastric inhibitory polypeptide comprising tildepagliflozin or derivatives thereof.

122. The method according to claim 119, wherein the additional agent or treatment comprises surgery including gastric sleeve, gastric bypass, adjustable gastric band, a ball placed in the stomach, or any combination thereof.

123. The method according to any one of claims 74 to 122, wherein the subject has not been diagnosed with cancer.

124. The method according to any one of claims 74 to 122, wherein the subject is a subject in need thereof.

125. The method according to any one of claims 74 to 122, wherein the subject has type I diabetes, type II diabetes, type IIIa diabetes, or metabolic syndrome.

126. The method according to any one of claims 74 to 122, wherein the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

127. The method according to claim 126, wherein the pharmaceutical composition is in unit dosage form.

128. The method according to any of the preceding claims, wherein the agent is a compound in Table 1 or a salt thereof.

129. The method according to any of the preceding claims, wherein the agent is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

130. The method according to claim 46, wherein the pharmaceutical composition is in unit dosage form.

131. i) a ferroptosis regulator, a glutamate-cysteine ligase (GCL) regulator, an agent that binds to GCL, or an agent that inhibits GCL; and ii) a GLP-1 agonist or a salt thereof, a gastric inhibitory polypeptide analog, naltrexone-bupropion or a salt thereof, orlistat or a salt thereof, phentermine or a salt thereof, and phentermine-topiramate or a salt thereof, or selenium or a salt thereof.

132. The composition according to claim 131, which is a pharmaceutical composition.

133. The pharmaceutical composition according to claim 132, which is in unit dosage form.

134. The composition according to any one of claims 131 to 133, for use in the treatment of a disease or condition.

135. The composition according to any one of claims 131 to 133, for use in the treatment of a kidney disease or condition.

136. The composition according to any one of claims 131 to 133, for use in the treatment of a symptom associated with a kidney disease or condition.

137. The composition for use according to claim 134, wherein the disease or condition is weight loss, weight maintenance, a disease or condition associated with iron metabolism, or a disease or condition associated with high, low, or abnormal cholesterol.

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