WO2019191030A1 - Procédés de détermination du dosage d'un agent thérapeutique en se basant sur les niveaux mesurés d'un métabolite - Google Patents

Procédés de détermination du dosage d'un agent thérapeutique en se basant sur les niveaux mesurés d'un métabolite Download PDF

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Publication number
WO2019191030A1
WO2019191030A1 PCT/US2019/023983 US2019023983W WO2019191030A1 WO 2019191030 A1 WO2019191030 A1 WO 2019191030A1 US 2019023983 W US2019023983 W US 2019023983W WO 2019191030 A1 WO2019191030 A1 WO 2019191030A1
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WIPO (PCT)
Prior art keywords
brequinar
subject
agent
dose
level
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PCT/US2019/023983
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English (en)
Inventor
Vikram S. Kumar
David P. Hesson
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Clear Creek Bio, Inc.
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Publication date
Application filed by Clear Creek Bio, Inc. filed Critical Clear Creek Bio, Inc.
Priority to EP19776042.4A priority Critical patent/EP3773523A4/fr
Priority to AU2019242626A priority patent/AU2019242626A1/en
Priority to CA3094104A priority patent/CA3094104A1/fr
Priority to JP2020552843A priority patent/JP2021519338A/ja
Publication of WO2019191030A1 publication Critical patent/WO2019191030A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests

Definitions

  • the invention relates generally to methods of determining dosing of a therapeutic agent based on measured levels of a metabolite in a pathway that is targeted by the therapeutic agent.
  • Dosing is often based on pharmacokinetic studies that examine the rate of metabolism and elimination of the drug from the body and yield data that represent the average rate of drug metabolism from a population of patients.
  • individual patients differ vastly in how they metabolize certain drugs, and those differences may be critical for drugs that have a narrow therapeutic window, i.e., a restricted range between the minimum dosage at which a drug is effective and the dosage at which it is toxic.
  • administration of the drug must be accompanied by ongoing measurement, i.e., monitoring, of one or more physiological parameters in the patient's body and adjustment of the drug dosage based on the measured values.
  • a complicating problem is that current methods of monitoring are flawed.
  • the physiological parameters that are measured in the patient's body such as the level of the API or a metabolite of the API, do not necessarily reflect whether a particular API has engaged its intended target. Because the kinetics of functional interaction between the API and its target often differ from the measured parameters, dosing schedules based on those measurements may lead to excessive side effects or fail to control the primary condition. Consequently, current methods for obtaining information on drug efficacy in real time and using that information to provide patient-tailored drug dosing are inadequate, and millions of individuals continue to suffer from improperly medicated ailments or unnecessary drug side effects.
  • the invention provides methods and devices that allow physicians to determine, optionally in real time, a therapeutically effective dose of a drug for an individual patient by examining levels of a metabolite in a pathway targeted by the drug.
  • the effectiveness of a drug containing an enzyme inhibitor is assessed by analyzing the level of the enzyme's substrate in a sample obtained from the patient. Because activity of the enzyme can be inferred from substrate levels, engagement of the API with its target can be evaluated, optionally in real time, and drug dosage can be adjusted accordingly.
  • the methods of the invention yield dosing schedules that afford better control of a variety of diseases, disorders, and conditions and decrease the risk harmful drug side effects.
  • the invention also provides devices that notify patients, in real time, of recommended adjustments to their dosing regimens based on measured levels of a metabolite in the pathway targeted by the drug.
  • the methods permit real-time adjustment of drug dosage to optimize therapeutic effectiveness, they are useful for treatment of various diseases, such as cancer.
  • control of dihydroorotate dehydrogenase (DHODH) in acute myeloid leukemia (AML) could selectively starve leukemia cells, so the DHODH inhibitor brequinar has potential as an anti cancer agent.
  • DHODH dihydroorotate dehydrogenase
  • AML acute myeloid leukemia
  • brequinar has potential as an anti cancer agent.
  • achieving a therapeutically effective dosing regimen of brequinar is problematic: when the drug is administered frequently, e.g., daily, it causes toxic side effects, and when it is administered too infrequently, e.g., biweekly or on a schedule that requires extended "off" periods between doses, it has no therapeutic benefit.
  • Methods of the invention solve this problem by monitoring levels of dihydroorotate (DHO), the substrate for DHODH, in the patient's body
  • the invention unlocks the therapeutic potential of brequinar and other drugs that have narrow therapeutic windows or high interindividual variability.
  • the invention also provides methods of evaluating the effectiveness of anti-cancer agents by assessing their effects on tumors, optionally in real time.
  • the methods involve analyzing properties of a tumor in the body, such as the flux or single point level of a nutrient, substrate, or metabolite in the tumor or the level of oxygenation of the tumor. By monitoring these properties in a patient who has been given a therapeutic agent, the impact of the drug on the tumor can be gauged, and the dosing regimen can be adjusted accordingly.
  • the invention provides methods for determining a therapeutically effective dose of an agent to treat a disorder in a subject.
  • the methods include receiving information regarding a measured level of a metabolite in a metabolic pathway in a sample from a subject having a disorder, comparing the received information to a reference that provides an association of a measured level of the metabolite with a recommended dosage adjustment of an agent, and determining, based on the comparing step, a dosage of the agent that results in the level of the metabolite being raised or maintained above a threshold level.
  • the threshold level is indicative that a sufficient amount of the agent is present in the subject to sufficiently alter the metabolic pathway to ameliorate, reduce, or eliminate at least one sign or symptom of the disorder.
  • the invention provides methods for determining a therapeutically effective dose of an agent to be provided to a subject to treat a disorder.
  • the methods include determining a therapeutically effective dose of an agent based on a measured level of a metabolite in a nucleotide synthesis pathway in a sample from a subject.
  • the therapeutically effective dose of the agent inhibits an enzyme within the nucleotide synthesis pathway to an extent that at least one sign or symptom of the disorder is ameliorated, reduced, or eliminated.
  • the recommend dosage adjustment may include a change in the dosage.
  • the recommend dosage adjustment may include an increase of the dosage by a certain value, a decrease of the dosage by a certain value, or no adjustment to the dosage.
  • the recommended dosage adjustment may include a change in the schedule of providing the dose.
  • the recommended dosage adjustment may include an increase in the interval between doses, a decrease in the interval between doses, or no change in the interval between doses.
  • the agent may be any therapeutic agent.
  • the agent may be PALA (N- phosphoacetyl-L-aspartate), brequinar, pyrazofurin, brequinar, a brequinar analog, a brequinar derivative, a brequinar prodrug, a micellar formulation of brequinar, or a brequinar salt.
  • the agent may inhibit an enzyme in the metabolic pathway.
  • the agent may inhibit aspartate transcarbamoylase, dihydrooratase, dihydroorotate dehydrogenase, orotidine 5'- monophosphate (OMP) decarboxylase, or orotate phosphoribosyl transferase.
  • the metabolite may be a substrate or product of an enzyme in the metabolic pathway targeted by the drug.
  • the metabolic pathway may be a nucleotide synthesis pathway, such as a pyrimidine synthesis pathway or a purine synthesis pathway.
  • the metabolite may be an intermediate in a nucleotide synthesis pathway.
  • the metabolite may be N- carbamoylaspartate, dihydroorotate, orotate, orotidine 5'-monophosphate (OMP), or uridine monophoshpate (UMP).
  • the disorder may be any disorder, disease, or condition for which altering the activity of a metabolic pathway can be of therapeutic benefit.
  • the disorder may be one in which inhibiting an enzyme in a metabolic pathway is of therapeutic benefit.
  • the disorder may be cancer or an autoimmune disorder.
  • the cancer may be leukemia, such as acute myeloid leukemia (AML), PTEN null prostate cancer, lung cancer, such as small cell lung cancer and non-small cell lung cancer, triple negative breast cancer (TNBC), glioma, multiple myeloma, acute lymphoblastic leukemia (ALL), neuroblastoma, or adult T cell leukemia/lymphoma (ATLL).
  • the autoimmune disorder may be arthritis or multiple sclerosis.
  • the methods may include additional steps.
  • the method may include measuring the level of the metabolite in a sample obtained from the subject or providing the agent to the subject at the determined dose.
  • the sample may be a body fluid sample.
  • the body fluid may be plasma, blood, serum, urine, sweat, saliva, interstitial fluid, feces, or phlegm
  • the invention provides methods for assessing the impact of a therapeutic agent on a tumor in real time.
  • the methods include monitoring in real time a molecule that is associated with a metabolic pathway as the molecule moves through the metabolic pathway in a tumor in a subject and assessing the impact on the tumor of a therapeutic agent that has been administered to a subject based on results of the monitoring.
  • the invention provides methods for assessing the impact of a therapeutic agent on tumor in real time. The methods include monitoring in real time an oxygenation level in a tumor and assessing the impact on the tumor of a therapeutic agent that has been administered to a subject based on results of the monitoring step.
  • the invention provides methods for assessing the impact of a therapeutic agent on tumor in real time.
  • the methods include monitoring in real time a molecule that is associated with a metabolic pathway as the molecule moves through the metabolic pathway in a tumor in a subject, monitoring in real time an oxygenation level in a tumor, and assessing the impact on the tumor of a therapeutic agent that has been administered to a subject based on results of the monitoring step.
  • the monitoring may include any suitable method.
  • monitoring the molecule in the tumor may include the use of hyperpolarization magnetic resonance imaging, and monitoring the oxygenation level of the tumor may include electron paramagnetic resonance (EPR) imaging.
  • EPR electron paramagnetic resonance
  • the molecule may be a carbon molecule.
  • the molecule may be or become associated with a metabolite in a metabolic pathway.
  • the metabolite may be N- carbamoylaspartate, dihydroorotate, orotate, orotidine 5'-monophosphate (OMP), or uridine monophoshpate (UMP).
  • the metabolic pathway may be any metabolic pathway, as described above.
  • the metabolic pathway may a nucleotide synthesis pathway.
  • the agent may be any therapeutic agent, as described above.
  • the methods may include quantifying the molecule. Quantifying the molecule may quantify the level of a metabolite in a metabolic pathway, such as dihydroorotate or orotate.
  • the methods may include determining, based on the levels of a metabolite, such as dihydroorotate or orotate, a dose of the therapeutic agent that is sufficient to inhibit an enzyme within the metabolic pathway, such as a nucleotide synthesis pathway, to an extent that at least one sign or symptom of the disorder is ameliorated, reduced, or eliminated.
  • a metabolite such as dihydroorotate or orotate
  • the methods may include repeating one or more of the monitoring, assessing, and determining steps at different points in time.
  • the methods may include adjusting the dose of the therapeutic agent based on results of the method from a subsequent point in time.
  • the invention provides devices for notifying a subject having a disorder that a dose of therapeutic agent that targets a metabolic pathway should be administered to the subject.
  • the devices include a processor coupled to a memory unit that causes the processor to receive data that includes a dose of the therapeutic agent and the time the dose was received by the subject, generate a reminder that includes the time the next dose should be administered to the subject, and output the reminder to the subject.
  • the time for administering the next dose to the subject is based on a relationship between the dose of the therapeutic agent and a threshold level of the metabolite, and administration of the next dose raises or maintains a level of the metabolite above the threshold.
  • the threshold level is indicative that a sufficient amount of the agent is present in the subject to sufficiently alter the metabolic pathway to ameliorate, reduce, or eliminate at least one sign or symptom of the disorder.
  • the reminder may be any type of notification that can be perceived by a human.
  • the reminder may be an audible signal, a visual signal, a tactile signal, a vibration, or a combination thereof.
  • the reminder may be outputted to a component of the device. Additionally, or alternatively, the reminder may be outputted to a remote device.
  • Each of the time when the dose was received by the subject and the time when the next dose should be administered may include any temporal component.
  • each of the times may include a date, day of the week, hour, minute, second, or time zone.
  • the device may store information related to the time when the dose was received by the subject, the time when the next dose should be administered, or both.
  • the information may be stored in the memory unit.
  • the process may perform calculations on the stored information. For example, the process may determine whether intervals between time points, such as times when individual doses are received by the subject, change over time.
  • the processor may determine that the subject has developed resistance or is developing resistance to a therapeutic agent based on the stored information. For example, the processor may determine that the subject has developed resistance or is developing resistance to a therapeutic agent based on a change in the intervals over time, such as a decrease in the intervals over time, a change in the dose over time, such as an increase in the dose over time, or both.
  • the processor may output a recommendation for adjusting a therapeutic course for the subject.
  • the recommendation may include altering, e.g., increasing or decreasing, a dose, or altering, e.g., increasing or decreasing, an interval between doses.
  • the recommendation may include administering a second therapeutic agent in addition to the first therapeutic agent.
  • the recommendation may include a dose for administration of the second therapeutic agent, a time for administration of the second therapeutic agent, or both.
  • the processor may output stored information to a physician.
  • the processor may output information on doses of the therapeutic agent received by the subject, time points when the therapeutic agent was received by the subject, or both to a physician.
  • the stored information may enable the physician to determine that the subject has developed or is developing resistance to the therapeutic agent.
  • the information may enable the physician to determine that the subject has developed resistance or is developing resistance to a therapeutic agent based on a change in the intervals of receiving the therapeutic agent over time, such as a decrease in the intervals over time, a change in the dose of the therapeutic agent over time, such as an increase in the dose over time, or both.
  • the stored information may enable the physician to adjust the therapeutic course for the subject. For example, the stored information may enable the physician to alter the dose of the therapeutic agent, the time when the therapeutic agent should be administered, or both.
  • Brequinar is an example of a drug that can target metabolic pathways, particularly de novo biosynthesis of pyrimidine.
  • this drug has failed in the past because it could not be delivered within an appropriate therapeutic window.
  • the invention recognizes that brequinar has failed in the past because it has not been dosed to achieve optimal enzyme inhibition.
  • the invention solves that problem by using a highly sensitive marker of target engagement, a metabolite, to tailor a patient’s dose to get an optimal Time Above Threshold (therapeutic window).
  • the claimed invention is based on measuring target engagement instead of drug metabolism. In that manner, proper dosing of brequinar is achieved to kill cancer cells without causing harmful and toxic side effects to patients.
  • pyrimidine is an essential metabolic pathway for nucleic acid synthesis. Although most cells meet their needs for nucleotides by reutilizing current ones through the salvage pathway, activated T cells and other rapidly proliferating cells, namely cancer cells are highly dependent on de novo nucleotide synthesis. Dihydroorotate
  • DHODH dehydrogenase
  • aspects of the invention are accomplished by measuring a trough dihydroorotate (DHO) level before a dose and using that level to dose adjust.
  • DHO dihydroorotate
  • the invention recognizes that measuring the DHO level provides an accurate indication of target engagement of brequinar. Accurately knowing target engagement allows for appropriate doses of brequinar to be achieved that maintains the dosing within the therapeutic window.
  • brequinar and more generally, inhibitors of dihydroorotate dehydrogenase, can be used to treat certain cancers, such as acute myeloid leukemia (AML).
  • AML afflicts over a million people worldwide. AML is incurable in the majority of cases and accounts for 1.8% of cancer deaths in the United States.
  • AML progress in treatment of AML has been less forthcoming: 90% of AML cases are treated with a therapeutic strategy that has remain unchanged for over 40 years.
  • the insights of the invention provide new compositions and methods for treating such cancers.
  • DHODH is present in all leukemic cells (essential enzyme). Differential metabolic sensitivity between leukemic cells and normal cells (i.e.,“Metabolic Therapeutic Window”) presents a treatment opportunity.
  • the compositions of the invention use inhibitors of dihydroorotate dehydrogenase (e.g., brequinar) on a novel dosing schedule to exploit the pro differentiation effects and tolerability (lower dose with long exposure) between leukemic cells and normal cells.
  • the compositions allow physicians to determine dosage of a drug based on engagement of the active pharmaceutical ingredient (API) with its target.
  • API active pharmaceutical ingredient
  • compositions are optimized to achieve prolonged exposure to the API at a level sufficient to starve leukemic cells and to avoid the need for higher dosing that can harm other cells.
  • the invention also provides methods of determining therapeutically effective doses of compositions that contain a DHODH inhibitor.
  • compositions and methods of the invention are more broadly useful for treating any diseases associated with unregulated or excessive DHODH activity, such as AML, arthritis, and multiple sclerosis.
  • the compositions are useful for treating diseases that require sustained inhibition of DHODH.
  • DHODH inhibitor brequinar decreases leukemia-initiating cell activity in mouse models of AML only when elevated levels of the compound are maintained in the plasma for extended periods.
  • compositions and methods of the invention also enable physicians to tailor dosing regimens to individual patients. Because the rate of metabolism and elimination of a given drug varies among patients, the degree of target engagement by the API will differ among patients who have received the same drug and dosage.
  • the level of DHO is a universal indicator of DHODH inhibition across all patients. Thus, by monitoring levels of DHO in individual patients, the dose of a drug can be adjusted to achieve a desired level of DHODH inhibition on a case-by-case basis.
  • the invention provides compositions containing an inhibitor of
  • DHODH in a therapeutically effective amount that raises or maintains a level of DHO above a threshold level in a subject for a period of more than 72 hours.
  • DHODH in a therapeutically effective amount that results in a level of DHO being at least about 25 ng/mL in a subject.
  • the invention provides an oral formulation containing an inhibitor of DHODH in a therapeutically effective amount that raises or maintains a level of DHO above a threshold level in a subject for a period of more than 72 hours.
  • the threshold level of DHO may be measured in a sample obtained from a subject.
  • the sample may be body fluid sample.
  • the body fluid may be plasma, blood, serum, urine, sweat, saliva, interstitial fluid, feces, or phlegm.
  • the threshold level of DHO may be a minimum level necessary for the DHODH inhibitor to provide a therapeutic benefit to a subject having a disorder.
  • the threshold level may be about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/mL, about 450 ng/mL, about 500 ng/mL, about 550 ng/mL, about 600 ng/mL, about 650 ng/mL, about 700 ng/mL, about 750 ng/mL, about 800 ng/mL, about 850 ng/mL, about 900 ng/mL, about 950 ng/mL, about 1000 ng/mL, about 1250 ng/ml, about 1500 ng/ml, about 1750 ng/ml, about 2000 ng
  • the threshold level of DHO may be a maximum level above which a subject experiences one or more side effects of the DHODH inhibitor.
  • the threshold level may be about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/mL, about 450 ng/mL, about 500 ng/mL, about 550 ng/mL, about 600 ng/mL, about 650 ng/mL, about 700 ng/mL, about 750 ng/mL, about 800 ng/mL, about 850 ng/mL, about 900 ng/mL, about 950 ng/mL, about 1000 ng/mL, about 1250 ng/ml, about 1500 ng/ml, about 1750 ng/ml, about 2000 ng/ml, about 2500 ng/ml, about 3000 ng/ml, about 3500 ng/
  • the threshold level of DHO may be a range of values.
  • the threshold level may from about 100 ng/mL to about 200 ng/mL, from about 150 ng/mL to about 200 ng/mL, from about 150 ng/mL to about 250 ng/mL, from about 200 ng/mL to about 250 ng/mL, or from about 200 ng/mL to about 300 ng/mL.
  • the DHODH inhibitor may be any agent that inhibits the activity of DHODH.
  • the DHODH inhibitor may be a small molecule, protein, peptide, antibody, or polypeptide.
  • the DHODH inhibitor may be brequinar, leflunomide, or teriflunomide. Brequinar may be in a modified form suitable for a therapeutic composition.
  • the DHODH inhibitor may be a brequinar analog, a brequinar derivative, a brequinar prodrug, a micellar formulation of brequinar, or a brequinar salt, such as a sodium salt.
  • the composition may contain the DHODH inhibitor at a defined amount.
  • the composition may contain brequinar sodium at about 400 mg/m , about 450 mg/m , about 500 mg/m 2 , about 550 mg/m 2 , about 600 mg/m 2 , about 650 mg/m 2 , about 700 mg/m 2 , about 750 mg/m 2 , or about 800 mg/m 2.
  • the composition may contain another form of brequinar in amount equivalent to brequinar sodium at about 400 mg/m 2 , about 450 mg/m 2 , about 500 mg/m 2 , about 550 mg/m , about 600 mg/m , about 650 mg/m , about 700 mg/m , about 750 mg/m , or about 800 mg/m 2 .
  • composition may be formulated for administration via a particular route.
  • the composition may be formulated for administration orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, subcutaneously, nasally, pulmonarily, or with or on an implantable medical device
  • the composition may contain a second therapeutic agent.
  • the second therapeutic agent may inhibit a target other than DHODH.
  • the second agent may inhibit a glutaminase, the PI3K pathway, or orotidine 5'-monophosphate (OMP) decarboxylase.
  • the therapeutically effective amount of the DHODH inhibitor may be an amount sufficient to raise or maintain a level of DHO in a subject to ameliorate, reduce, or eliminate one or more signs or symptoms of a disorder in the subject.
  • the therapeutically effective amount of the DHODH inhibitor may be an amount sufficient to raise or maintain a level of DHO in a subject above a threshold level, such as a threshold level described above.
  • the therapeutically effective amount of the DHODH inhibitor may be an amount sufficient to raise or maintain a level of DHO in a subject for a period of time, such as 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, 10 days, 2 weeks, or more.
  • the therapeutically effective amount of the DHODH inhibitor may be an amount that does not result in the subject developing a side effect.
  • the therapeutically effective amount of the DHODH inhibitor may be an amount that does not result in the subject developing one or more of a blood disorder, nausea, vomiting, stomatitis, mucositis, skin rash, phlebitis, photosensitivity reactions, angioneurotic edema, and localized secondary hyperpigmentation of inflamed skin.
  • the composition may be provided as a single unit dosage.
  • the composition may be provided as divided dosages.
  • the invention provides methods of determining a therapeutically effective dose of a DHODH inhibitor to be provided to a subject to treat a disorder.
  • the therapeutically effective dose inhibits DHODH to an extent that at least one sign or symptom of the disorder is reduced or eliminated.
  • the methods include determining a therapeutically effective dose of a DHODH inhibitor based on a measured level of DHO in a sample from a subject.
  • the therapeutically effective dose of the DHODH inhibitor may be a dose that raises or maintains a level of DHO above a threshold level in a sample obtained from the subject for a period of more than 72 hours.
  • the threshold level may be any threshold level, such as those described above.
  • the sample may be any sample, such as those described above.
  • the invention provides methods of adjusting a dosing regimen of a DHODH inhibitor to treat a disorder in a subject that is currently on the dosing regimen.
  • the methods include receiving information regarding a measured level of dihydroorotate (DHO) in a sample from a subject, comparing the received information to a reference that provides an association of a measured level of DHO with a recommended dosage adjustment of a DHODH inhibitor, and adjusting the dosing regimen of the DHODH inhibitor so that a next dose of the DHODH inhibitor in the dosing regimen results in a level of DHO being raised or maintained above a threshold level indicative that the amount of the DHODH inhibitor in the subject is sufficient to reduce or eliminate at least one sign or symptom of the disorder.
  • DHO dihydroorotate
  • the DHODH inhibitor may be any DHODH inhibitor, such as those described above.
  • the disorder may be any disease, disorder, or condition for which a DHODH inhibitor would provide a therapeutic benefit.
  • the disorder may be cancer, such as leukemia (e.g., acute myeloid leukemia) or prostate cancer, or an autoimmune disease, such as multiple sclerosis or arthritis (e.g., rheumatoid arthritis or psoriatic arthritis).
  • the methods may include determining a time point when the therapeutically effective dose of the DHODH inhibitor should be provided to the subject.
  • the methods may include providing the DHODH inhibitor to the subject at the therapeutically effective dose.
  • the recommended dosage adjustment may be an increase the dosage, decrease in the dosage, or no change in the dosage.
  • the recommendation may include a value by which the dosage should be increased or decreased.
  • the information regarding a measured level of DHO in a sample from a subject may include a measured level from one sample obtained from the subject or measured levels from multiple samples obtained from the subject.
  • the information may include a time point indicating when each sample was obtained from the subject.
  • the dosing regimen may be adjusted in any manner.
  • the dosing regimen may be adjusted by adjusting the dose, the time for delivering the dose, or both.
  • the adjustment may include determining a time point for delivering the dose.
  • the methods may include providing the DHODH inhibitor to the subject at the determined dose.
  • the DHODH inhibitor may be provided orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, subcutaneously, nasally, pulmonarily, or with or on an implantable medical device.
  • the DHODH inhibitor may be provided as a single unit dosage, or it may be provided as divided dosages.
  • the invention provides methods of making a 2-(2’ -halo- 1-1’-biphenyl- 4-yl)-quinoline carboxylic acid.
  • the methods include incubating a compound of formula (I) with a compound of formula (II) in a mixture containing a base and adding an acid to the mixture, thereby creating a compound of formula (III) according to following reaction:
  • Ri, R 2 , R 3 , and R 4 are independently H, F, Cl, Br, I, CH 3 , CF 3 , SCH 3 or CH 2 CH 3 , at least two of Ri, R , R 3 , and R 4 being H;
  • R 5 is H, alkoxy of 1-3 carbon atoms, or alkyl of 1-2 carbon atoms;
  • R 6 and R 7 are independently H, F, Cl, Br, alkyl of 1-5 carbon atoms, N0 2 , OH, CF 3 or
  • X is a halogen
  • the incubating step includes at least one of:
  • the incubating step may include one or more of incubating the mixture at a temperature of from about 60° C to about 70° C, using a mixture containing a molar ratio of the base to the compound of formula (II) of from about 5:1 to about 8:1, and incubating the mixture for from about 15 hours to about 30 hours.
  • the method may include a minimum yield of the compound of formula (III).
  • the yield of the compound of formula (III) may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • the base may be any suitable base.
  • the base may be KOH, NaOH, or
  • the alcohol may be any suitable alcohol.
  • the alcohol may be methanol, ethanol, l-propanol, 2-propanol, butanol, 2-methyl- 1 -propanol, or pentanol.
  • the acid may be any suitable acid.
  • the acid may be HC1 or acetic acid.
  • the compound of formula (III) may be brequinar.
  • the compound of formula (III) may have the structure represented by formula (IV):
  • the invention also recognizes that each year about 25,000 people in the United States are newly diagnosed with some form of brain cancer.
  • the five-year survival is about 35% for all patients with malignant brain tumors and about 5% for patients with glioblastoma multiforme, the most common type of primary brain tumor.
  • Brain cancer is costly in financial terms as well.
  • a 2011 study found that the average estimated lifetime economic cost of a case of brain cancer is 1.9 million Australian dollars, the highest of any type of cancer.
  • Treatment typically includes surgery, radiation therapy, chemotherapy, or some combination of these three approaches.
  • Surgery achieves the best outcomes, but many brain tumors are intractable to surgery due to their anatomical location.
  • craniotomy the most common surgical approach to treat brain cancer, carries a high risk of infection, and patients experience significant pain during recovery.
  • Radiotherapy to the brain is relatively painless for the patient but can cause swelling of the brain, which produces its own set of symptoms that may require treatment, and long-term cognitive decline.
  • Chemotherapy is ineffective for treating most brain cancers because many chemotherapeutic drugs do not traverse the blood-brain barrier.
  • the invention provides methods of treating brain cancer, such as gliomas of neuroepithelial tissue and neuroblastoma, by providing an inhibitor of an enzyme in a metabolic pathway. Due to their rapid growth rate, cancer cells are more dependent on certain metabolic pathways, such as those involved in nucleotide synthesis, than are normal cells. Therefore, by providing an agent that reduces the activity of such pathways, cancer cells can be selectively killed.
  • enzyme inhibitors that pass through blood-brain barrier represent a potent new class of anti-cancer agents for treatment of brain cancer.
  • An exemplary method of the invention entails treating brain cancer using an inhibitor of dihydroorotate dehydrogenase (DHODH), and enzyme involved in synthesis of uridine monophosphate (UMP).
  • DHODH inhibitors such as brequinar, kill cancer cells and have minimal adverse effect on healthy tissue when provided at appropriate dosages.
  • the invention further recognizes that engagement of a DHODH inhibitor with the enzyme can be monitored by analysis of levels of DHO, a substrate of DHODH, in samples obtained from the patient. Therefore, methods of the invention enable physicians to ensure that a DHODH inhibitor is administered in a therapeutically effective amount to treat brain cancer.
  • Methods of the invention provide therapeutic strategies for treating brain cancer that overcome many of the limitations of prior methods. Significantly, the methods avoid the high risk of infection associated with surgery. In addition, in contrast to surgery and radiotherapy, the methods are not constrained by the number and anatomical location of tumors. Compared to prior chemotherapeutic approaches, the methods of the invention are more broadly applicable and thus can be used to treat a variety of types of brain cancer.
  • the invention provides methods of treating brain cancer in a subject by providing to the subject an agent that crosses the blood-brain barrier and that inhibits a metabolic pathway in a cancerous cell in the brain of the subject.
  • any metabolic pathway may be targeted, provided that cancer cells are more sensitive to activity of the pathway than are normal cells.
  • the metabolic pathway may be nucleotide synthesis pathway, such as a pyrimidine synthesis pathway or a purine synthesis pathway.
  • the metabolic pathway may be a pathway for the synthesis of UMP.
  • the enzyme may be any enzyme in the metabolic pathway.
  • the enzyme may be DHODH or orotidine 5'-monophosphate (OMP) decarboxylase.
  • the agent may be any agent that inhibits an enzyme in the metabolic pathway.
  • the agent may be a small molecule, protein, peptide, antibody, or polypeptide.
  • the agent may be brequinar, leflunomide, or teriflunomide.
  • Brequinar may be in a modified form suitable for a therapeutic composition.
  • the agent may be a brequinar analog, a brequinar derivative, a brequinar pro-drug, a micellar formulation of brequinar, or a brequinar salt, such as a sodium salt.
  • the brain cancer may be any cancer of the brain or central nervous system.
  • the brain cancer may include a tumor of neuroepithelial tissue, cranial or paraspinal nerves, the meninges, the hematopoietic system, germ cells, or the sellar region.
  • the brain cancer may include cancer cells derived from neuroepithelial cells, meningeal cells, or hematopoietic cells.
  • the brain cancer may be astrocytoma, glioma, meningioma, or neuroblastoma.
  • the methods may include receiving a measured level of a metabolite in the metabolic pathway in a sample from the subject.
  • the measured level of the metabolite may be received prior to, during, or subsequent to providing the agent.
  • the measured level of the metabolite may be compared to a threshold level, and measured levels below the threshold level may indicate that one or more additional doses of the agent are required.
  • the methods may include using the measured level of the metabolite to determine a dose of the agent required to raise or maintain the measured level of the metabolite above the threshold level.
  • the methods may include providing the agent in the determined dose.
  • the metabolite may be a substrate or product of the enzyme that is inhibited by the agent.
  • the metabolite may be dihydroorotate or orotate.
  • the sample may be body fluid sample.
  • the body fluid may be plasma, blood, serum, urine, sweat, saliva, interstitial fluid, feces, or phlegm.
  • the invention provides methods of treating brain cancer in a subject by providing a DHODH inhibitor to the subject.
  • the DHODH inhibitor is an agent that crosses the blood-brain barrier.
  • the DHODH inhibitor may be a small molecule, protein, peptide, antibody, or polypeptide.
  • the DHODH inhibitor may be brequinar, leflunomide, or teriflunomide. Brequinar may be in a modified form suitable for a therapeutic composition.
  • the DHODH inhibitor may be a brequinar analog, a brequinar derivative, a brequinar pro-drug, a micellar formulation of brequinar, or a brequinar salt, such as a sodium salt.
  • the brain cancer may be any cancer of the brain or central nervous system, such as those described above.
  • the methods may include receiving a measured level of a metabolite in the metabolic pathway in a sample from the subject.
  • the measured level of the metabolite may be received prior to, during, or subsequent to providing the DHODH inhibitor.
  • the measured level of the metabolite may be compared to a threshold level, and measured levels below the threshold level may indicate that one or more additional doses of the DHODH inhibitor are required.
  • the sample may be any sample obtained from a subject, such as those described above.
  • the sample may be a plasma sample.
  • the methods may include using the measured level of the metabolite to determine a dose of the DHODH inhibitor required to raise or maintain the measured level of the metabolite above the threshold level.
  • the methods may include providing the agent in the determined dose.
  • the metabolite may be a metabolite is in a nucleotide synthesis pathway.
  • the metabolite may be dihydroorotate or orotate.
  • FIG. 1 is a series of graphs showing levels of brequinar and DHO in three patients that have received a single dose of brequinar according to the same dosing regimen.
  • FIG. 2 is a series of graphs showing levels of brequinar and DHO in three patients that have received multiple doses of brequinar according to the same dosing regimen.
  • FIG. 3 is a flow chart illustrating an example of determining dose of a DHODH inhibitor for a patient according to an embodiment of the invention.
  • FIG. 4 is a scatter plot illustrating the concentration of brequinar in subject plasma over time when administered twice weekly.
  • FIG. 5 is a scatter plot illustrating the bioavailability of an IV formulation of brequinar as compared to an oral dosage form.
  • FIG. 6 is a scatter plot illustrating the concentration of brequinar in mice at a dose of 50 mg/kg over time.
  • FIG. 7 is a scatter plot illustrating the baseline DHO levels in random cancer patients and healthy patients, as reported in Table 5.
  • FIG. 8 is a scatter plot illustrating the concentrations of pyrazofurin and orotate in murine plasma over time when pyrazofurin is administered as a single dose (20 mg/kg).
  • FIG. 9 is a scatter plot illustrating the concentrations of pyrazofurin and orotate in murine plasma over time when pyrazofurin is administered as a single dose (20 mg/kg) on a log scale.
  • FIG. 10 is a graph showing the therapeutic benefit of a drug that targets a metabolic pathway as a function of levels of a metabolite that is an intermediate in the pathway.
  • the invention provides methods that allow real-time determination of therapeutically effective dosing regimens of drugs that include an enzyme inhibitor as an active pharmaceutical ingredient (API).
  • the methods are based on the insight that the extent to which the target enzyme is engaged by the inhibitor can be evaluated based on measured levels of a metabolite in a pathway in which the enzyme functions. In particular, target engagement can be assessed from levels of a substrate of the enzyme. From the measured level of a metabolite in a sample obtained from a patient, the methods allow a physician to determine an appropriate amount of drug that contains an enzyme inhibitor to administer to the patient to alleviate a sign or symptom of a disorder and minimize undesirable side effects of the drug.
  • the methods of the invention greatly improve the utility of drugs that have large interpatient variability in drug metabolism or a narrow therapeutic window, i.e., drugs for which the range between doses necessary to achieve therapeutic effect and doses that cause toxicity is small.
  • Administration of such drugs requires precise dosing and typically includes monitoring of their effects on patients. Monitoring often involves measurement of the level of the API or a metabolic product of the API in the patient's body.
  • patients vary widely in their ability to metabolize drugs and in how drugs affect targets in their bodies, so analysis of the API or a metabolic product thereof provides an incomplete readout of the efficacy of a given drug in an individual patient.
  • the invention overcomes this limitation by using levels of a metabolite in an enzymatic pathway as a metric of engagement of the API with its target enzyme.
  • levels of a metabolite in the pathway of the API's target are universal indicators of target engagement.
  • the methods of the invention afford greater precision in the dosage and timing of drug administration. Consequently, the methods enable the safe and effective treatment of a variety of conditions using therapeutic agents that are ineffective or too dangerous under prior methods.
  • drug dosage is determined based on real-time measured levels of a metabolite in a patient.
  • the levels may be measured in a sample, such as plasma sample, obtained from a patient.
  • the methods permit rapid, convenient monitoring of patients.
  • levels of the metabolite may be measured in a tumor in vivo.
  • the invention also provides methods that allow direct, real-time assessment of the effect of a therapeutic agent on a tumor in the patient's body.
  • the invention further provides devices, such as wearable electronic devices, that provide reminders to a patient regarding drug dosing, such as the dosage of a drug or time for administration.
  • drug dosing such as the dosage of a drug or time for administration.
  • the notifications that the devices provide are based on one or more measured values of a metabolite in a sample obtained from the patient.
  • Methods of the invention include determining the dosage of a drug based on a measured level of a metabolite in a sample obtained from a subject.
  • the metabolite may be any molecule that provides an indication of target engagement by the API of the drug.
  • the API is an inhibitor of an enzyme in a metabolic pathway, and the metabolite is an intermediate the pathway.
  • the metabolite the API is an inhibitor of an enzyme in a metabolic pathway, and the metabolite is a substrate of the enzyme.
  • Nucleotide synthesis pathways are of particular therapeutic interest.
  • the high proliferation rate of cancer cells often places increased demand on nucleotide synthesis pathways. Consequently, enzymes that function in such pathways are useful targets for antineoplastic drugs.
  • drugs that inhibit enzymes require for nucleotide synthesis have been investigated for treating cancer. Therefore, levels of metabolites in nucleotide synthesis pathways are useful for evaluating the extent to which the APIs in such drugs are engaging their targets in vivo.
  • Pyrimidine biosynthesis involves a sequence of step enzymatic reactions that result in the conversion of glutamine to uridine monophosphate as shown below:
  • aspartate carbamoyltransferase also known as aspartate transcarbamoylase or ATCase
  • DHODH dihydroorotate dehydrogenase
  • OMPD OMP decarboxylase
  • One element of the invention is recognition of the utility of DHO as an indicator of target engagement by DHODH inhibitors.
  • DHO is that cell membranes are permeable to the molecule. DHODH is localized to the mitochondrial inner membrane within cells, making direct measurement of enzyme activity difficult. However, DHO, which accumulates when DHODH is inhibited, diffuses out of cells and into the blood, which can be easily sampled.
  • Another insight of the invention is that DHO is sufficiently stable that levels of the metabolite can be measured reliably. Previously, DHO was considered too unstable at ambient temperatures to be quantified accurately and was thus deemed unsuitable as an indicator of DHODH inhibition. However, the methods provided herein permit detection of DHO in plasma samples. Thus, by analyzing levels of DHO in blood or blood products, one can readily assess target engagement of a DHODH inhibitor.
  • orotate and OMP can serve as indicators for target engagement of OMP decarboxylase inhibitors.
  • inhibition of OMP decarboxylase leads to increased plasma levels of orotate, so measurement of plasma orotate levels is useful for assessing the effect of agents that target OMP decarboxylase.
  • the methods of the invention are applicable for therapeutic agents that regulate the activity of other metabolic pathways as well.
  • examples of such pathways include the purine synthesis pathway, which is targeted by methotrexate and 6-mercaptopurine and in which an enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) may be targeted; the anandamide degradation pathway, including the enzyme fatty acid amide hydrolase, which is targeted by a variety of inhibitors and activators; and glycolysis, the citric acid cycle, and the balance between the two, which are targeted by various drug candidates; the pentose phosphate pathway; and the beta-oxidation pathway.
  • IMPDH enzyme inosine-5'-monophosphate dehydrogenase
  • Methods of the invention include analysis of a measured level of metabolite in a sample.
  • the methods may include measurement of the metabolite.
  • the metabolite is measured by mass spectrometry, optionally in combination with liquid chromatography.
  • Molecules may be ionized for mass spectrometry by any method known in the art, such as ambient ionization, chemical ionization (Cl), desorption electrospray ionization (DESI), electron impact (El), electrospray ionization (ESI), fast-atom bombardment (FAB), field ionization, laser ionization (LIMS), matrix-assisted laser desorption ionization (MALDI), paper spray ionization, plasma and glow discharge, plasma-desorption ionization (PD), resonance ionization (RIMS), secondary ionization (SIMS), spark source, or thermal ionization (TIMS).
  • ambient ionization chemical ionization
  • DESI desorption electrospray ionization
  • El electron impact
  • ESI electrospray ionization
  • FAB fast-atom bombardment
  • LIMS laser
  • a sample can be directly ionized without the need for use of a separation system.
  • mass spectrometry is performed in conjunction with a method for resolving and identifying ionic species. Suitable methods include chromatography, capillary electrophoresis-mass spectrometry, and ion mobility. Chromatographic methods include gas chromatography, liquid chromatography (LC), high-pressure liquid chromatography (HPLC), hydrophilic interaction chromatography (HILIC), ultra-performance liquid
  • LC-MS liquid chromatography-mass spectrometry
  • a sample may be obtained from any organ or tissue in the individual to be tested, provided that the sample is obtained in a liquid form or can be pre-treated to take a liquid form.
  • the sample may be a blood sample, a urine sample, a serum sample, a semen sample, a sputum sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a plasma sample, a pus sample, an amniotic fluid sample, a bodily fluid sample, a stool sample, a biopsy sample, a needle aspiration biopsy sample, a swab sample, a mouthwash sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a synovial fluid sample, a phlegm sample, a saliva sample, a sweat sample, or a combination of such samples.
  • the sample may also be a solid or semi-solid sample, such as a tissue sample, feces sample, or stool sample, that has been treated to take a liquid form by, for example, homogenization, sonication, pipette trituration, cell lysis etc.
  • a sample is from plasma, serum, whole blood, or sputum.
  • the sample may be kept in a temperature-controlled environment to preserve the stability of the metabolite.
  • DHO is more stable at lower temperatures, and the increased stability facilitates analysis of this metabolite from samples.
  • samples may be stored at 4° C, -20° C, or -80° C.
  • a sample is treated to remove cells or other biological particulates.
  • Methods for removing cells from a blood or other sample are well known in the art and may include e.g., centrifugation, sedimentation, ultrafiltration, immune selection, etc.
  • the subject may be an animal (such as a mammal, such as a human).
  • the subject may be a pediatric, a newborn, a neonate, an infant, a child, an adolescent, a pre-teen, a teenager, an adult, or an elderly patient.
  • the subject may be in critical care, intensive care, neonatal intensive care, pediatric intensive care, coronary care, cardiothoracic care, surgical intensive care, medical intensive care, long-term intensive care, an operating room, an ambulance, a field hospital, or an out-of-hospital field setting.
  • the sample may be obtained from an individual before or after administration to the subject of an agent that alters activity of a metabolic pathway, such as inhibitor of an enzyme in the pathway.
  • the sample may be obtained 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more before administration of an agent, or it may be obtained 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more after administration of an agent.
  • Methods of the invention include determining a dosing regimen of an agent that alters a metabolic pathway, such as an inhibitor of an enzyme in the pathway, for a subject.
  • the dosing regimen may include a dose, i.e., an amount, of the agent that should be administered.
  • the dosing regimen may include a time point for administration of a dose of the agent to the subject. Because the dosing regimen is based on one or more measured levels of a metabolite in a sample obtained from the subject, the dosing regimen is tailored to an individual subject, e.g., a patient. Consequently, the methods of the invention provide customized dosing regimens that account for variability in pharmacokinetic properties, i.e., metabolism of the API by the subject, and pharmacodynamics properties, effect of the API on its target, among individuals.
  • the dosing regimen may be determined by comparing a measured level of a metabolite in a sample obtained from a subject to a reference that provides an association between the measured level and a recommended dosage adjustment of the agent.
  • the reference may provide a relationship between administration of the agent and levels of the metabolite in the subject. The relationship can be empirically determined from a known dose and time of administration of the agent and measured levels of the metabolite at one or more subsequent time points.
  • the reference may include a relationship between measured levels of the agent or a metabolic product of the agent and measured levels of the metabolite.
  • a dosing regimen may then be determined.
  • the dosing regimen may include a dosage of the agent, a time for administration of the dosage, or both.
  • the dosing regimen may be determined de novo, or it may comprise an adjustment to a previous dosing regimen, such as an adjustment in the dosage, the interval between administration of dosages, or both.
  • the dosing regimen is designed to deliver the agent to the subject in an amount that achieves a therapeutic effect.
  • the therapeutic effect may be a sign or symptom of a disease, disorder, or condition.
  • the therapeutic effect may be inhibition of an enzyme in the metabolic pathway, or it may be a change in an indicator of inhibition of an enzyme in a metabolic pathway.
  • the indicator may be a metabolite in the pathway, and the therapeutic effect may be an increase or decrease in levels of the metabolite.
  • the therapeutic effect may be a decrease in number of cancer cells, a decrease in proliferation of cancer cells, an increase in differentiation of pre-cancerous cells, such as myeloblasts, complete remission of cancer, complete remission with incomplete hematologic recovery, morphologic leukemia-free stat, or partial remission. Increased differentiation of myeloblasts may be assessed by one or more of expression of CD 14, expression of CDl lb, nuclear morphology, and cytoplasmic granules.
  • the dosing regimen may ensure that levels of a metabolite are raised or maintained a minimum threshold required to achieve a certain effect.
  • the dosing regimen may raise or maintain levels of the metabolite above a threshold level in the subject for a certain time period.
  • the time period may include a minimum, a maximum, or both.
  • the dosing regimen may raise or maintain levels of the metabolite above the threshold level for at least 6 hours, 12, hours, 24 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, or more.
  • the dosing regimen may raise or maintain levels of the metabolite above the threshold level for not more than 24 hours, not more than 36 hours, not more than 48 hours, not more than 60 hours, not more than 72 hours, not more than 84 hours, not more than 96 hours, not more than 5 days, not more than 6 days, not more than 7 days, not more than 10 days, or not more than 2 weeks.
  • the dosing regimen may raise or maintain levels of the metabolite above the threshold level for at least 72 hours but not more than 96 hours, for at least 72 hours but not more than 5 days, for at least 72 hours but not more than 6 days, for at least 72 hours but not more than 7 days, for at least 96 hours but not more than 7 days.
  • the dosing regimen may ensure that levels of a metabolite do not exceed or are maintained below a maximum threshold that is associated with toxicity. Levels of the metabolite above a maximum threshold may indicate that the agent is causing or is likely to cause an adverse event in the subject.
  • adverse events include abdominal pain, anemia, anorexia, blood disorders, constipation, diarrhea, dyspepsia, fatigue, fever, granulocytopenia, headache, infection, leukopenia, mucositis, nausea, pain at the injection site, phlebitis, photosensitivity, rash, somnolence, stomatitis, thrombocytopenia, and vomiting.
  • the dosing regimen may include a time point for administration of one or more subsequent doses to raise or maintain levels of the metabolite above a threshold level for a certain time period.
  • the time point for administration of a subsequent dose may be relative to an earlier time point.
  • the time point for administration of a subsequent dose may be relative to a time point when a previous dose was administered or a time point when a sample was obtained from a subject.
  • the dosing regimen may include a schedule for administration of doses.
  • doses may be administered at regular intervals, such as every 24 hours, every 36 hours, every 48 hours, every 60 hours, every 72 hours, every 84 hours, every 96 hours, every 5 days, every 6 days, every week, every 2 weeks, every 3 weeks, or every 4 weeks.
  • doses may be administered according to a schedule that does not require precisely regular intervals. For example, doses may be administered once per week, twice per week, three times per week, four times per week, once per month, twice per month, three times per month, four times per month, five times per month, or six times per month.
  • a dosing regimen for administration of a therapeutic agent, such brequinar, e.g., brequinar sodium, to a human subject may be as follows: 100 mg/m , administered intravenously twice weekly; 125 mg/m , administered intravenously twice weekly; 150 mg/m 2 , administered intravenously twice weekly; 200 mg/m 2 , administered intravenously twice weekly; 250 mg/m 2 , administered intravenously twice weekly; 275 mg/m 2 , administered intravenously twice weekly; 300 mg/m , administered intravenously twice weekly;
  • 650 mg/m 2 administered intravenously twice weekly; 700 mg/m 2 , administered intravenously twice weekly; 750 mg/m 2 , administered intravenously twice weekly; 800 mg/m 2 , administered intravenously twice weekly; 100 mg/m 2 , administered intravenously every 72 hours; 125 mg/m 2 , administered intravenously every 72 hours; 150 mg/m , administered intravenously every 72 hours; 200 mg/m 2 , administered intravenously every 72 hours; 250 mg/m 2 , administered intravenously every 72 hours; 275 mg/m , administered intravenously every 72 hours; 300 mg/m 2 , administered intravenously every 72 hours; 350 mg/m 2 , administered intravenously every
  • 96 hours 350 mg/m 2 , administered intravenously every 96 hours; 400 mg/m 2 , administered intravenously every 96 hours; 425 mg/m , administered intravenously every 96 hours; 450 mg/m 2 , administered intravenously every 96 hours; 500 mg/m 2 , administered intravenously every
  • 96 hours 550 mg/m 2 , administered intravenously every 96 hours; 600 mg/m 2 , administered intravenously every 96 hours; 650 mg/m , administered intravenously every 96 hours; 700 mg/m 2 , administered intravenously every 96 hours; 750 mg/m 2 , administered intravenously every
  • Minimum and maximum threshold levels of a metabolite depend on a variety of factors, such as the type of subject, metabolite, therapeutic agent, and type of sample. Minimum and maximum threshold levels may be expressed in absolute terms, e.g., in units of concentration, or in relative terms, e.g., in ratios relative to a baseline or reference value. For example, the minimum threshold (below which a patient may receive a dose increase or additional dose) could also be calculated in terms of increase from a pre-treatment DHO level or baseline level.
  • Minimum threshold levels of DHO or orotate in a human plasma sample may be about 0 ng/ml, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/mL, about 450 ng/mL, about 500 ng/mL, about 550 ng/mL, about 600 ng/mL, about 650 ng/mL, about 700 ng/mL, about 750 ng/mL, about 800 ng/mL, about 850 ng/mL, about 900 ng/mL, about 950 ng/mL, about 1000 ng/mL, about 1250 ng/ml, about 1500 ng/ml, about 1750 ng/ml, about 2000 ng/ml, about 2500 ng/ml, about
  • Maximum threshold levels of DHO or orotate in a human plasma sample may be about 50 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about 400 ng/mL, about 450 ng/mL, about 500 ng/mL, about 550 ng/mL, about 600 ng/mL, about 650 ng/mL, about 700 ng/mL, about 750 ng/mL, about 800 ng/mL, about 850 ng/mL, about 900 ng/mL, about 950 ng/mL, about 1000 ng/mL, about 1250 ng/ml, about 1500 ng/ml, about 1750 ng/ml, about 2000 ng/ml, about 2500 ng/ml, about 3000 ng/ml, about 3500 ng/ml, about 4000 ng/ml
  • the minimum threshold of DHO or orotate may be about 1.5 times the baseline level, about 2 times the baseline level, about 2.5 times the baseline level, about 3 times the baseline level, about 4 times the baseline level, about 5 times the baseline level, about 10 times the baseline level, about 20 times the baseline level, about 50 times the baseline level, about 100 times the baseline level, about 200 times the baseline level, about 500 times the baseline level, about 1000 times the baseline level, about 2000 times the baseline level, or about 5000 times the baseline level.
  • the minimum threshold may include any ratio that falls between those recited above. Thus, the minimum threshold may be any ratio between 1.5 times the baseline level and 5000 times the baseline level.
  • the maximum threshold of DHO or orotate may be about 2 times the baseline level, about 2.5 times the baseline level, about 3 times the baseline level, about 4 times the baseline level, about 5 times the baseline level, about 10 times the baseline level, about 20 times the baseline level, about 50 times the baseline level, about 100 times the baseline level, about 200 times the baseline level, about 500 times the baseline level, about 1000 times the baseline level, about 2000 times the baseline level, about 5000 times the baseline level, or about 10,000 times the baseline level.
  • the maximum threshold may include any ratio that falls between those recited above. Thus, the maximum threshold may be any ratio between 2 times the baseline level and 10,000 times the baseline level.
  • the agent may be any agent that alters activity of a metabolic pathway.
  • the agent is an inhibitor of an enzyme in a metabolic pathway.
  • Inhibitors of DHODH include brequinar, lefhmomide, and teriflunomide.
  • Brequinar which has the systematic name 6-fluoro-2-(2’- fluoro-l,l’ biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid, has the following structure:
  • Brequinar and related compounds are described in, for example, U.S. Patent Nos. 4,680,299 and 5,523,408, the contents of which are incorporated herein by reference.
  • the use of brequinar to treat leukemia is described in, for example, U.S. Patent No. 5,032,597 and WO 2017/037022, the contents of which are incorporated herein by reference.
  • Leflunomide, N-(4'- trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide (I) is described in, for example, U.S. Patent No. 4,284,786, the contents of which are incorporated herein by reference.
  • OMP decarboxylase inhibitors include pyrazofurin. Pyrazofurin, 5-[(2S,3R,4S,5R)- 3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4-hydroxy- lH-pyrazole-3-carboxamide, has the following structure:
  • ATCase inhibitors include N-(phosphonacetyl)-L-aspartate (PALA).
  • PALA is described in, for example, Swyryd et al, N-(Phosphonacetyl)-L-Aspartate, a Potent Transition State Analog Inhibitor of Aspartate Transcarbamylase, Blocks Proliferation of Mammalian Cells in Culture, J. Biol. Chem. Vol. 249, No. 21, Issue of November 10, pp. 6945-6950, 1974.
  • Dosing of the agent may account for the formulation of the agent.
  • therapeutic agents such as brequinar, pyrazofurin, leflunomide, teriflunomide, and PALA
  • prodrugs such as brequinar, pyrazofurin, leflunomide, teriflunomide, and PALA
  • prodrugs such as a micellar formulation.
  • Any of the aforementioned chemical forms may be provided in a pharmaceutically acceptable formulation, such as a micellar formulation.
  • Dosage of the agent also depends on factors such as the type of subject and route of administration.
  • the dosage may fall within a range for a given type of subject and route of administration, or the dosage may adjusted by a specified amount for a given type of subject and route of administration.
  • dosage of brequinar for oral or intravenous administration to a subject may be about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, or about 100 mg/kg.
  • Dosage of brequinar for oral or intravenous administration to a subject, such as human or mouse, may be adjusted by about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 50 mg/kg.
  • Dosage of brequinar for oral or intravenous administration to an animal subject, such as a human or mouse, may be about 50 mg/m , about 100 mg/m , about 200 mg/m 2 , about 300 mg/m 2 , about 350 mg/m 2 , about 400 mg/m 2 , about 500 mg/m 2 , about 600 mg/m 2 , about 700 mg/m 2 , about 750 mg/m 2 , about 800 mg/m 2 , or about 1000 mg/m 2 .
  • Dosage of brequinar for oral or intravenous administration to an animal subject, such as a human or mouse, may be adjusted by about 50 mg/m , about 100 mg/m , about 200 mg/m , about 300 mg/m , about 350 mg/m , or about 400 mg/m .
  • FIG. 1 is a series of graphs showing levels of brequinar and DHO in three patients that have received a single dose of brequinar according to the same dosing regimen.
  • the graph on the left is from patient #1
  • the graph in center is from patient #2
  • the graph on the right is from patient #3.
  • Levels of brequinar are shown in dark green
  • levels of DHO are shown in red.
  • Metabolism of brequinar is faster than average in patient #1, average in patient #2, and slower than average in patient #3.
  • Inhibition of DHODH leads to accumulation of DHO, a substrate of DHODH.
  • analysis of brequinar levels alone provides an incomplete picture of the efficacy of brequinar. Because analysis of DHO levels gives a more accurate representation of target engagement, DHO is a superior biomarker.
  • FIG. 2 is a series of graphs showing levels of brequinar and DHO in three patients that have received a multiple doses of brequinar according to the same dosing regimen.
  • the graph on the top is from patient #2, the graph in center is from patient #1, and the graph on the bottom is from patient #3.
  • Levels of brequinar are shown in dark green, levels of DHO are shown in red, and the dashed line represents a threshold level above which brequinar provides sufficient inhibition of DHODH.
  • patient #2 i.e., a patient with an average rate of brequinar metabolism
  • the dosing regimen provides periods of sustained inhibition of DHODH interspersed with short recovery periods.
  • This dosing regimen is optimal for patient #2 because the prolonged inhibition of DHODH kills leukemia cells that are sensitive to uridine starvation, while the recovery period allows an adequate supply of pyrimidines to support survival of normal cells. In patient #1, however, the duration of DHODH inhibition is not sufficient to kill leukemia cells, so this dosing regimen does not provide a therapeutic benefit. Conversely, in patient #3, the second and subsequent doses of brequinar are provided too shortly after DHODH activity is restored following the previous dose, and the pyrimidine pool is not adequately restored to support survival of normal cells. Consequently, this dosing regimen is toxic to patient #3.
  • FIG. 3 is a flow chart illustrating an example of determining a dose a of DHODH inhibitor for a patient according to an embodiment of the invention.
  • a pre-treatment DHO level is measured to determine the DHO baseline for the patient.
  • the patient is given a starting dose for 2 weeks and examined for the presence of adverse events (AE). If adverse events occur, subsequent doses are withheld to see whether the adverse events resolve within 7 days. If adverse events resolve, dosage is decreased by 75 mg/m and dosing is resumed. If no adverse events occur, DHO levels are analyzed at 84 hours post-administration. If DHO levels are below 100 ng/mL or two times the baseline, dosage of brequinar is increased by 150 mg/m but not to exceed a maximum dosage of 800 mg/m . If DHO levels are above 100 ng/mL, the dosing is maintained for 2 weeks. The process can be repeated to optimize the dosing to achieve sustained elevation of DHO levels above the threshold level without adverse events.
  • AE adverse events
  • the methods are useful for providing guidance on dosing of therapeutic agents for individuals. Therefore, the methods may be performed by any party that wishes to provide such guidance. For example and without limitation, the methods may be performed by a clinical laboratory; a physician or other medical professional; a supplier or manufacturer of a therapeutic agent; an organization that provides analytical services to a physician, clinic, hospital, or other medical service provider; or a healthcare consultant.
  • the methods of the invention are useful for determining the dosage of drugs that affect that alter the activity of a metabolic pathway to treat or prevent a disorder.
  • the drug inhibits an enzyme in the metabolic pathway.
  • the drug inhibits an enzyme in a related metabolic pathway, such as a pathway that regulates, compensates for, or antagonizes the pathway in which the target enzyme functions.
  • the disorder may be any disease, disorder, or condition for which enzyme inhibition provides a therapeutic benefit.
  • one disorder that can be treated by methods of the invention is acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • myeloblasts arrested in an early stage of differentiation proliferate in an uncontrolled manner and interfere with the development of other blood cells in the bone marrow.
  • Inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme involved in pyrimidine synthesis cause differentiation of myeloblasts and prevent their leukemia-initiating activity.
  • DHODH dihydroorotate dehydrogenase
  • DHODH Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia, Cell 167, 171-186, September 22, 2016; dx.doi.org/l0.l0l6/j.cell.2016.08.057, the contents of which are incorporate herein by reference.
  • DHODH inhibitors to treat AML require a precise dosing regimen. Care must be taken to avoid excessive inhibition of DHODH.
  • DHODH is an essential enzyme, and homozygous recessive mutations in DHODH cause Miller syndrome, a disorder characterized by multi-organ dysfunction.
  • daily administration of high doses of the DHODH inhibitor brequinar lead to weight loss, anemia, and thrombocytopenia.
  • sustained exposure to brequinar is necessary to inhibit DHODH for sufficient periods to produce a therapeutic effect in the mouse AML model.
  • malignant cells display an increased sensitivity to DHODH inhibition.
  • normal cells may be able to tolerate periods of nucleotide starvation that kill cancer cells due to the elevated metabolic needs of the latter.
  • brequinar was evaluated for treatment of solid tumor malignancies and found to be ineffective when administered over a 5 -day period followed by a 3 -week gap or once per week for three weeks followed by a l-week gap.
  • brequinar Phase I clinical and pharmacokinetic trial of Brequinar sodium (DuP 785; NSC 368390) Cancer Res. 49, 4648- 4653; Burris, H.A., et al. (1998) Pharmacokinetic and phase I studies of brequinar (DUP 785; NSC 368390) in combination with cisplatin in patients with advanced malignancies, Invest.
  • brequinar may be effective for treatment of other cancers if the drug is administered in a manner that provides sustained DHODH inhibition.
  • the disorder may be one in which inhibiting an enzyme in a metabolic pathway is of therapeutic benefit.
  • the disorder may be cancer.
  • the cancer may include a solid tumor or hematological tumor.
  • the cancer may be acute lymphoblastic leukemia (ALL), adult T cell leukemia/lymphoma (ATLL), bladder cancer, breast cancer, such as triple negative breast cancer (TNBC), glioma, head and neck cancer, leukemia, such as AML, lung cancer, such as small cell lung cancer and non-small cell lung cancer, lymphoma, multiple myeloma, neuroblastoma, osteosarcoma, ovarian cancer, prostate cancer, or renal cell cancer.
  • the disorder may have a genetic mutation such as MYC amplification or PTEN loss that leads to increased dependence on the metabolic pathway, such as increased "addiction" to glutamine.
  • the disorder may be an inflammatory or autoimmune disorder, such as arthritis, hepatitis, chronic obstructive pulmonary disease, multiple sclerosis, or tendonitis.
  • the disorder may be a psychiatric disorder, such as anxiety, stress, obsessive- compulsive disorder, depression, panic disorder, psychosis, addiction, alcoholism, attention deficit hyperactivity, agoraphobia, schizophrenia, or social phobia.
  • the disorder may be a neurological or pain disorder, such as epilepsy, stroke, insomnia, diskinesia, peripheral neuropathic pain, chronic nociceptive pain, phantom pain, deafferentation pain, inflammatory pain, joint pain, wound pain, post-surgical pain, or recurrent headache pain, appetite disorders, or motor activity disorders.
  • the disorder may be a neurodegenerative disorder, such as
  • Alzheimer’s disease Parkinson’s disease, or Huntington’s disease.
  • Brain tumors may be classified as primary, i.e., originating in the brain or secondary, i.e., originating in other organs and metastasizing into the brain.
  • a commonly used scheme for classification of tumors of the central nervous system (CNS) is provided by the World Health Organization (WHO) and described in, for example, Louis DN, et ah, (Aug 2007) "The 2007 WHO Classification of Tumours of the Central Nervous System". Acta Neuropathol. 114 (2): 97-109,
  • CNS tumors includes tumors of
  • neuroepithelial tissue such as astrocytic tumors (astrocytomas), oligodendroglial tumors, oligoastrocytic tumors, ependymal tumors, choroid plexus tumors, other neuroepithelial tumors, neuronal and mixed neuronal-glial tumors, tumors of the pineal region, and embryonal tumors, including neuroblastoma; tumors of cranial and paraspinal nerves, such as schwannoma, neurofibroma, perineurioma, and malignant peripheral nerve sheath tumors; tumors of the meninges, such as tumors of meningothelial cells, mesenchymal tumors, rimary melanocytic lesions, and other neoplasms related to the meninges; tumors of the hematopoietic system, such as malignant lymphomas, plasmacytoma, and granulocytic sarcoma; germ cell tumors, such as
  • the methods of the invention are used to treat tumors derived from neuroepithelial cells. In some embodiments, the methods of the invention are used to treat astrocytoma, glioma, meningioma, or neuroblastoma.
  • the brain cancer may be associated with a genetic mutation such as MYC amplification or PTEN loss that leads to increased dependence on the metabolic pathway, such as increased "addiction" to glutamine.
  • the disorder may include a class or subset of patients having a disease, disorder, or condition.
  • AML cases are classified based on cytological, genetic, and other criteria, and AML treatment strategies vary depending on classification.
  • One AML classification system is provided by the World Health Organization (WHO).
  • the WHO classification system includes subtypes of AML provided in Table 1 and is described in Falini B, et al. (October 2010) "New classification of acute myeloid leukemia and precursor-related neoplasms: changes and unsolved issues" Discov Med. 10 (53): 281-92, PMID 21034669, the contents of which are incorporated herein by reference.
  • AML French- American-British
  • the FAB classification system includes the subtypes of AML provided in Table 2 and is described in Bennett JM, et al. (March 1976). "Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group" Br. J. Haematol. 33 (4); 451-8, doi: 10.111 l/j.1365-2141. l976.tb03563.x. PMID 188440; and Bennett JM, et al. (June 1989) "Proposals for the classification of chronic (mature) B and T lymphoid leukaemias.
  • the disorder may include a sub-population of patients.
  • the patients may be pediatric, newborn, neonates, infants, children, adolescent, pre-teens, teenagers, adults, or elderly.
  • the patients may be in critical care, intensive care, neonatal intensive care, pediatric intensive care, coronary care, cardiothoracic care, surgical intensive care, medical intensive care, long-term intensive care, an operating room, an ambulance, a field hospital, or an out-of-hospital field setting.
  • Methods of the invention may include providing a therapeutic agent to a subject according to a dosing regimen or dosage determined as described above.
  • Providing the agent to the subject may include administering it to the subject.
  • a dose may be administered as a single unit or in multiple units.
  • the agent may be administered by any suitable means.
  • the agent may be administered orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, intravenously, subcutaneously, nasally, pulmonarily, or with or on an implantable medical device (e.g., stent or drug-eluting stent or balloon equivalents).
  • an implantable medical device e.g., stent or drug-eluting stent or balloon equivalents.
  • the methods include assessing a metabolite level in a sample from a subject, and determining whether that level is within a threshold range (e.g., above a minimal threshold and/or below a potential toxicity threshold) that warrants dosing, and/or that warrants dosing at a particular level or in a particular amount.
  • a threshold range e.g., above a minimal threshold and/or below a potential toxicity threshold
  • the methods may include administering at least one dose of the agent to a subject whose plasma metabolite level has been determined and is below a pre-determined threshold (e.g., a pre-determined potential toxicity threshold and/or a pre-determined potential efficacy threshold).
  • a pre-determined threshold e.g., a pre-determined potential toxicity threshold and/or a pre-determined potential efficacy threshold.
  • the predetermined threshold reflects percent inhibition of a target enzyme in the subject relative to a baseline determined for the subject.
  • the baseline is determined by an assay.
  • multiple doses of the agent may be administered.
  • multiple doses of the agent may be administered.
  • dosing of the agent can occur at different times and in different amounts.
  • the present disclosure encompasses those methods that can maintain inhibition of the target enzyme at a consistent level at or above the efficacy threshold throughout the course of treatment.
  • the amount of inhibition of the target enzyme is measured by the amount of metabolite in the plasma of a subject.
  • the method further comprises a step of re-determining the subject’s plasma metabolite level after administration of the at least one dose.
  • the subject’s plasma metabolite level is re-determined after each dose.
  • the method further comprises administering at least one further dose of the agent after the subject’s plasma metabolite level has been determined again (e.g., after administering a first or previous dose), and is below the pre-determined threshold. If the subject’s plasma metabolite level is determined to be above a pre-determined threshold, dosing can be discontinued. In some embodiments, therefore, no further dose of the agent is administered until the subject’s plasma metabolite level has been determined to again be below a pre-determined threshold.
  • the methods may include administering an agent to a subject at a dosage level at or near a cell-lethal level. Such dosage can be supplemented with a later dose at a reduced level, or by discontinuing of dosing.
  • the present disclosure provides a method of administering a dihydroorotate dehydrogenase inhibitor to a subject in need thereof, the method comprising: administering a plurality of doses of an agent, according to a regimen characterized by at least first and second phases, wherein the first phase involves administration of at least one bolus dose of an agent at a cell-lethal level; and the second phase involves either: administration of at least one dose that is lower than the bolus dose; or absence of administration of an agent.
  • an agent is not administered during a second phase.
  • a second phase involves administration of uridine rescue therapy.
  • a bolus dose is or comprises a cell lethal dose.
  • a cell lethal dose is an amount of an agent that is sufficient to cause apoptosis in normal (e.g., non-cancerous) cells in addition to target cells (e.g., cancer cells).
  • the first phase and the second phase each comprise administering an agent. In some embodiments, the first phase and the second phase are at different times. In some embodiments, the first phase and the second phase are on different days. In some embodiments, the first phase lasts for a period of time that is less than four days. In some embodiments, the first phase comprises administering an agent, followed by a period of time in which no agent is administered. In some embodiments, the period of time in which no agent is administered is 3 to 7 days after the dose during the first phase. In some embodiments, the first phase comprises administering more than one dose.
  • an agent is administered during a second phase. In some embodiments, an agent is administered sub-cell-lethal levels during the second phase. In some embodiments, the first phase is repeated after the second phase. In some embodiments, both the first and second phases are repeated.
  • the present disclosure provides a method of administering an agent to a subject in need thereof, according to a multi-phase protocol comprising: a first phase in which at least one dose of the agent is administered to the subject; and a second phase in which at least one dose of the agent is administered to the subject, wherein one or more doses administered in the second phase differs in amount and/or timing relative to other doses in its phase as compared with the dose(s) administered in the first phase.
  • a metabolite level is determined in a sample from the subject between the first and second phases.
  • the sample is a plasma sample.
  • the timing or amount of at least one dose administered after the metabolite level is determined or differs from that of at least one dose administered before the metabolite level was determined.
  • the amount of agent that is administered to the patient is adjusted in view of the metabolite level in the subject’s plasma.
  • a first dose is administered in the first phase.
  • metabolite level is determined at a period of time after administration of the first dose.
  • the amount of agent administered in a second or subsequent dose is increased and/or the interval between doses is reduced.
  • the amount of agent administered may be increased, for example, by 100 mg/m .
  • the amount of agent administered in a second or subsequent dose is increased by 150 mg/m .
  • the amount of agent administered in a second or subsequent dose is increased by 200 mg/m .
  • the amount of agent administered may be increased by an adjustment amount determined based on change in metabolite levels observed between prior doses of different amounts administered to the subject.
  • the amount of agent administered in a second or subsequent dose is the same as the amount administered in the first or previous dose and/or the interval between doses is the same.
  • the amount of agent in a second or subsequent dose is decreased and/or the interval between doses is increased.
  • the amount of agent administered may be decreased, for example, by 50 mg/m .
  • the amount of agent in a second or subsequent dose is decreased by 75 mg/m .
  • the amount of agent in a second or subsequent dose is decreased by 100 mg/m .
  • the amount of agent administered may be decreased by an adjustment amount determined based on change in metabolite levels observed between prior doses of different amounts administered to the subject.
  • the present disclosure provides a method of administering a later dose of an agent to a patient who has previously received an earlier dose of the agent, wherein the patient has had a level of metabolite assessed subsequent to administration of the earlier dose, and wherein the later dose is different than the earlier dose.
  • the later dose may be different from the earlier dose in amount of agent included in the dose, time interval relative to an immediately prior or immediately subsequent dose, or combinations thereof.
  • the amount of agent in the later dose may be less than that in the earlier dose.
  • the method may include administering multiple dose of the agent, separated from one another by a time period that is longer than 2 days and shorter than 8 days
  • the time period may be about 3 days.
  • the metabolite level is determined in a sample from the subject before each dose is administered, and dosing is delayed or skipped if the determined metabolite level is above a pre-determined threshold.
  • the metabolite level may be determined about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours after administration of an agent
  • the method may include administering the agent according to a regimen approved in a trial in which a level of metabolite was measured in a patients between doses of the agent
  • the regimen may include multiple doses whose amount and timing were determined in the trial to maintain the metabolite level within a range determined to indicate a degree of target enzyme inhibition below a toxic threshold and above a minimum threshold.
  • the regimen may include determining the metabolite level in the subject after administration of one or more doses of the agent.
  • the regimen includes a dosing cycle in which an established pattern of doses is administered over a first period of time. In some embodiments, the regimen comprises a plurality of the dosing cycles. In some embodiments, the regimen includes a rest period during which the agent is not administered between the cycles.
  • compositions of the invention include DHODH inhibitors.
  • DHODH inhibitors are known in the art.
  • inhibitors of DHODH include brequinar, leflunomide, and teriflunomide.
  • Brequinar which has the systematic name 6-fluoro-2-(2’- fluoro-l,l’ biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid, has the following structure:
  • Brequinar and related compounds are described in, for example, U.S. Patent Nos. 4,680,299 and 5,523,408, the contents of which are incorporated herein by reference.
  • the use of brequinar to treat leukemia is described in, for example, U.S. Patent No. 5,032,597 and WO 2017/037022, the contents of which are incorporated herein by reference.
  • Leflunomide, N-(4'- trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide (I) is described in, for example, U.S. Patent No. 4,284,786, the contents of which are incorporated herein by reference.
  • the DHODH inhibitor may be provided as a prodrug, analog, derivative, or salt.
  • the DHODH inhibitor may be provided in a micellar formulation.
  • the DHODH inhibitors may be provided as pharmaceutical compositions.
  • a pharmaceutical composition may be in a form suitable for oral use, for example, as tablets, troches, lozenges, fast-melts, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs.
  • Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the compounds in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example com starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated, or they may be coated by known techniques to delay disintegration in the stomach and absorption lower down in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U.S. Patents 4,256,108, 4,166,452 and 4,265,874, to form osmotic therapeutic tablets for control release. Preparation and administration of compounds is discussed in U.S. Pat. No. 6,214,841 and U.S. Pub. No. 2003/0232877, the contents of each of which are incorporated by reference herein.
  • Formulations for oral use may also be presented as hard gelatin capsules in which the compounds are mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the compounds are mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example calcium carbonate, calcium phosphate or kaolin
  • an oil medium for example peanut oil, liquid paraffin or olive oil.
  • An alternative oral formulation where control of gastrointestinal tract hydrolysis of the compound is sought, can be achieved using a controlled-release formulation, where a compound of the invention is encapsulated in an enteric coating.
  • Aqueous suspensions may contain the compounds in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such a polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.
  • suspending agents for example sodium carboxymethylcellulose, methylcellulose
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the compounds in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compounds in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent, suspending agent and one or more preservatives Suitable dispersing or wetting agents and suspending agents are exemplified, for example sweetening, flavoring and coloring agents, may also be present.
  • the pharmaceutical compositions may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soya bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and agents for flavoring and/or coloring.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in l,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • compositions may include other pharmaceutically acceptable carriers, such as sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin (glycerol), erythritol, xylitol.
  • sugars such as lactose, glucose and sucrose
  • starches such as corn starch and potato starch
  • cellulose, and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate
  • the pharmaceutically acceptable carrier may be an encapsulation coating.
  • the encapsulation coating may contain carrageenan, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate trimellitate, collagen, gelatin, hydroxypropyl methyl cellulose acetate, a methyl acrylate-methacrylic acid copolymer, polyvinyl acetate phthalate shellac, sodium alginate, starch, or zein.
  • N-acylethanolamide compounds including prodrugs, analogs, and derivatives thereof, may be provided as pharmaceutically acceptable salts, such as nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate,
  • benzenesulfonate benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate
  • a pharmaceutically acceptable salt may be found in, for example, Remington, The Science and Practice of Pharmacy (20th ed. 2000).
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • a pharmaceutically acceptable salt is an alkali salt.
  • a pharmaceutically acceptable salt is a sodium salt.
  • a pharmaceutically acceptable salt is an alkaline earth metal salt.
  • pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
  • the invention provides methods of making a 2-(2’-halo-l-l’-biphenyl-4-yl)-quinoline carboxylic acid, such as brequinar.
  • the methods include incubating a compound of formula (I) with a compound of formula (II) in a mixture containing a base and adding an acid to the mixture, thereby creating a compound of formula (III) according to following reaction:
  • R 5 is H, alkoxy of 1-3 carbon atoms, or alkyl of 1-2 carbon atoms;
  • R 6 and R 7 are independently H, F, Cl, Br, alkyl of 1-5 carbon atoms, N0 2 , OH, CF 3 or
  • X is a halogen
  • the incubating step includes at least one of:
  • An insight of the invention is that optimizing the conditions of the first step, i.e., incubating compounds of formula (I) and formula (II) in the presence of a base, improves yield of the product.
  • One key variable is the molar ratio of the base to the compound of formula (II). Higher yields are achieved with when this molar ratio is optimized.
  • the molar ratio of the base to the compound of formula (II) may be from about 5:1 to about 8:1, from about 6.5:1 to about 7.5:1, or about 7:1.
  • the base is KOH, NaOH, and NH 4 OH.
  • the alcohol may be methanol, ethanol, l-propanol, 2-propanol, butanol, 2-methyl- 1 -propanol, or pentanol.
  • the temperature is Another important variable in the incubation step. A minimum temperature is required for the reaction to occur, but temperatures that are too high result in increased generation of undesired side products. Thus, the temperature may be from about 60° C to about 70° C, from about 60° C to about 65° C, or about 60° C.
  • the length of incubation may be from about 15 hours to about 30 hours, from about 15 hours to about 25 hours, or from about 15 hours to about 20 hours.
  • the reaction outlined above can be performed using one or more of an optimized molar ratio of the base to the compound of formula (II) as described above, an optimized temperature as described above, and an optimized incubation time as described above.
  • the reaction may include one, two, or three of the optimized variables described above.
  • the acid may be any suitable acid.
  • the acid may be HC1 or acetic acid.
  • the method may provide a minimum yield of the compound of formula (III).
  • the yield of the compound of formula (III) may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • the compound of formula (III) may be brequinar.
  • the compound of formula (III) may have the structure represented by formula (IV):
  • the invention also provides methods for assessing the effects of therapeutic agents on tumors in vivo in real time. This information obtained from such in vivo analysis may be used to determine or make adjustments to dosing regimens.
  • One modality for assessing the effect of an agent on a tumor is to monitor within the tumor the flux of a metabolite through a pathway whose activity is altered by the agent, such as the pathways and agents described above.
  • Activity of metabolic pathways in vivo can be analyzed in real-time by hyperpolarization magnetic resonance imaging, as described in, for example, Miloushev, VZ et al., Hyperpolarization MRI: Preclinical Models and Potential Applications in Neuroradiology, Top Magn Reson Imaging 2016 Feb; 25(1): 31-37, doi:
  • the methods entail injection of an isotopically-labeled metabolite, which can be imaged by magnetic resonance, into a subject and tracking movement of the isotope through the body.
  • the metabolite may be a carbon-containing molecule, such as an intermediate in the pyrimidine synthesis pathway, that is enriched for an isotope of carbon, such as C, or nitrogen, such as 15 N.
  • the therapeutic agent may be an agent that inhibits an enzyme in a pathway through which the metabolite passes. Analysis may include comparison of metabolism of the labeled metabolite when the subject has been provided the therapeutic agent with metabolism in an untreated subject, either the same subject or a different subject having similar characteristics.
  • the methods are useful for analysis of tumors due to the increase flux through certain metabolic pathways, such as the pyrimidine synthesis pathway, in tumor cells. For example, a subject having a tumor with increased glutamine flux (determined by isotopically-labeled glutamine) may be given a DHODH inhibitor, e.g., brequinar, and isotopically-labeled DHO. If the level of DHODH inhibition is high, accumulation of the metabolite can be detected at the site of the tumor.
  • a DHODH inhibitor e.g., brequinar
  • Another way to assess the effect of an agent on a tumor in vivo in real time is to analyze oxygenation of the tumor.
  • Many solid tumors contain regions of poor oxygenation due to the inability of the vasculature to keep pace with the rapid growth of tumor cells.
  • tumor cells often alter their metabolism to derive more energy from glucose metabolism and become less dependent on oxygen.
  • Methods of measuring oxygenation levels of tissue that contains tumors is known in the art and described in, for example, Zhao, D., et ah, Measuring changes in tumor oxygenation, Methods Enzymol.
  • tumor oxygenation may be measured by electron paramagnetic resonance imaging (EPR).
  • EPR is known in the art and described in, for example, Abramovic Z., et ah, (eds) l lth Mediterranean Conference on Medical and Biomedical Engineering and Computing 2007. IFMBE Proceedings, vol 16.
  • the invention also includes a device or assay to rapidly measure levels of a metabolite of interest, for e.g., DHO.
  • Plasma from a patient is run on the assay with the objective to determine the level of metabolite in the plasma.
  • set levels of the target enzyme are added with known activity.
  • the assay quantifies the amount of metabolite present in plasma by colorimetric changes, a competitive assay, or other techniques known in the field.
  • the objective is to quantify the amount of DHO after a dose of brequinar.
  • a patient plasma specimen is collected.
  • the plasma is run on the assay containing set amount of DHODH.
  • Patient DHO may compete with colored DHO in the assay and cause a change in color that can be read out as a measure of DHO level in the plasma.
  • substrate and DHODH could be lyophilized in a blood collection tube. Blood drawn into the tube could provide a visible change in color to determine if DHO is below, at or above a specified threshold. This would enable point of care monitoring of metabolite levels for rapid adjustments in dose as needed.
  • the invention also includes devices for notifying a subject concerning a dosing regimen, such as a dosage of a therapeutic agent, timing for administration of a dose, timing for collection of a metabolite to determine dose adjustments, or any combination thereof, or an adjustment to a dosing regimen.
  • the devices include a processor coupled to a memory unit.
  • the memory unit drives the processor to receive data about a dose of a therapeutic agent, collect data from laboratory or point of care analysis of the metabolite tested, generate a notification about a dosing regimen or a change to the dosing regimen, and output the reminder to the subject.
  • the data received by the processor may contain any information related to a dose of an agent provided to a subject.
  • the data may include information about the agent, such as the name of the agent, a classification the agent, the dose or amount of the agent provided to the subject, the concentration, the formulation, and the like.
  • the data may include the route of administration, such as oral or intravenous administration.
  • the data may include the when the dose was administered to the subject, including the day, date, hour, minute, second, time zone, or any other temporal component.
  • the data may include information concerning multiple doses of the agent that were administered to the subject.
  • the data may include information concerning multiple agents that were administered to the subject.
  • the data may include a metabolite level and whether a specified threshold has been reached.
  • the notification may include any type of reminder to the subject concerning the dosing regimen or adjustments thereto.
  • the notification may include a time for
  • the notification may include adjustments to any of the aforementioned parameters.
  • the notification may include information provided in absolute terms or relative terms.
  • the notification may include a time component that indicate that the next dose should be provided at a certain number of hours, e.g., 72 hours, following the previous dose, or it may indicate an objective time and/or date for administration of the next dose.
  • the notification may indicate that the dosage should be adjusted by a defined amount, e.g., increased by 75 ng/mL, by a relative amount, e.g., increased by 50%.
  • the dosing regimen or adjustment to the dosing regimen is based on a measured level of a metabolite in a sample obtained from the subject, as described above.
  • the notification may also recommend the time for an additional blood collection for metabolite analysis based on a trend analysis of historic drug and metabolite levels, a change in disease, or new evidence for an alternative blood sampling schedule.
  • the device may provide the notification in any manner that can be perceived by the subject.
  • output of the notification may include an audible signal, a visual signal, a tactile signal, a vibration, or any combination thereof.
  • the device may output the notification to a component of the device, such as a display, or it may output the notification to a remote device.
  • the device may output the notification to a third party, such as health care professional, e.g., a physician, nurse, or other practitioner.
  • the memory unit may enable the processor to perform additional processes.
  • the processor may determine a dosing regimen or an adjustment to a dosing regimen, as described above.
  • the processor may use information stored in the memory unit to determine whether the subject has developed or is developing resistance to a therapeutic agent.
  • Resistance of a subject to a therapeutic agent can become manifest when the interval between time points of dose administration to achieve the same effect, e.g., level of metabolite, become smaller over the course of therapy, i.e., when the subject requires more frequent doses.
  • Resistance of a subject to a therapeutic agent can become manifest when higher dosages are required to achieve the same effect, e.g., level of metabolite, over the course of therapy.
  • the processor may determine that intervals between time points for administration of the agent have changed, e.g., grown smaller or larger, over the course of therapy, that dosages have changed, e.g., increased or decreased, over the course of therapy, or a combination of the two.
  • the processor may output a recommended adjustment in the dosing regimen to the subject.
  • the recommended adjustment may include administration of a second or additional therapeutic agent.
  • the device may be, or be a part of, a portable or wearable electronic device, such as a phone, watch, belt, armband, legband, article of clothing, handheld device, or the like.
  • a portable or wearable electronic device such as a phone, watch, belt, armband, legband, article of clothing, handheld device, or the like.
  • Methods of the invention include determining a dosing regimen that includes providing an agent that alters activity of a metabolic pathway in a tumor that is specifically dependent on that metabolic pathway. For example, tumor cells bearing a mutation that affects the activity of a first pathway may rely more heavily on the activity of a second pathway that compensates for or counteracts the altered activity of the first pathway. A change in the activity of the second pathway that may therefore be deadly to tumor cells but not to normal cells, a phenomenon called synthetic lethality.
  • Examples of tumors with altered pathways for which a DHODH inhibitor, such as brequinar, may be synthetically lethal include tumors that have phosphatase and tensin homolog (PTEN) low, Myc protein family member amplification, a Notch protein family member mutations, and activating mutations of Ras protein family members.
  • PTEN phosphatase and tensin homolog
  • Methods of the invention include determining a dosing regimen that includes providing an agent that alters activity of a metabolic pathway, as described above, in combination with one or more other therapeutic agents.
  • the methods may also include providing both therapeutic agents in such combination dosing regimens.
  • Combination therapies are useful, for example, for treating autoimmune toxicity and cytokine-associated toxicity.
  • Autoimmune toxicity may result from an antigen- specific attack on host tissues when the targeted tumor associated antigen is expressed on nonmalignant tissue. It may result due to increased immune activation due to immunoncology (IO) therapy. It may preferentially affect patients with pre-existing autoimmune disease such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis.
  • IO immunoncology
  • Cytokine release syndrome ( CRS )
  • Cytokine associated toxicity also referred to as cytokine release syndrome (CRS) or cytokine storm
  • CRS cytokine release syndrome
  • IO cytokine storm
  • CRS is clinically observed in cases where large numbers of lymphocytes (B cells, T cells, and/or natural killer cells) and/or myeloid cells (macrophages, dendritic cells, and monocytes) become activated and release inflammatory cytokines including IL-lbeta, TNFalpha, IFNbeta, IFNgamma, IL-6, and IL-8.
  • CRS is caused by a hyperactivated T-cell response which is not tissue specific and thus causes reactivity with normal issue. This results in the production of high levels of CD4 T-helper cell cytokines or increased migration of cytolytic CD8 T cells within normal tissues. Weber, J.
  • CRS can lead to serious organ damage and failure; such injury includes pulmonary infiltrates, lung injury, acute respiratory distress syndrome, cardiac dysfunction, cardiovascular shock, neurologic toxicity, disseminated intravascular coagulation (DIC), hepatic failure, or renal failure.
  • CRS has been reported following the administration of IO therapies including HSCT, cancer vaccines (either alone or in combination with adoptive T-cell therapy), mAbs, and CAR-T cells.
  • CRS is a potentially life-threatening toxicity, with some patients requiring extensive intervention and life support. Patients have experienced neurological damage and/or death. Diagnosis and management of CRS in response to immune cell-based therapies is routinely based on clinical parameters and symptoms.
  • Lee et al. has described a revised CRS grading system, shown below in Table 3.
  • Grades 2-4 refer to CTCA.E v4.0 grading
  • immunosuppressive drugs e.g., anti-cytokine antibodies such as tocilizumab and
  • CRS corticosteroids
  • the present disclosure relates particularly to methods of improving the safety of immuno- oncology (IO) treatments while maintaining efficacy.
  • Cancer or autoimmune disease may be viewed as the result of a dysfunction of the normal immune system.
  • the goal of IO is to utilize a patient’s own immune system to effect treatment of a disorder.
  • IO treatments may include hematopoietic stem cell transplantation (HSCT), cancer vaccines, monoclonal antibodies (mAbs), and adoptive T-cell immunotherapy
  • therapeutic agents that can be used in combination dosing regimens are described below.
  • the second or additional therapeutic agent may target a metabolic pathway different from the pathway targeted by the primary therapeutic agent.
  • the second agent may inhibit a glutaminase, the PI3K pathway, or orotidine 5'-monophosphate (OMP) decarboxylase.
  • OMP orotidine 5'-monophosphate
  • the second or additional therapeutic agent may be an anti-cancer agent used to treat brain cancer.
  • the second agent may be carboplatin, carmustine, cisplatin, cyclophosphamide, etoposide, irinotecan, lomustine, methotrexate, procarbazine, temozolomide, or vincristine.
  • Adoptive T-cell immunotherapy may be performed with either natural T-cells or with engineered T-cells.
  • Engineered T-cells can include T-cells which have been engineered to express chimeric antigen receptors (CARs) on their surface (CAR-T cells).
  • CARs chimeric antigen receptors
  • Autologous adoptive cell transfer involves the collection, modification, and return of a patient's immune cells, offering a promising immunotherapeutic approach for the treatment of different types of cancers.
  • leukocytes are isolated, usually by well established density barrier centrifugation, and T lymphocytes are expanded ex vivo using cell culture methods, often relying on the immunomodulatory action of interleukin- 2.
  • T lymphocytes are expanded ex vivo using cell culture methods, often relying on the immunomodulatory action of interleukin- 2.
  • the cells are administered intravenously to the patent in an activated state.
  • effector T cells Such cells are referred to as effector T cells.
  • a combination of anti-CD3 and anti-CD28 antibodies may be used as a surrogate for antigen presentation with appropriate co- stimulation cues to promote the proliferation of T cells in culture.
  • TCR CD4 + and CD8 + T cell receptor
  • Co- stimulation is achieved naturally by the interaction of CD28, a co- stimulatory cell surface receptor on T cells, with a counter-receptor on the surface of the APC, e.g., CD80 and/or CD86.
  • An APC may also be used for the antigen-dependent activation of T cells.
  • APCs must also express on their surface a co-stimulatory molecule. Such APCs are capable of stimulating T cell proliferation, inducing cytokine production, and acting as targets for cytolytic T lymphocytes (CTL) upon direct interaction with the T cell.
  • CTL cytolytic T lymphocytes
  • CARs chimeric antigen receptors
  • CAR-T cells can be cultured and expanded in the laboratory, then re-infused to patients in a similar manner to that described above for adoptive transfer of native T cells.
  • the CAR directs the CAR T-cell to a target cell expressing an antigen to which the CAR is specific.
  • the CAR T cell binds the target and through operation of a stimulatory domain activates the CAR T-cell.
  • the stimulatory domain is selected from CD28, 0X40, CD27, CD2, CD5, ICAM-l, LFA-l (CDl la/CDl8), 4-1BB, or a combination thereof.
  • CARs may be specific for any tumor antigen.
  • a CAR comprises an extracellular binding domain specific for a tumor antigen.
  • a tumor antigen is selected from TSHR, CD19, CD123, CD22, CD30, CD171, CS-l, CLL-l, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-l3Ra2, Mesothelin, IL-llRa, PSCA, PRSS21, VEGFR2, LewisY,
  • a CAR comprises an extracellular binding domain specific for a tumor targeting antibody.
  • an extracellular binding domain specific for a tumor targeting antibody binds an Fc portion of a tumor targeting antibody.
  • an extracellular binding domain specific for a tumor targeting antibody comprises an Fc receptor or an Fc binding portion thereof.
  • an Fc receptor is an Fc- gamma receptor, an Fc-alpha receptor, or an Fc epsilon receptor.
  • an extracellular binding domain can be an extracellular ligand-binding domain of CD 16 (e g., CD16A or CD16B), CD32 (e g., CD32A, or CD32B), or CD64 (e g., CD64A, CD64B, or CD64C).
  • a CAR comprises a transmembrane domain.
  • a transmembrane domain is selected from CD8a, CD8P, 4-1BB, CD28, CD34, CD4, FceRfy, CD16 (e g., CD16A or CD16B), 0X40, O ⁇ 3z CD3e, CD3y, CD35, TCRa, CD32 (e g., CD32A or CD32B), CD64 (e g., CD64A, CD64B, or CD64C), VEGFR2, FAS, and FGFR2B, or a combination thereof.
  • the transmembrane domain is not CD8a.
  • a transmembrane domain is a non-naturally occurring hydrophobic protein segment.
  • a CAR comprises a co- stimulatory domain for T-cell activation.
  • a co- stimulatory domain is selected from CD28, 0X40, CD27, CD2, CD5, ICAM-l, FFA-l (CDl la/CDl8), 4-1BB, GITR, HVEM, TIM1, FFA1, or CD2, a functional fragment thereof, or a combination thereof.
  • a CAR comprises two or more co-stimulatory domains.
  • the two or more co-stimulatory domains are selected from CD28, 0X40, CD27, CD2, CD5, ICAM-l, LFA-l (CDl la/CDl8), 4-1BB, GITR, HVEM, TIM1, LFA1, or CD2.
  • Cytokine release syndrome is a common and potentially lethal complication of CAR-T cell therapy. It is a non-antigen specific toxicity that can occur as a result of the high- levels of CAR-T cell expansion and immune activation typically required to mediate clinical benefit using modem immunotherapies such as CAR-T cell transfer. Timing of symptom onset and CRS severity depends on the inducing agent and the magnitude of immune cell activation. Symptom onset typically occurs days to occasionally weeks after T cell infusion, coinciding with maximal in vivo T-cell expansion.
  • CRS following CAR-T therapy has recently been reported to be greater in patients having large tumor burdens. Without wishing to be bound by any theory, it is believe that this is due to the expression of production of pro-inflammatory cytokines such as TNF-a by the adoptively transferred expanding and activated CAR-T cell populations.
  • CRS following CAR-T therapy has been consistently associated with elevated IFNy, IL-6, and TNF-a levels, and increases in IL-2, granulocyte macrophage-colony- stimulating factor (GM-CSF), IL-10, IL-8, IL-5, and fracktalkine have also been reported.
  • GM-CSF granulocyte macrophage-colony- stimulating factor
  • an immune-oncology therapy is a cancer vaccine.
  • a cancer vaccine is an immunogenic composition which stimulates a patient’s immune system to produce anti - tumor antibodies, thereby enabling the immune system to target and destroy cancerous cells.
  • a cancer vaccine is a peptide vaccine.
  • a cancer vaccine is a conjugate vaccine.
  • a cancer vaccine is used in combination with adoptive T cell therapy.
  • a cancer vaccine is administered to a patient, after which tumor specific T cells are obtained from the patient, isolated, expanded ex vivo, and then administered to the patient.
  • the ex vivo expansion of tumor specific T cells provides for a method of obtaining a greater number of T cells which may attack and kill cancerous cells than what could be obtained by vaccination alone.
  • adoptive T cell therapy comprises culturing tumor infiltrating lymphocytes.
  • one particular T cell or clone is isolated and expanded ex vivo prior to administration to a patient.
  • a T cell is obtained from a patient who has received a cancer vaccine.
  • Administration of cancer vaccines, either alone or in combination with adoptive T cell transfer has been reported to result in CRS.
  • HSCT Human stem cell transplantation
  • HSCT is the transplantation of stem cells to reestablish hematopoietic function in a patient with defective bone marrow or immune system.
  • the stem cells are autologous.
  • the stem cells are allogeneic.
  • the transplant is performed by intravenous infusion.
  • autologous HSCT may be used to treat multiple myeloma, non- Hodgkin lymphoma, Hodgkin disease, acute myeloid leukemia, neuroblastoma, germ cell tumors, autoimmune disorders (e.g., systemic lupus erythematosus [SLE], systemic sclerosis), or amyloidosis.
  • autoimmune disorders e.g., systemic lupus erythematosus [SLE], systemic sclerosis
  • amyloidosis e.g., amyloidosis.
  • allogeneic HSCT may be used to treat acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin lymphoma, Hodgkin disease, aplastic anemia, pure red-cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis, inborn errors of metabolism, Epidermolysis Bullosa, severe congenital neutropenia, Shwachman- Diamond syndrome, Diamond-Blackfan anemia, or leukocyte adhesion deficiency.
  • SCID severe combined immunodeficiency
  • stem cells are obtained from a donor for administration to a patient.
  • the donor is an identical twin of the patient.
  • the donor is a matched donor related to the patient. In some embodiments, the donor is a matched donor unrelated to the patient. In some embodiments, the donor is a mismatched donor related to the patient. In some embodiments, the donor is haploidentical to the patient.
  • stem cells are obtained from bone marrow, peripheral blood, or umbilical cord blood.
  • HSCT may result in graft vs. host disease (GvHD), which remains a major cause of morbidity and mortality in patients undergoing HSCT.
  • GvHD graft vs. host disease
  • CRS Inflammatory cytokine release
  • CRS activation of T-cells is one step in this complex process.
  • Monoclonal antibodies are useful in the treatment of various cancers.
  • mAb cancer treatments utilize natural immune system functions to attack cancerous cells.
  • Administration of mAbs specific for tumor antigens can be useful in targeting the tumor cells for destruction by the immune system.
  • mAbs can trigger lysis of cancer cells, block cancer cell growth/replication, prevent angiogenesis, act as checkpoint inhibitors, and in some cases act to bind a tumor antigen while also activating specific immune cells.
  • a monoclonal antibody is monospecific.
  • a monoclonal antibody is bispecific.
  • a monoclonal antibody is a checkpoint inhibitor.
  • a mAb may be used in combination with CAR-T therapy.
  • T-cell surface receptors When activated by therapeutic monoclonal antibodies, T-cell surface receptors can cause CRS.
  • antibodies which may induce CRS include anti-CD3 antibodies, anti-CD20 antibodies, anti-CD28 antibodies, anti-CTLA-4 antibodies, anti-PD-l antibodies, and anti-PD-Ll antibodies.
  • antibodies which may induce CRS include alemtuzumab, muromonab-CD3, rituximab, tosituzumab, CP- 870,893, LO-CD2a/BTI-322, TGN1412, pembrolizumab, nivolumab, or ipilimumab.
  • the methods of the invention may combine the use of an agent that alters the activity of a metabolic pathway, such as the agents described above, with another therapeutic approach, such as surgery or radiotherapy.
  • FIG. 4 is a scatter plot illustrating the concentration of brequinar in subject plasma over time when administered twice weekly.
  • FIG. 5 is a scatter plot illustrating the bioavailability of an IV formulation of brequinar as compared to an oral dosage form.
  • the concentration of DHO in a subject’s plasma is correlated with the concentration of DHODH inhibitor in the plasma.
  • the disclosed methods provide, in some embodiments, administering the DHODH inhibitor when the DHO concentration in the plasma is either at least a particular efficacy threshold or below a potential toxic threshold (i.e., a pre determined level).
  • FIG. 6 is a scatter plot illustrating the concentration of brequinar in mice at a dose of 50 mg/kg over time.
  • the dashed line illustrates that about 100 ng/mL concentration of DHO remains in the plasma at about 84 hours.
  • Brequinar was administered intravenously to 209 subjects once a week with a median number of doses per patient of 4 (range 1 to 24) at a median dose of 1200 mg/m (range 588 to 3110). Adverse events that were observed in more than 3% of subjects are reported in Table 4, below:
  • Example 3 Determining DHO Levels in Plasma Samples Using DHO as a Standard
  • the plasma samples Prior to analysis the plasma samples are deproteinized by centrifugation through a 50 kD Amicon ultrafilter. 10 pL of a plasma sample is spiked with 5 pL of a standard solution of (S)- 4,5-dihydroorotic-4,5,6-carboxy- 13 C4 acid ( 13 C4-DHO) and then diluted with 35 pL of 0.1% (w/w) formic acid. Samples are injected into a reverse-phase 4 pm C18 column (Synergy Hydro RP-80A, 3 pm, 150 x 3 mm; Phenomenex, Australia).
  • Chromatography is performed at 30° C with a total flow rate of 0.3 mL/min, using solvent A (aqueous 5 mM ammonium acetate, 0.05% (w/v) formic acid) and solvent B (0.05% (w/v) formic acid in methanol) in a linear gradient elution from A:B 98:2 (v/v) to 85:15 (v/v) over 11 minutes, the 40:60 (v/v) for 1 minute, before returning to initial conditions for a further 6 minutes of equilibration.
  • solvent A aqueous 5 mM ammonium acetate, 0.05% (w/v) formic acid
  • solvent B 0.05% (w/v) formic acid in methanol
  • Tandem mass spectrometry (LC/MS/MS) is performed using an Applied Biosystems API 4000 QTRAP mass spectrometer equipped with a Turbo-V-Spray source with the gas temperature set at 500° C.
  • the source operated an electrospray interface (ESI) with switching ionization polarity (between +5000 V and -4000 V) during the run (18 min).
  • ESI electrospray interface
  • the eluent is monitored by specific ion transitions for DHO and the internal standard. All data is quantified using Applied Biosystems software.
  • Example 4 Determining DHO Acid levels in Plasma Samples Using Orotic Acid as a Standard
  • the plasma samples Prior to analysis the plasma samples are deproteinized by centrifugation through a 50 kD Amicon ultrafilter. 10 pL of a plasma sample is spiked with 5 pL of a standard solution of 15N2- orotic acid and then diluted with 35 pL of 0.1% (w/w) formic acid. Samples are injected into a reverse-phase 4 pm C18 column (Synergy Hydro RP-80A, 3 pm, 150 x 3 mm; Phenomenex, Australia).
  • Chromatography is performed at 30° C with a total flow rate of 0.3 mL/min, using solvent A (aqueous 5 mM ammonium acetate, 0.05% (w/v) formic acid) and solvent B (0.05% (w/v) formic acid in methanol) in a linear gradient elution from A:B 98:2 (v/v) to 85:15 (v/v) over 11 minutes, the 40:60 (v/v) for 1 minute, before returning to initial conditions for a further 6 minutes of equilibration.
  • solvent A aqueous 5 mM ammonium acetate, 0.05% (w/v) formic acid
  • solvent B 0.05% (w/v) formic acid in methanol
  • Tandem mass spectrometry (LC/MS/MS) is performed using an Applied Biosystems API 4000 QTRAP mass spectrometer equipped with a Turbo- V-Spray source with the gas temperature set at 500° C.
  • the source operated an electrospray interface (ESI) with switching ionization polarity (between +5000 V and -4000 V during the run (18 min).
  • ESI electrospray interface
  • the eluent is monitored by specific ion transitions for DHO and the internal standard. All data was quantified using Applied Biosystems SCIEX Multiquant software.
  • the concentration of dihydroorotic acid in human K2EDTA plasma samples was determined by reversed-phase high performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). Plasma samples (50 pL) were spiked with 5 pL of a 1.0 pg/mL solution of (S)-4,5-dihydroorotic-4,5,6,carboxy- 13 C4 acid ( 13 C4-DHO) in water, which was used as the internal standard (IS), then vigorously mixed with acetonitrile (200 pL) for 5 min.
  • LC-MS/MS reversed-phase high performance liquid chromatography with tandem mass spectrometric detection
  • Nitrogen was used as the nebulizing gas (30 p.s.i.) and drying gas (10 L/min, 350° C). With a transfer capillary potential of 1,500 V, negative ions resulting from the m/z 157 113 transition for dihydroorotic acid and the m/z 161 l 17 transitions for the IS were measured by multiple reaction monitoring (dwell time, 150 msec; fragmentor potential, 70 V; collision energy, 4 V; collision cell accelerator voltage, 4 V). Quantitation was based upon integrating the extracted ion chromatograms for both transitions to provide peak areas and calculating the ratio of the analyte peak area to the IS peak area for each sample.
  • Table 5 provides data of DHO concentration for samples from certain random cancer patients, samples from healthy subjects, and samples from mice.
  • Table 6 provides patient data for 20 anonymous cancer patients whose DHO acid concentration was measured.
  • Table 7 provides baseline endogenous DHO acid concentration in plasma samples from the set of 20 cancer patients.
  • FIG. 7 is a scatter plot illustrating the baseline DHO levels in random cancer patients and healthy patients, as reported in Table 5.
  • Example 6 Clinical Dosing Regimens Previously Tested for Brequinar in Patients with Refractory Solid Tumors
  • Brequinar for phase II evaluation is 250 mg/m for good risk patients and 135 mg/m for poor risk patients.”
  • Burris reported“investigating the pharmacokinetic and toxicity of brequinar in combination with cisplatin” where patients were initially treated with weekly brequinar, in combination with an every-three-week administration of cisplatin. See Burris, et ah,
  • Schwartsmann reported dosing brequinar in 43 patients who“received 110 courses of Brequinar sodium by short-term intravenous (i.v.) infusion” every 3 weeks.” See Schwartsmann, et ah,“Phase I study of Brequinar sodium (NSC 368390) in patients with solid malignancies,” Cancer Chemother. Pharmacol., 25(5):345-35l (1990).
  • Example 7 Exemplary Clinical Dosing in Accordance with the Present Disclosure
  • the interval from prior leukemiadirected therapy to time of study initiation will be at least 7 days for cytotoxic or non-cytotoxic
  • Hydrea is allowed up to 48 hours prior to the first dose for patients with rapidly proliferative disease.
  • Intrathecal chemotherapy for prophylactic use or maintenance of controlled CNS leukemia • Intrathecal chemotherapy for prophylactic use or maintenance of controlled CNS leukemia. • Use of hydroxyurea may be allowed during the first 2 weeks of therapy if in the best interest of the participant and is approved by the medical monitor.
  • GVHD graft versus host disease
  • APL acute promyelocytic leukemia
  • HBV hepatitis B
  • HCV hepatitis C
  • Prior malignancy unless it has not been active or has remained stable for at least 5 years. Participants with treated non-melanoma skin cancer, in situ carcinoma or cervical intraepithelial neoplasia, regardless of the disease-free duration, are eligible if definitive treatment for the condition has been completed. Participants with organ-confined prostate cancer with no evidence of recurrent or progressive disease are eligible if hormonal therapy has been initiated or the malignancy has been surgically removed or treated with definitive radiotherapy.
  • Another example dosing schema is:
  • the dosing sequence (i.e. every 3.5 days) will be subject to revision after review of preliminary efficacy, toxicity, and PK data within this clinical trial.
  • PK data from patients treated at dose level 0 will be used to evaluate the anticipated minimally effective dose, to adjust the dose and schedule, if necessary, in subsequent dose level cohorts.
  • the following assay protocol is useful for measuring the concentration of analytes such as pyrazofurin, orotate (i.e., orotate), orotidylate monophosphate (OMP), and uradilyate monophosphate (UMP) in serum samples of subjects.
  • analytes such as pyrazofurin, orotate (i.e., orotate), orotidylate monophosphate (OMP), and uradilyate monophosphate (UMP) in serum samples of subjects.
  • AMP adenosine monophosphate
  • the acetonitrile: methanol solution is evaporated at 50° C with nitrogen and reconstituted with 150 pL of water for injection. Samples are injected into a reverse-phase Waters Atlantis T3 2.1 mm x 100 mm, 3 pm column.
  • Chromatography is performed, using solvent A (aqueous 10 mM ammonium acetate, pH 4.8) and solvent B (0.1% (w/v) formic acid in methanol) in a linear gradient elution from A:B 98:2 (v/v) to 85:15 (v/v) over 11 minutes, the 40:60 (v/v) for 1 minute, before returning to initial conditions for a further 6 minutes of equilibration.
  • solvent A aqueous 10 mM ammonium acetate, pH 4.8
  • solvent B 0.1% (w/v) formic acid in methanol
  • Tandem mass spectrometry (LC/MS/MS) is performed using an Applied Biosystems API 5000 QTRAP mass spectrometer equipped with a Turbo-V-Spray source with the gas temperature set at 500° C.
  • the source operated an electrospray interface (ESI) with switching ionization polarity (between +5000 V and -4000 V) during the run (18 min).
  • ESI electrospray interface
  • the eluent is monitored by specific ion transitions for DHO and the internal standard. All data is quantified using Applied Biosystems software.
  • OMP decarboxylase inhibitor pyrazofurin was administered to mice by oral gavage.
  • concentration (ng/mL) of analytes selected from pyrazofurin (PYR), orotic acid (i.e., orotate), orotidylate monophosphate (OMP), and uradilyate monophosphate (UMP) in the serum samples were measured according to the assay methods reported in Example 1. The results are reported in Table 9:
  • FIG. 8 is a scatter plot illustrating the concentrations of pyrazofurin and orotate in murine plasma over time when pyrazofurin is administered as a single dose (20 mg/kg).
  • FIG. 9 is a scatter plot illustrating the concentrations of pyrazofurin and orotate in murine plasma over time when pyrazofurin is administered as a single dose (20 mg/kg) on a log scale.
  • Example 10 Prior Dosing Regimens
  • pyrazofurin “by iv bolus at a dose level ranging from 100 to 300 mg/m of estimated body surface area.” Further,“five patients with acute leukemia were given [pyrazofurin] by infusion at doses ranging from 250 mg/m 2 /24 hours to 1500 mg/m 2 /144 hours.”
  • Ohnuma and Holland “Initial Clinical Study with Pyrazofurin,” Cancer Treatment Reports, 6l(3):389-l34 (May/June 1977).
  • Example 11 Optimized dosage based metabolite levels
  • FIG. 10 is a graph showing the therapeutic benefit of a drug, such as brequinar, that targets a metabolic pathway as a function of levels of a metabolite, such as DHO, that is an intermediate in the pathway.
  • a drug such as brequinar
  • levels of the metabolite are below a minimum threshold, and target engagement of the drug is insufficient to have a therapeutic effect.
  • levels of the metabolite are above a minimum threshold but below a maximum threshold, so the drug has sufficiently engaged its target to provide a therapeutic effect but has not caused effects that are deleterious to healthy cells.
  • levels of the metabolite are above the maximum threshold, and the effects of the drug cause harm to healthy cells. Adjustments to the dosing regimen based on the relationship between therapeutic benefit and metabolite levels are illustrated in Table 10.
  • Example 12 Effect of brequinar-containing composition on patient with AML
  • AML acute myeloid leukemia
  • Example 13 Effect of brequinar-containing composition on patient with AML
  • the effect of a composition containing brequinar was analyzed on second patient a with AML. After administration of a dose of the composition, the patient achieved a DHO plasma level threshold of 2,900 ng/mL in less 24 hours and remained above that threshold for 84 hours. This patient showed a positive response to the disease with a lowering of peripheral blasts and increase in absolute neutrophil count, along with greater differentiation of peripheral blasts.
  • Example 14 Effect of brequinar-containing composition on patient with AML
  • the effect of a composition containing brequinar was analyzed on second patient a with AML. After administration of a dose of the composition, the patient achieved a DHO plasma level threshold of 133 ng/mL in less than 2 hours and remained above that threshold for 84 hours. This patient showed a positive response as indicated by a trend towards differentiation of his peripheral blasts.

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Abstract

L'invention concerne des procédés de détermination d'une dose thérapeutiquement efficace d'un agent qui cible une voie métabolique en se basant sur les niveaux mesurés d'un métabolite dans la voie. Les procédés, qui peuvent comprendre la fourniture de l'agent dans une dose thérapeutiquement efficace, sont utiles pour le traitement de troubles tels que le cancer chez un sujet. L'invention concerne également des procédés d'évaluation de l'impact d'un agent thérapeutique sur une tumeur chez un sujet par surveillance en temps réel du métabolisme d'une molécule dans la tumeur, l'oxygénation de la tumeur, ou les deux. L'invention concerne en outre des dispositifs qui déterminent une dose thérapeutiquement efficace d'un agent qui cible une voie métabolique en se basant sur les niveaux mesurés d'un métabolite dans la voie et notifient un sujet d'administrer la dose.
PCT/US2019/023983 2018-03-26 2019-03-26 Procédés de détermination du dosage d'un agent thérapeutique en se basant sur les niveaux mesurés d'un métabolite WO2019191030A1 (fr)

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WO2021188504A1 (fr) * 2020-03-18 2021-09-23 Flow Pharma, Inc. Formulation injectable de microsphères d'acide poly(lactique-co-glycolique) (plga) encapsulant du siltuximab

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