JP2023518419A - Stable polymorphic compositions of brequinal sodium and methods of use and preparation thereof - Google Patents

Abstract

The present invention provides compositions containing stable crystalline forms of brequinar and methods of making such compositions. The invention also provides methods of treating conditions in a subject using such compositions. The present invention recognizes that crystalline brequinal sodium salt exists in multiple polymorphic forms, one of these forms, polymorphic Form C, being the most stable under ambient conditions. Thus, the stable crystalline forms of brequinal sodium salt described herein allow the drug to be formulated into pharmaceutical compositions amenable to commercial production and distribution (e.g., solid pharmaceutical compositions for oral administration). allow it to be transformed.

Description

FIELD OF THE INVENTION The present invention relates generally to pharmaceutical compositions containing crystalline forms of brequinal sodium salt, methods of making such compositions, and methods of using such compositions to treat a condition in a subject. .

BACKGROUND Brequinar is a compound that acts as a potent and selective inhibitor of the enzyme dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine synthesis. This drug blocks the synthesis of pyrimidine-based nucleotides in the body, thus inhibiting cell growth. Brequinar has been investigated for a number of different indications.

SUMMARY The present invention recognizes that crystalline brequinal sodium salt exists in multiple polymorphic forms, one of these forms, polymorphic Form C, being the most stable under ambient conditions. . Accordingly, the stable crystalline forms of brequinal sodium salt described herein are useful for formulation of pharmaceutical compositions (e.g., solid pharmaceutical compositions for oral administration) that are amenable to commercial production and distribution of the drug. allow it to be Prior to the insight of the present invention, the production of crystalline brequinal sodium salt appears to be too unpredictable for large scale production, such as the production of solid oral dosage forms such as tablets and capsules. It was The composition of the present invention overcomes previous stability and consistency problems in the manufacturing process to provide the most stable form of brequinal sodium. The polymorphic forms of brequinar sodium salt described herein enable the manufacture and distribution of solid dosage forms that can be readily self-administered by patients. The present invention also provides a method of making polymorphic Form C crystals of brequinal sodium salt. The present invention also provides methods of treating various conditions using compositions containing polymorphic Form C of brequinal sodium salt. Notably, polymorphic Form C of brequinar sodium salt can be formulated into any type of pharmaceutical formulation for any route of delivery.

In one aspect, the invention provides crystals containing polymorphic Form C of brequinal sodium salt. Polymorphic Form C may contain brequinar sodium salt hydrate. In another aspect, the invention provides a pharmaceutical composition containing a solid form of brequinal sodium salt, wherein at least 75% of the brequinal sodium salt is polymorphic Form C. The composition is a solid form of brequinal sodium salt and contains at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of brequinal sodium salt. % or at least 99.5% is polymorphic Form C. Polymorphic Form C of brequinal sodium salt can include brequinal sodium salt hydrate. A composition can be formulated for administration by a particular route. For example, compositions can be formulated for oral, intravenous, enteral, parenteral, dermal, buccal, topical, transdermal, subcutaneous, nasal or pulmonary administration. The composition can be formulated for administration by injection or with or on an implantable medical device.

In another aspect, the invention provides a method of treating a condition in a subject having the condition by providing the subject with a pharmaceutical composition comprising brequinal sodium salt, wherein at least 75% of the brequinal sodium salt comprises Polymorphic Form C is provided. The method comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the brequinal sodium salt is in the polymorphic form. C, providing a composition containing brequinal sodium salt. Polymorphic Form C of brequinal sodium salt can include brequinal sodium salt hydrate. Compositions containing polymorphic Form C of brequinal sodium salt can be administered in any type of pharmaceutical composition suitable for any route of administration. The compositions may be administered orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, subcutaneously, nasally, pulmonally or implanted. It can be provided with a medical device or on an implantable medical device.

The compositions described herein can be used to treat any condition associated with brequinar, such as cancer or viral infections or autoimmune disorders. In certain embodiments, the condition can be a viral infection. Viral infections can include adenovirus, coronavirus, enterovirus, human metapneumovirus, human parainfluenza virus, human respiratory syncytial virus, influenza virus or rhinovirus. The coronavirus may be Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The influenza virus can be influenza A, influenza B, influenza C or influenza D. Influenza A viruses can be H1N1, H3N2, N9N2 or H5N1 strains. Infections can affect specific tissues, organs or systems. Infections can affect one or more of the alveoli, bronchi, bronchioles, larynx, lungs, nasal cavities, nose, pharynx, respiratory system, sinuses and trachea.

In other embodiments, the condition can be cancer. Cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adult T-cell leukemia/lymphoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, Glioma, head and neck cancer, renal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer cancer or uterine cancer.

The condition can be an autoimmune disorder. The autoimmune disorder can be arthritis, hepatitis, chronic obstructive pulmonary disease, multiple sclerosis or tendonitis.

In another aspect, the present invention provides a method of making a crystal containing polymorphic Form C of brequinar sodium salt, comprising a solid form of brequinar acid, sodium hydroxide (NaOH) and a solvent. In combination, a method is provided comprising the steps of forming a solution and crystallizing brequinal sodium salt from the solution to produce crystals comprising polymorphic Form C of brequinal sodium salt. Polymorphic Form C of brequinal sodium salt can include brequinal sodium salt hydrate.

Solvents can contain isopropanol, water, or both. The solvent can contain isopropanol and water in a defined ratio. Solvents are from about 90:10 to about 99:1, from about 92:8 to about 99:1, from about 95:5 to about 99:1, from about 96:4 to about 99:1, from about 97:3 to about 99:1 :1, about 90:10 to about 98:2, about 92:8 to about 98:2, about 95:5 to about 98:2, about 96:4 to about 98:2, about 97:3 to about 98 :2, about 95:5, about 96:4, about 97:3 or about 98:2 isopropanol and water.

A solid form of brequinaric acid can be combined with NaOH in a defined ratio. The solid forms of NaOH and brequinaric acid are about 0.25 to about 4.0, about 0.5 to about 4.0, about 0.75 to about 4.0, about 1.0 to about 4.0, about 0.25 to about 2.0, about 0.5 to about 2.0, about 0.75 to about 2.0, about 1.0 to about 2.0, about 0.25 to about 1.6, about 0.5 to about 1.6, about 0.75 to about 1.6, about 1.0 to about 1.6, about 0.25 to about 1.3, about 0.5 to about 1.3, about 0.75 to about 1.3, about 1.0 to about 1.3, about 0.25 to about 1.1, about 0.5 to about 1.1, about 0.75 to about 1.1, about They can be combined in molar equivalents of 1.0 to about 1.1, about 1.0, about 1.05 or about 1.1.

The combining step comprises heating the combined brequinaric acid, NaOH and solvent to about 60°C to about 90°C, about 65°C to about 90°C, about 70°C to about 90°C, about 73°C to about 90°C, about 60°C. to about 85°C, about 65°C to about 85°C, about 70°C to about 85°C, about 73°C to about 85°C, about 60°C to about 80°C, about 65°C to about 80°C, about 70°C to about 80°C, about 73°C to about 80°C, about 60°C to about 77°C, about 65°C to about 77°C, about 70°C to about 77°C, about 73°C to about 77°C, about 73°C, about 75°C or may include incubating at about 77°C.

Combining can include removing insoluble materials from the solution or combined brequinaric acid, NaOH and solvent. Insoluble material can be removed by filtration.

The combining step comprises heating the combined brequinaric acid, NaOH and solvent to about 50° C. to about 70° C., about 55° C. to about 70° C., about 50° C. to about 65° C., about 55° C. to about 65° C., about 55° C. , at about 60°C or about 65°C.

Combining can include two or more of the above steps performed in a particular order. The combining step can include two or more of the following steps performed in the following order: incubating the combined brequinaric acid, NaOH and solvent at a first temperature; and incubating the combined brequinal, NaOH and solvent at a second temperature that can be lower than the first temperature.

The crystallizing step can comprise adding seed crystals of polymorphic Form C of brequinal sodium salt to the solution to form a seeded mixture. The crystallizing step heats the seeded mixture to about 15° C. to about 35° C., about 20° C. to about 35° C., about 15° C. to about 30° C., about 20° C. to about 30° C., about 20° C., about Incubating at 25°C or about 30°C can be included. Crystallizing can include adding an alkane to the solution or seeded mixture to produce an alkane-containing mixture. The alkane can be n-heptane. Crystallizing can include incubating the alkane-containing mixture for a defined period of time. Crystallizing can include incubating the alkane-containing mixture for about 6 hours to about 24 hours. Crystallizing can include removing the solvent from polymorphic Form C of brequinal sodium salt. Removing the solvent can include filtering polymorphic Form C of brequinal sodium salt, drying polymorphic Form C of brequinal sodium salt, or both.

Crystallizing can include two or more of the above steps performed in a particular order. The crystallizing step can comprise two or more of the following steps performed in the following order: adding seed crystals of polymorphic Form C of brequinal sodium salt to the solution; incubating the seeded mixture at a defined temperature; adding an alkane to the solvent or the seeded mixture; incubating the alkane-containing mixture for a defined period of time. and removing the solvent from polymorphic Form C of brequinar sodium salt.

FIG. 1 is an X-ray powder diffraction (XRPD) pattern of brequinal sodium salt from batch PS04375-1-E-P4.

FIG. 2 is a differential scanning calorimetry (DSC) thermogram of brequinal sodium salt from batch PS04375-1-E-P4.

FIG. 3 is a thermogravimetric analysis (TGA) thermogram of brequinal sodium salt from batch PS04375-1-E-P4.

FIG. 4 is an overlay of XRPD scan patterns AJ of brequinar sodium salt from batch PS04375-1-E-P4.

FIG. 5 is a DSC thermogram of polymorphic Form C of brequinar sodium salt from batch FR00514-2-SL-B9.

FIG. 6 is a TGA thermogram of polymorphic Form C of brequinar sodium salt from batch FR00514-2-SL-B9.

FIG. 7 is a graph of the ¹ H NMR spectrum of polymorphic Form C of brequinal sodium salt from batch FR00514-2-SL-B9.

FIG. 8 is an X-ray powder diffraction (XRPD) pattern of polymorphic Form C of brequinal sodium salt prepared according to the method of the present invention.

DETAILED DESCRIPTION Brequinar has been investigated as a potential therapeutic agent for treating a variety of conditions. Brequinar was identified over 30 years ago, but one of the limitations in its therapeutic utility has been the difficulty in formulating the compound for commercial scale manufacturing and production. The present invention overcomes this problem by identifying crystalline forms of brequinar sodium salt that are stable and consistently reproducible in commercial manufacturing processes. The present invention shows that crystals of brequinal sodium salt can exist in various polymorphic forms and that only one of those forms, polymorphic form C, is stable under ambient conditions. recognize. The present invention also provides a method of producing crystals containing polymorphic Form C of brequinal sodium salt. Thus, the compositions and methods of the present invention enable the production of stable solid forms of brequinal sodium that are suitable for large-scale manufacturing, distribution and storage. The compositions herein can be formulated for any pharmaceutical composition for any route of administration.

を有する。 Compositions Containing Polymorphic Form C of Brequinal Sodium Salt The present invention provides crystals of polymorphic Form C of brequinal sodium salt and pharmaceutical compositions containing such crystals. Brequinar has the systematic name 6-fluoro-2-(2′-fluoro-1,1′biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid and the following structure:

have

Brequinar and related compounds are described, for example, in US Pat. Nos. 4,680,299 and 5,523,408, the contents of which are incorporated herein by reference.

Brequinar is an inhibitor of dihydroorotate dehydrogenase (DHODH), a key enzyme involved in pyrimidine synthesis. DHODH converts dihydroorotate (DHO) to orotate, and inhibition of DHODH activity leads to accumulation of DHO. Since cell membranes are permeable to DHO, inhibition of DHODH in the body results in elevated levels of DHO in blood and other body fluids. Therefore, the activity of brequinar at its target can be monitored in vivo by analysis of DHO levels in body fluid samples. The use of metabolites such as DHO to assess in vivo target engagement of DHODH inhibitors such as brequinar is known in the art, e.g. It is described in publication numbers WO2019/191030 and WO2019/191032.

Crystals of brequinar sodium salt exist in several different polymorphic forms. As described in more detail in the Examples, the inventors identified ten distinct X-ray powder diffraction (XRPD) patterns, patterns AJ, in crystals of brequinal sodium salt. Seven of these patterns, patterns B, D, E, F, G, I and J, are unstable polymorphs that spontaneously convert to polymorphic form C under conditions of ambient temperature and humidity. Represents gender. Pattern A represents a metastable form, which also changes over time to other forms when stored at ambient conditions. In contrast, polymorphic form C is stable under ambient conditions. As a result, polymorphic Form C of brequinal sodium salt retains its density and does not tend to swell or shrink during manufacture, distribution and storage of the drug, thus making it useful in solid oral dosage formulations such as tablets. most desirable for

In crystals containing polymorphic form C of brequinal sodium salt, the crystals may contain brequinal sodium salt hydrate.

In the pharmaceutical compositions of the present invention, the compositions may contain all or almost all of brequinal sodium salt in polymorphic form C. By way of example and without limitation, the composition contains at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of brequinal sodium salt. , brequinal sodium salt that is at least 99% or at least 99.5% polymorphic Form C.

Compositions can be formulated for administration by any route. By way of example and without limitation, compositions are formulated for oral, intravenous, enteral, parenteral, dermal, buccal, topical, transdermal, subcutaneous, nasal or pulmonary administration. be able to. The composition can be formulated for administration by injection or with or on an implantable medical device. By way of example and not limitation, compositions for oral administration may be formulated as caplets, capsules, pills or tablets.

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 be pharmaceutically elegant and palatable. In order to provide palatable preparations, one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents may be included. Tablets contain the compound in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. Such excipients are, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binding agents such as starch. , gelatin or acacia, and lubricants such as 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 down the gastrointestinal tract, thereby providing a sustained action over a longer period of time. . For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Tablets are described in U.S. Pat. Nos. 4,256,108, 4,166,452 and 4,265,874 to form osmotic therapeutic tablets for controlled release. It may be coated by techniques. The preparation and administration of compounds are described in US Pat. No. 6,214,841 and US Patent Application Publication No. 2003/0232877, the contents of each of which are incorporated herein by reference.

Formulations for oral use may be presented as hard gelatin capsules in which the compound is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin.

The pharmaceutical composition contains sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; Other pharmaceutically acceptable carriers such as excipients can be included.

A pharmaceutically acceptable carrier can be an encapsulating coating. For example, encapsulating coatings include carrageenan, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate trimellitate, collagen, gelatin, hydroxypropyl methylcellulose acetate, methyl acrylate-methacrylic acid copolymer, polyvinyl acetate phthalate shellac, sodium alginate, starch. Or it can contain zein.

The composition can be formulated for non-oral routes of administration. By way of example and not limitation, compositions may be formulated for intravenous, enteral, parenteral, dermal, buccal, topical, transdermal, subcutaneous, nasal or pulmonary administration. The composition can be formulated for administration by injection or with or on an implantable medical device.

The composition can be formulated for direct, non-oral administration. Alternatively, the compositions can be formulated to be mixed, dissolved, or converted to another format prior to administration. By way of example and not limitation, brequinar can be administered as a solution, suspension, emulsion, cream, paste, ointment, or any other format suitable for non-oral administration.

The composition can be formulated for a single unit dosage. Compositions may be formulated for divided doses.

Methods of Making Crystals of Polymorphic Form C of Brequinal Sodium Salt The present invention provides methods of making crystals containing polymorphic Form C of brequinal sodium salt. The method comprises combining a solid form of brequinaric acid, sodium hydroxide (NaOH) and a solvent to form a solution, and crystallizing brequinar from the solution to form polymorphic Form C crystals of brequinar sodium salt. and the step of As described in more detail below, the method may include additional steps, and the steps described above may include steps or substeps therein.

Solvents can contain organic solvents, water, or both. The organic solvent can be an alcohol such as ethanol or isopropanol. The solvent can contain an alcohol such as isopropanol and water in a defined ratio. By way of example and not limitation, the solvents are from about 90:10 to about 99:1, from about 92:8 to about 99:1, from about 95:5 to about 99:1, from about 96:4 to about 99:1 :1, about 97:3 to about 99:1, about 90:10 to about 98:2, about 92:8 to about 98:2, about 95:5 to about 98:2, about 96:4 to about 98 :2, about 97:3 to about 98:2, about 95:5, about 96:4, about 97:3 or about 98:2 isopropanol and water.

A strong base such as NaOH can be added to the mixture to facilitate dissolution of the solid brequinaric acid in the solvent. A solid form of brequinaric acid can be combined with NaOH in a defined ratio. By way of example and not limitation, solid forms of NaOH and brequinal are about 0.25 to about 4.0, about 0.5 to about 4.0, about 0.75 to about 4.0, about 1 .0 to about 4.0, about 0.25 to about 2.0, about 0.5 to about 2.0, about 0.75 to about 2.0, about 1.0 to about 2.0, about 0 .25 to about 1.6, about 0.5 to about 1.6, about 0.75 to about 1.6, about 1.0 to about 1.6, about 0.25 to about 1.3, about 0 .5 to about 1.3, about 0.75 to about 1.3, about 1.0 to about 1.3, about 0.25 to about 1.1, about 0.5 to about 1.1, about 0 .75 to about 1.1, about 1.0 to about 1.1, about 1.0, about 1.05 or about 1.1 molar equivalents.

The mixture can be heated, ie, incubated, at a first defined temperature to facilitate dissolution of the solid brequinaric acid in the solvent. By way of example and not limitation, the mixture may be heated from about 60° C. to about 90° C., from about 65° C. to about 90° C., from about 70° C. to about 90° C., from about 73° C. to about 90° C., from about 60° C. to about 85°C, about 65°C to about 85°C, about 70°C to about 85°C, about 73°C to about 85°C, about 60°C to about 80°C, about 65°C to about 80°C, about 70°C to about 80°C , about 73° C. to about 80° C., about 60° C. to about 77° C., about 65° C. to about 77° C., about 70° C. to about 77° C., about 73° C. to about 77° C., about 73° C., about 75° C., or about Incubate at 77°C.

It may be necessary to remove insoluble materials from the mixture. For example, insoluble material can be removed by filtration.

After heating and/or filtering, the mixture or solution can be cooled, ie incubated, at a second defined temperature that is lower than the first defined temperature. By way of example and not limitation, the solution can be at about 50° C. to about 70° C., about 55° C. to about 70° C., about 50° C. to about 65° C., about 55° C. to about 65° C., about 55° C., about It can be incubated at 60°C or about 65°C.

The above steps involved in making a brequinal solution can be performed in a specific order. By way of example and not limitation, two or more of the steps can be performed in the following order: a solid form of brequinaric acid and a solvent, optionally including a strong base such as NaOH. heating the mixture by incubating it at a first temperature described above; removing insoluble material from the mixture; and heating it at a second temperature described above. cooling the mixture by incubating.

The crystallizing step can comprise adding seed crystals of polymorphic Form C of brequinal sodium salt to the solution to form a seeded mixture.

Crystallizing can include incubating the seeded mixture at a defined temperature. By way of example and not limitation, the seeded mixture can be at about 15° C. to about 35° C., about 20° C. to about 35° C., about 15° C. to about 30° C., about 20° C. to about 30° C., about Incubation can be at 20°C, about 25°C, or about 30°C.

Crystallizing can include adding an agent that facilitates precipitation of brequinal sodium salt. The agent can be an organic compound such as an alkane. The alkane can be n-heptane.

Crystallizing can include incubating the alkane-containing mixture for a defined period of time. Crystallizing can include incubating the alkane-containing mixture for about 6 hours to about 24 hours.

Crystallizing can include removing the solvent from the precipitated brequinal sodium salt. Removing the solvent can include filtering the brequinal sodium salt, drying the brequinal sodium salt, or both.

Crystallizing can include two or more of the above steps performed in a particular order. By way of example and not limitation, two or more of the steps can be performed in the following order: Seed crystals of polymorphic Form C of brequinal sodium salt are added to the solution. incubating the seeded mixture at a defined temperature; adding an alkane to the seeded mixture; incubating the alkane-containing mixture for a defined period of time. and removing the solvent from polymorphic Form C of brequinar.

Further details regarding the preparation of polymorphic Form C of brequinar are provided in the Examples.

Method of Treating a Condition with Polymorphic Form C of Brequinar Condition The present invention provides a method of treating a condition in a subject by providing the subject with a composition comprising polymorphic Form C of brequinar sodium salt. do. The condition can be any disease, disorder or condition for which brequinar provides therapeutic benefit.

The condition can be cancer. Cancer can include solid tumors or hematologic tumors. By way of example and without limitation, cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult T-cell leukemia/lymphoma (ATLL), bladder cancer, triple-negative breast cancer ( Breast cancer such as TNBC), cervical cancer, colorectal cancer, gastric cancer, glioblastoma, glioma, head and neck cancer, renal cancer, leukemia, liver cancer, small cell lung cancer and non-small cell lung cancer lung cancer such as lymphoma, melanoma, Merkel cell carcinoma, myeloma such as multiple myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer or uterine cancer.

In AML, myeloblasts that are arrested early in differentiation proliferate in an uncontrolled manner and interfere with the development of other blood cells in the bone marrow. Inhibitors of dihydroorotate dehydrogenase (DHODH) cause differentiation of myeloblasts and prevent their leukemia-initiating activity. The role of DHODH in AML has been reviewed by Sykes et al. , Inhibition of Dihydrorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia, Cell 167, 171-186, September 22, 2016; dx. doi. org/10.1016/j. cell. 2016.08.057.

A condition can be or include one or more subtypes of AML. AML cases are classified based on cytological, genetic and other criteria, and AML treatment strategies vary according to classification. One AML classification system is provided by the World Health Organization (WHO). The WHO classification system includes the subtypes of AML provided in Table 1, the contents of which are incorporated herein by reference, 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.

An alternative classification scheme for AML is the French-American-British (FAB) classification system. The FAB classification system includes the subtypes of AML provided in Table 2, the contents of each of which are incorporated herein by reference, Bennett JM, et al. (August 1976). "Proposals for the classification of the acute leukemias. French-American-British (FAB) co-operative group" Br. J. Haematol. 33 (4): 451-8, doi: 10.1111/j. 1365-2141.1976. tb03563. x. PMID 188440; and Bennett JM, et al. (June 1989) "Proposals for the classification of chronic (mature) B and T lymphoid leukemias. French-American-British (FAB) Cooperative Group"J. Clin. Pathol. 42 (6): 567-84, doi: 10.1136/jcp. 42.6.567, PMC 1141984, PMID 2738163.

The condition can be an inflammatory or autoimmune disorder. By way of example and without limitation, the inflammatory or autoimmune disorder can be arthritis, hepatitis, chronic obstructive pulmonary disease, multiple sclerosis or tendonitis.

The condition can be a psychiatric disorder. By way of example and without limitation, the psychiatric disorder is anxiety, stress, obsessive-compulsive disorder, depression, panic disorder, psychosis, addiction, alcoholism, attention deficit hyperactivity disorder, agoraphobia, schizophrenia or social phobia. could be.

The condition can be an infection. The infection can be a viral infection. By way of example and without limitation, viral infections are infections by adenovirus, coronavirus, enterovirus, human metapneumovirus, human parainfluenza virus, human respiratory syncytial virus, influenza virus or rhinovirus. obtain. The coronavirus may be Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The influenza virus can be influenza A, influenza B, influenza C or influenza D. Influenza A viruses can be H1N1, H3N2, N9N2 or H5N1 strains.

Infections can affect specific tissues, organs or systems. Infections can affect one or more of the alveoli, bronchi, bronchioles, larynx, lungs, nasal cavities, nose, pharynx, respiratory system, sinuses and trachea.

The condition can include genetic mutations such as MYC amplification or PTEN loss that result in increased dependence in metabolic pathways such as increased "addiction" to glutamine.

A condition may involve or affect a subpopulation of patients. For example, the patient can be a child, newborn, infant, infant, infant, child, adolescent, pre-teen, teenager, adult or geriatric. Patients are treated in critical care, intensive care, neonatal intensive care, pediatric intensive care, coronary intensive care, cardiothoracic care, surgical intensive care, medical intensive care, long-term intensive care, surgery It can be placed in a room, ambulance, field hospital or out-of-hospital field setting.

Monitoring Metabolite Levels in a Sample Methods of treating a condition in a subject can include monitoring levels of a metabolite, such as DHO, in a sample obtained from the subject. Monitoring levels of metabolites can include receiving information about measured levels of metabolites. Monitoring the level of metabolites can include measuring the metabolites.

In some embodiments, metabolites are measured by mass spectrometry optionally combined with liquid chromatography. Molecules can be subjected to ambient ionization, chemical ionization (CI), desorption electrospray ionization (DESI), electron impact (EI), 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) for mass spectrometry by any method known in the art. Methods of mass spectrometry are known in the art, for example US Pat. No. 8,895,918; US Pat. No. 9,546,979, the contents of each of which are incorporated herein by reference. U.S. Pat. No. 9,761,426; Hoffman and Strobant, Mass Spectrometry: Principles and Applications (2nd ed.). John Wiley and Sons (2001), ISBN 0-471-48566-7; Dass, Principles and practices of biological mass spectrometry, New York: John Wiley (2001) ISBN 0 -471-33053-1; and Lee, ed. , Mass Spectrometry Handbook, John Wiley and Sons, (2012) ISBN: 978-0-470-53673-5.

In certain embodiments, the sample can be directly ionized without the need for use of a separation system. In other embodiments, mass spectrometry is performed in conjunction with methods for resolving and identifying ion 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 chromatography (UPLC) and Including reverse phase liquid chromatography (RPLC). In preferred embodiments, liquid chromatography-mass spectrometry (LC-MS) is used. Methods for coupling chromatography and mass spectrometry are known in the art, see, for example, Holcapek and Brydwell, eds. Handbook of Advanced Chromatography/Mass Spectrometry Techniques, Academic Press and AOCS Press (2017), ISBN 9780128117323; Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry, The Clinical Biochemist Reviews. 30(1): 19-34 (2017) ISSN 0159-8090; Niessen, Liquid Chromatography-Mass Spectrometry, Third Edition. Boca Raton: CRC Taylor & Francis. pp. 50-90. (2006) ISBN 9780824740825; Ohnesorge et al. , Quantitation in capillary electrophoresis-mass spectrometry, Electrophoresis. 26 (21): 3973-87 (2005) doi: 10.1002/elps. 200500398; Kolch et al. , Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery, Mass Spectrom Rev. 24 (6): 959-77. (2005) doi: 10.1002/mas. 20051; Kanu et al. , Ion mobility-mass spectrometry, Journal of Mass Spectrometry, 43 (1): 1-22 (2008) doi: 10.1002/jms. 1383.

A sample can be obtained from any organ or tissue in the individual to be examined, provided that the sample is obtained in liquid form or can be pretreated to obtain liquid form. . By way of example and without limitation, samples may include blood samples, urine samples, serum samples, semen samples, sputum samples, lymph samples, cerebrospinal fluid samples, plasma samples, pus samples, amniotic fluid samples, biological fluid samples, faeces. samples, biopsy samples, needle aspiration biopsy samples, swab samples, mouthwash samples, cancer samples, tumor samples, tissue samples, cell samples, synovial fluid samples, phlegm samples, saliva samples, It can be a sweat sample, or a combination of such samples. The sample can also be a solid or semi-solid sample, such as a tissue sample, stool sample or stool sample that has been treated to assume liquid form, for example by homogenization, sonication, pipetting, cell lysis, or the like. . For the methods described herein, samples are preferably derived from plasma, serum, whole blood or sputum.

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

In some embodiments, samples are treated to remove cells or other biological particulates. Methods for removing cells from blood or other samples are well known in the art and can include, for example, centrifugation, sedimentation, ultrafiltration, immunoselection, and the like.

The sample can be obtained from the individual before or after administration to the subject of an agent that alters the activity of a metabolic pathway, such as an inhibitor of an enzyme in the pathway. For example, samples may be administered at 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 after administration of the drug. or can be obtained before or 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 hours of administration of the drug It can be obtained in days, 6 days, 7 days or later.

Dosing Regimens Including Determination of Dosing Regimens Based on Metabolite Levels Methods of treating a condition can include providing a composition comprising polymorphic Form C of brequinal sodium salt to a subject according to a dosing regimen. . A composition containing polymorphic Form C of brequinal sodium salt in solid form can be administered in its solid form, or can be mixed, dissolved or converted to another format prior to administration. . Such compositions may be administered orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, subcutaneously, nasally, pulmonally, or can be provided with or on an implantable medical device.

A dosing regimen can include dosage amounts and schedules of administration. The dosage of brequinar includes the amount of drug. The amount of brequinal can be expressed in absolute terms, eg, in terms of mass of brequinal. The amount of brequinal can be expressed in terms of an amount for an object, eg, brequinal mass per object mass, or brequinal mass per object volume. The amount of brequinar can be expressed, for example, as the amount of brequinar that achieves a target concentration in a subject-derived tissue or sample, in terms of indicating the effect of the drug. Dosages can include the time period over which the amount should be administered to a subject. Thus, dosages can include amounts of brequinar per unit of time. A dosage can comprise a single dose, ie, the entire amount can be provided at once. Alternatively, the dosage may comprise multiple, eg, 2, 3, 4, 6 or 8, doses which together achieve the total dosage. The schedule of administration may be by intervals between doses, e.g., every 24 hours, every 48 hours, etc., or by the number of doses administered in a given period, e.g., once a week, weekly It can be described as twice and so on.

A dosing regimen can include a schedule for administration of doses. For example, doses can be administered 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, weekly It can be administered at regular intervals, such as every, every two weeks, every three weeks or every four weeks. Alternatively, doses can be administered according to a schedule that does not require precise periodic intervals. For example, the dosage can be once a week, twice a week, three times a week, four times a week, once a month, twice a month, three times a month, monthly It can be administered 4 times, 5 times a month or 6 times a month.

By way of example and not limitation, dosages of brequinar range from about 10 mg to about 180 mg, from about 26 mg to about 180 mg, from about 51 mg to about 180 mg, from about 76 mg to about 180 mg, from about 101 mg to about 180 mg, from about 126 mg to about 180 mg, about 151 mg to about 180 mg, about 10 mg to about 150 mg, about 26 mg to about 150 mg, about 51 mg to about 150 mg, about 76 mg to about 150 mg, about 101 mg to about 150 mg, about 126 mg to about 150 mg, about 10 mg to about 125 mg, about 26 mg to about 125 mg, about 51 mg to about 125 mg, about 76 mg to about 125 mg, about 101 mg to about 125 mg, about 10 mg to about 100 mg, about 26 mg to about 100 mg, about 51 mg to about 100 mg, about 76 mg to about 100 mg, about 10 mg to about 75 mg, about 26 mg to about 75 mg, about 51 mg to about 75 mg, about 10 mg to about 50 mg, about 26 mg to about 50 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg or about It can be 180 mg.

By way of example and not limitation, dosages of brequinar range from about 10 mg to about 4000 mg, from about 26 mg to about 4000 mg, from about 51 mg to about 4000 mg, from about 76 mg to about 4000 mg, from about 101 mg to about 4000 mg, from about 151 mg to about 4000 mg, about 201 mg to about 4000 mg, about 10 mg to about 2000 mg, about 26 mg to about 2000 mg, about 51 mg to about 2000 mg, about 76 mg to about 2000 mg, about 101 mg to about 2000 mg, about 151 mg to about 2000 mg, about 201 mg to about 2000 mg, about 10 mg to about 1000 mg, about 26 mg to about 1000 mg, about 51 mg to about 1000 mg, about 76 mg to about 1000 mg, about 101 mg to about 1000 mg, about 151 mg to about 1000 mg, about 201 mg to about 1000 mg, about 10 mg to about 500 mg, about 26 mg to about 500 mg, about 51 mg to about 500 mg, about 76 mg to about 500 mg, about 101 mg to about 500 mg, about 151 mg to about 500 mg, about 201 mg to about 500 mg, about 10 mg to about 300 mg, about 26 mg to about 300 mg, about 51 mg to about 300 mg, about 76 mg to about 300 mg, about 101 mg to about 300 mg, about 151 mg to about 300 mg, about 201 mg to about 300 mg, about 10 mg to about 200 mg, about 26 mg to about 200 mg, about 51 mg to about 200 mg, about 76 mg to about 200 mg, about 101 mg to about 200 mg, about 151 mg to about 200 mg, about 10 mg to about 150 mg, about 26 mg to about 150 mg, about 51 mg to about 150 mg, about 76 mg to about 150 mg, about 101 mg to about 150 mg, about 10 mg to about 100 mg, about 26 mg about 100 mg, about 51 mg to about 100 mg, about 76 mg to about 100 mg, about 50 mg, about 75 mg, about 100 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 1000 mg, about 2000 mg, or about 4000 mg obtain.

By way of example and without limitation, a dosage regimen for administration of brequinar, e.g., brequinal sodium, to a human subject can be: 100 mg/ ^m2 (orally twice weekly); 125 mg/ ^m2. (p.o. twice weekly); 150 mg/ ^m2 (p.o. twice weekly); 200 mg/ ^m2 (p.o. twice weekly); 250 mg/ ^m2 (p.o. twice weekly); m ² (p.o. twice weekly); 300 mg/m ² (p.o. twice weekly); 350 mg/m ² (p.o. twice weekly); 400 mg/m ² (p.o. twice weekly); 425 mg/m ² (orally administered twice weekly); 450 mg/m ² (orally administered twice weekly); 500 mg/m ² (orally administered twice weekly); 550 mg/m ² (orally administered twice weekly) 600 mg/m ² (p.o. twice weekly); 650 mg/m ² (p.o. twice weekly); 700 mg/m ² (p.o. twice weekly); 750 mg/m ² (p.o. twice weekly); 100 mg/m ² (orally administered every 72 hours); 125 mg/m ² (orally administered every 72 hours); 150 mg/m ² (orally administered every 72 hours) ^; 200 mg/m ² (orally every 72 hours); 250 mg/m ² (orally every 72 hours); 275 mg/m ² (orally every 72 hours); 300 mg/m ² (orally every 72 hours); ⁴⁰⁰ mg/m ² (orally every 72 hours); 425 mg/m ² (orally every 72 hours); 450 mg/m ² (oral every 72 hours); 500 mg/m ² (orally every 72 hours); 550 mg/m ² (orally every 72 hours); 600 mg/m ² (orally every 72 hours); 700 mg/m ² (orally every 72 hours) ^; 750 mg/m ² (orally every 72 hours); 800 mg/m ² (orally every 72 hours) 100 mg/m ² (orally every 84 hours); 125 mg/m ² (orally every 84 hours); 150 mg/m ² (orally every 84 hours); 200 mg/m ² (orally every 84 hours); 250 mg/m ² (orally administered every 84 hours); 275 mg ^/ m ² (orally administered every 84 hours); 300 mg/m ² (orally administered every 84 hours); 400 mg/m ² (orally every 84 hours); 425 mg/m ² (orally every 84 hours); 450 mg/m ² (orally every 84 hours); 500 mg/m ² (orally every 84 hours); 550 mg/m ² (orally every 84 hours); 600 mg/m ² (orally every 84 hours); 650 mg/m ² (orally every 84 hours); 700 mg/m ² (PO every 84 hours); 750 mg/ ^m2 (PO every 84 hours); 800 mg/ ^m2 (PO every 84 hours); 100 mg/ ^m2 (PO every 96 hours); m ² (oral every 96 hours); 150 mg/m ² (orally every 96 hours); 200 mg/m ² (orally every 96 hours); 250 mg/m ² (orally every 96 hours); 275 mg/m ² (orally every 96 hours); 300 mg/m ² (orally every 96 hours); 350 mg/m ² (orally every 96 hours); 400 mg/m ² (orally every 96 hours); 425 mg/m ² (orally every 96 hours); 450 mg/m ² (orally every 96 hours); 500 mg/m ² (orally every 96 hours); 550 mg/m ² (orally every 96 hours); 600 mg/m ² (orally administered every 96 hours); 650 mg/m ² (orally administered every 96 hours); 700 mg/m ² (orally administered every 96 hours); 750 mg/m ² (orally administered every 96 hours); or 800 mg/m ² (orally every 96 hours).

By way of example and without limitation, dosages of brequinar over a 24 hour period are at least 0.01 μg/mL, at least 0.03 μg/mL, at least 0.1 μg/mL, at least 0.2 μg/mL, at least 0.3 μg/mL, at least 0.375 μg/mL, at least 0.4 μg/mL, at least 0.5 μg/mL, at least 0.6 μg/mL, at least 0.8 μg/mL, at least 1 μg/mL, at least 1. The amount may be sufficient to maintain a concentration of brequinar in the lungs of the subject of 5 μg/mL or at least 2 μg/mL.

A dosing regimen can contain a single dosage. Alternatively, a dosing regimen can contain multiple dosages. For example, a dosing regimen can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more dosages. A dosing regimen can include multiple doses provided on a continual basis. For example, a dosage can comprise a defined amount of brequinar provided over a defined period of time, e.g. For example, it can include dosages provided over a period of several days or 24 hours.

In dosing regimens comprising multiple doses, each dose may be the same, ie, each contains the same amount of brequinar. Alternatively, a dosing regimen can include dosages that are not all the same. In some embodiments, the dosing regimen comprises multiple successive doses, with the first 1, 2, 3 or 4 doses being higher than subsequent doses. In some embodiments, the dosing regimen comprises multiple successive doses with the first 1, 2, 3 or 4 doses being less than subsequent doses. In the above embodiments, the subsequent dosages can all be the same or different from each other. Various other dosage variations are possible within the scope of this invention. By way of example and without limitation, a dosing regimen can include any of the following sequences of dosages: alternating high and low dosages; gradual reduction of individual dosages. or increasing; one or more steps stepwise decreasing or increasing, including two or more dosages that are the same; A pattern that is repeated or interspersed in the . Each dosage can be independently selected from any of the dosages listed above. For example, the first dosage can be 100 mg and subsequent dosages can be 25 mg, 50 mg or 75 mg.

A dosing regimen can include drug-free periods during which the subject does not receive brequinar or a pharmaceutically acceptable salt thereof. The drug-free period is at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 5 days, at least 7 days, at least 10 days, at least 14 days, about 12 hours, about It can be 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 5 days, about 7 days, about 10 days or about 14 days.

A drug-free period can follow one dose. The drug-free period can be followed by multiple doses provided over a continuous 24-hour period. The drug-free period consists of multiple doses provided over 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive 24-hour periods. can continue.

The method can include determining a dosing regimen for the subject. Dosing regimens are determined by comparing the measured levels of the metabolite, e.g., DHO, in a sample obtained from the subject to a reference that provides a relationship between the measured levels and recommended dosage adjustments of brequinar. be able to. For example, a reference can provide the relationship between administration of a brequinar composition and levels of metabolites in a subject. Relationships can be determined empirically from known doses and times of administration of brequinar and measured levels of metabolites at one or more subsequent time points. The reference can include measured levels of brequinal or a metabolite of brequinal and relationships between measured levels of metabolites. Methods of dosing brequinar based on measured metabolite levels are known in the art and described, for example, in International Patent Publication Nos. WO2019/191030 and WO2019/191032, the contents of which are incorporated herein by reference. It is

From the comparison between the measured levels of the metabolites and the reference, a dosing regimen can then be determined. A dosing regimen can include the dosage of brequinar, the time for administration of the dosage, or both. Dosing regimens can be determined de novo or can include adjustments to previous dosing regimens, such as adjustments in dosage, intervals between administration of dosages, or both.

Dosage regimens are designed to deliver brequinar to a subject in amounts that achieve a therapeutic effect. A therapeutic effect can be a sign or symptom of a disease, disorder or condition. The therapeutic effect may be inhibition of DHODH or a change in the index of inhibition of DHODH. The indicator can be a metabolite such as DHO and the therapeutic effect can be an increase or decrease in the level of the metabolite. Therapeutic effect is accompanied by reduction in cancer cell number, reduction in cancer cell proliferation, increased differentiation of precancerous cells such as myeloblasts, complete cancer remission, and incomplete hematologic recovery There may be complete remission, morphologic leukemia-free stat, or partial remission. Increased differentiation of myeloblasts can be assessed by one or more of CD14 expression, CD11b expression, nuclear morphology and cytoplasmic granules.

A dosing regimen can ensure that levels of metabolites such as DHO are elevated or maintained at the minimum threshold required to achieve a particular effect. For example, a dosing regimen can elevate or maintain metabolite levels above a threshold level in a subject for a specified period of time. A time period can include a minimum value, a maximum value, or both. For example, the dosing regimen may be 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 hours The level of the metabolite can be elevated or maintained above the threshold level for days, at least two weeks or longer. Dosing regimen is ≤24 hours, ≤36 hours, ≤48 hours, ≤60 hours, ≤72 hours, ≤84 hours, ≤96 hours, ≤5 days, ≤6 days, ≤7 days, ≤10 days or 2 weeks Levels of metabolites can be elevated or maintained above threshold levels for: The dosing regimen is above the threshold level for at least 72 hours and 96 hours or less, at least 72 hours and 5 days or less, at least 72 hours and 6 days or less, at least 72 hours and 7 days or less, at least 96 hours and 7 days or less can increase or maintain levels of metabolites

The dosing regimen can ensure that levels of metabolites such as DHO do not exceed or remain below the maximum threshold associated with toxicity. Metabolite levels above the maximum threshold can indicate that brequinar is causing or likely to cause an adverse event in the subject. By way of example and without limitation, adverse events include abdominal pain, anemia, anorexia, blood disorders, constipation, diarrhea, dyspepsia, fatigue, fever, granulocytopenia, headache, infections, leukopenia, mucous membranes. Inflammation, nausea, pain at the injection site, phlebitis, photophobia, rash, somnolence, stomatitis, thrombocytopenia and vomiting.

A dosing regimen can include time points for administration of one or more subsequent doses to elevate or maintain levels of metabolites such as DHO above threshold levels for a specified period of time. . Time points for administration of subsequent doses may be relative to earlier time points. For example, the time points for administration of subsequent doses can be relative to the time the previous dose was administered or the time the sample was obtained from the subject.

A dosing regimen can include a schedule for administration of doses. For example, doses can be administered 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, weekly It can be administered at regular intervals, such as every, every two weeks, every three weeks or every four weeks. Alternatively, doses can be administered according to a schedule that does not require precise periodic intervals. For example, the dosage can be once a week, twice a week, three times a week, four times a week, once a month, twice a month, three times a month, monthly It can be administered 4 times, 5 times a month or 6 times a month.

By way of example and without limitation, a dosage regimen for administration of brequinar, e.g., brequinal sodium, to a human subject can be: 100 mg/ ^m2 (iv twice weekly); ² (iv twice weekly); 150 mg/m ² (iv twice weekly); 200 mg/m ² (iv twice weekly); 250 mg/m ² (iv twice weekly); 275 mg/m ² (twice weekly intravenous administration); 300 mg ^/ m ² (twice weekly intravenous administration); 350 mg/m ² (twice weekly intravenous administration); 425 mg/m ² (twice weekly iv); 450 mg/m ² (twice weekly iv); 500 mg/m ² (twice weekly iv) 550 mg/m ² (iv twice weekly); 600 mg/m ² (iv twice weekly); 650 mg/m ² (twice weekly iv); 700 mg/m ² (twice weekly); 750 mg/m ² (twice weekly intravenous); 800 mg/m ² (twice weekly intravenous); 100 mg/m ² (every 72 hours intravenously); 125 mg 150 mg/ ^{m 2 (} iv every 72 hours); 200 mg/m ² (iv every 72 hours); 250 mg/m ² (iv every 72 hours); 275 mg/ ^m2 (IV every 72 hours); 300 mg/ ^m2 (IV every 72 hours); 350 mg/ ^m2 (IV every 72 hours); 400 mg/m ² (iv every 72 hours); 425 mg/m ² (iv every 72 hours); 450 mg/m ² (iv every 72 hours) ^; 550 mg/m ² (IV every 72 hours); 600 mg/m ² (IV every 72 hours); 650 mg/m ² (IV every 72 hours); 700 mg/m ² (IV every 72 hours); 750 mg/ ^m2 (IV every 72 hours); 800 mg/ ^m2 (IV every 72 hours); 100 mg/ ^m2 (IV every 84 hours) 125 mg/m ² (IV every 84 hours); 150 mg/m ² (IV every 84 hours); 200 mg/m ² (IV every 84 hours); 250 mg/m ² (IV every 84 hours); 275 mg/m ² (IV every 84 hours); 300 mg/m ² (IV every 84 hours); 350 mg/m ² (IV every 84 hours); 400 mg 425 mg/m ² (IV every 84 hours); 450 mg/m ² (IV every 84 hours ⁾ ; 500 mg/m ² (IV every 84 hours); 550 mg/ ^m2 (IV every 84 hours); 600 mg/ ^m2 (IV every 84 hours); 650 mg/ ^m2 (IV every 84 hours); 700 mg/m ² (iv every 84 hours); 750 mg/m ² (iv every 84 hours); 800 mg/m ² (iv every 84 hours); 100 mg/m ² (iv every 96 hours) 125 mg/m ² (IV every 96 hours); 150 mg/m ² (IV every 96 hours); 200 mg/m ² (IV every 96 hours); 250 mg/m ² (IV every 96 hours); 275 mg/ ^m2 (IV every 96 hours); 300 mg/ ^m2 (IV every 96 hours); 350 mg/ ^m2 (IV every 96 hours) 400 mg/ ^m2 (IV every ⁹⁶ hours); 425 mg/ ^m2 (IV every 96 hours); 450 mg/ ^m2 (IV every 96 hours); 550 mg/m ² (IV every 96 hours); 600 mg/m ² (IV every 96 hours); 650 mg/m ² (IV every 96 hours); 700 mg 750 mg/ ^{m 2 (} IV every 96 hours); 800 mg/m ² (IV every 96 hours); 100 mg/m ² (IV every 96 hours); 125 mg/m ² (orally twice weekly); 150 mg/m ² (orally twice weekly); 200 mg/m ² (orally twice weekly); 250 mg/m ² (twice weekly); 275 mg/m ² (twice weekly oral dose); 300 mg/m ² (twice weekly oral dose); 350 mg/m ² (twice weekly oral dose); 400 mg/m ² (twice weekly oral dose); 425 mg/m ² (oral twice weekly); 450 mg/m ² (orally twice weekly); 500 mg/m ² (orally twice weekly); 550 mg/m ² (PO twice weekly); 600 mg/ ^m2 (PO twice weekly); 650 mg/ ^m2 (PO twice weekly); 700 mg/ ^m2 (PO twice weekly); 800 mg/m ² (orally administered twice weekly); 100 mg/m ² (orally administered every 72 hours) ^; 125 mg/m ² (orally administered every 72 hours) 150 mg/m ² (orally every 72 hours); 200 mg/m ² (orally every 72 hours); 250 mg/m ² (orally every 72 hours); 275 mg/m ² (orally every 72 hours); 300 mg/m ² (orally administered every 72 hours); 350 mg/m ² (orally administered every 72 hours); 400 mg/m ² (orally administered every 72 hours); 425 mg/m ² (every 72 hours); 450 mg/m ² (orally every 72 hours); 500 mg/m ² (orally every 72 hours); 550 mg/m ² (orally every 72 hours); 600 mg/m ² (orally every 72 hours); 650 mg/m ² (orally every 72 hours); 700 mg/m ² (orally every 72 hours); 750 mg/m ² (orally every 72 hours); 800 mg/m ² 100 mg/m ² (orally every 84 hours); 125 mg/m ² (orally every 84 hours); 150 mg/m ² (orally every 84 hours); 250 mg/ ^m2 (oral every 84 hours); 275 mg/ ^{m2 (} orally every 84 hours); 300 mg/ ^m2 (orally every 84 hours); 350 mg/m ² (orally every 84 hours); 400 mg/m ² (orally every 84 hours); 425 mg/m ² (orally every 84 hours); 450 mg/m ² (orally every 84 hours); 500 mg/m ² (orally every 84 hours); 550 mg/m ² (orally every 84 hours); 600 mg/m ² (orally every 84 hours); 650 mg/m ² (orally every 84 hours); 700 mg/m ² (orally administered every 84 hours); 750 mg/m ² (orally administered every 84 hours); 800 mg/m ² (orally administered every 84 hours); 100 mg/m ² (orally administered every 84 hours); 125 mg/m ² (orally every 96 hours); 150 mg/m ² (orally every 96 hours); 200 mg/m ² (orally every 96 hours); 250 mg/m ² (orally every 96 hours); 275 mg/m ² (orally every 96 hours); 300 mg/m ² (orally every 96 hours); 350 mg/m ² (orally every 96 hours); 400 mg/m ² (oral every 96 hours); 425 mg/m ² (orally every 96 hours); 450 mg/m ² (orally every 96 hours); 500 mg/m ² (orally every 96 hours); 600 mg/m ² (oral every 96 hours); 650 mg/m ² (oral every 96 hours); 700 mg/m ² ( ^oral every 96 hours) 750 mg/m ² (orally administered every 96 hours); or 800 mg/m ² (orally administered every 96 hours).

The minimum and maximum threshold levels of metabolites depend on various factors such as the type of metabolite and sample. Minimum and maximum threshold levels can be expressed absolutely, eg, in units of concentration, or relatively, eg, as a ratio compared to a baseline or reference value. For example, a minimum threshold, below which a patient may receive a dose escalation or additional doses, can also be calculated in terms of pretreatment DHO levels or increases from baseline levels.

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

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

Minimum threshold for DHO or orotate was approximately 1.5 times baseline level, approximately 2 times baseline level, approximately 2.5 times baseline level, approximately 3 times baseline level, and approximately 4 times 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, It can be 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 can include any value that falls between the values listed above. Thus, the minimum threshold can include any value between 1.5 times the baseline level and 5000 times the baseline level.

DHO or orotate maximal thresholds were approximately 2x baseline levels, 2.5x baseline levels, 3x baseline levels, 4x baseline levels, 5x baseline levels, Approximately 10 times baseline level, Approximately 20 times baseline level, Approximately 50 times baseline level, Approximately 100 times baseline level, Approximately 200 times baseline level, Approximately 500 times baseline level, Baseline It can be about 1000 times the level, about 2000 times the baseline level, about 5000 times the baseline level or about 10,000 times the baseline level. The maximum threshold can include any value that falls between the values listed above. Thus, the maximum threshold can include any value between 2 times the baseline level and 10,000 times the baseline level.

The dosage of brequinar will also depend on factors such as subject type and route of administration. The dosage can fall within a range for a given type of subject and route of administration, or the dosage can be adjusted by a specified amount for a given type of subject and route of administration. For example, dosages of brequinar for oral or intravenous administration to a subject such as a human or mouse are 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. The dosage of brequinar for oral or intravenous administration to a subject such as a human or mouse is about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about It can be adjusted by 15 mg/kg, about 20 mg/kg, about 25 mg/kg or about 50 mg/kg. Dosages of brequinar for oral or intravenous administration to animal subjects such as humans or mice are about 50 mg/m ² , about 100 mg/m ² , about 200 mg/m ² , about 300 mg/m ² , about 350 mg/m 2 . 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 . Dosages of brequinar for oral or intravenous administration to animal subjects such as humans or mice are about 50 mg/m ² , about 100 mg/m ² , about 200 mg/m ² , about 300 mg/m ² , about 350 mg/m 2 . m ² or about 400 mg/m ² can be adjusted.

The method provides that the level of the metabolite is within a threshold range that warrants dosing and/or warrants dosing at a particular level or amount (e.g., above a minimum threshold and/or below a potential toxicity threshold). below). Methods for determining metabolite levels and adjusting brequinal dosing regimens based on the determined levels of metabolites are known in the art, e.g. It is described in publication numbers WO2019/191030 and WO2019/191032.

The method comprises administering at least one dose of brequinar to subjects whose plasma metabolite levels have been determined and are below a predetermined threshold (e.g., a predetermined potential toxicity threshold and/or a predetermined potential efficacy threshold). providing. A predetermined threshold reflects the percent inhibition of DHODH in a subject compared to a baseline determined for the subject. A baseline can be determined by assay.

Multiple doses of brequinar can be administered to a subject to maintain inhibition of DHODH at the efficacy threshold. Dosing of brequinar can occur at different times and in different amounts. The present disclosure encompasses methods that can maintain inhibition of the target enzyme at a consistent level at or above the efficacy threshold throughout the course of treatment. In some embodiments, the amount of inhibition of DHODH is measured by the amount of metabolite in the subject's plasma.

The method can include redetermining the subject's plasma metabolite levels after administration of at least one dose. In some embodiments, the subject's plasma metabolite levels are redetermined after each dose. The method comprises administering at least one additional dose of brequinar after the subject's plasma metabolite level is again determined to be below a predetermined threshold (e.g., after administering a first or previous dose). can contain. If the subject's plasma metabolite levels are determined to be above a predetermined threshold, dosing can be discontinued. Accordingly, no further doses of brequinar will be administered until the subject's plasma metabolite levels are again determined to be below the predetermined threshold.

The method can comprise administering brequinar to the subject at a dosage level at or near cell-lethal levels. Such dosages can be replenished with later doses at reduced levels, or the dosage can be discontinued. For example, the method can comprise administering multiple doses of brequinar according to a regimen characterized by at least first and second phases, the first phase comprising at least one bolus at a cytolethal level A dose of brequinar is administered, and the second phase comprises administration of either at least one dose lower than the bolus dose or no brequinar.

In some embodiments, brequinar is not administered in the second phase. In some embodiments, the second phase comprises administration of uridine rescue therapy. In some embodiments, the bolus dose is or comprises a cytocidal dose. In some embodiments, a cytolethal dose is an amount of brequinar sufficient to cause apoptosis in target cells (eg, cancer cells) as well as normal (eg, non-cancerous) cells.

In some embodiments, the first phase and the second phase each comprise administration of brequinar. In some embodiments, the first phase and the second phase are done at different times. In some embodiments, the first and second phases are done on different days. In some embodiments, the first phase lasts for a period of less than 4 days. In some embodiments, the first phase comprises administration of brequinal, followed by a period in which brequinal is not administered. In some embodiments, the period during which brequinar is not administered is 3-7 days after dosing in the first phase. In some embodiments, the first phase comprises administration of more than one dose.

In some embodiments, brequinar is administered in the second phase. In some embodiments, brequinar is administered at subcytotoxic levels in the second phase. In some embodiments, the first phase is repeated after the second phase. In some embodiments, both the first and second phases are repeated.

In some embodiments, the present disclosure provides a multiphasic treatment comprising a first phase in which at least one dose of brequinar is administered to the subject and a second phase in which at least one dose of brequinar is administered to the subject. A method of administering brequinar to a subject in need thereof, according to a protocol, wherein one or more doses administered in the second phase are compared to the dose(s) administered in the first phase to provide a method that differs in amount and/or timing relative to other doses in that phase.

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

In some embodiments, the amount of brequinar administered to a patient is adjusted to take into account metabolite levels in the subject's plasma. For example, in some embodiments the first dose is administered in the first phase. In some embodiments, the metabolite level is determined during the period after administration of the first dose.

In some embodiments, if metabolite levels are below a predetermined level, the amount of brequinar administered in a second or subsequent dose is increased and/or the interval between doses is shortened. For example, in some such embodiments, the amount of brequinar administered can be increased, eg, by 100 mg/m ² . In some embodiments, the amount of brequinar administered in the second or subsequent dose is increased by 150 mg/ ^m2 . In some embodiments, the amount of brequinar administered in the second or subsequent dose is increased by 200 mg/ ^m2 . In some embodiments, the amount of brequinar administered can be increased by an adjustment amount determined based on changes in metabolite levels observed between different amounts of previous doses administered to a subject.

In some embodiments, if the metabolite level is above a predetermined level, the amount of brequinar administered in the second or subsequent dose is the same as the amount administered in the first or previous dose. and/or the intervals between doses are the same.

In some embodiments, if metabolite levels are above a predetermined level, the amount of brequinar in the second or subsequent dose is decreased and/or the interval between doses is lengthened. For example, in some such embodiments, the amount of brequinar administered can be reduced by, for example, 50 mg/m ² . In some embodiments, if metabolite levels are above a predetermined level, the amount of brequinar in the second or subsequent dose is decreased by 75 mg/m ² . In some embodiments, the amount of brequinar in the second or subsequent dose is reduced by 100 mg/m ² if metabolite levels are above a predetermined level. In some embodiments, the amount of brequinar administered can be decreased by an adjustment amount determined based on changes in metabolite levels observed between different amounts of previous doses administered to a subject.

In some embodiments, the present disclosure is a method of administering a later dose of brequinar to a patient who has previously received an earlier dose of brequinar, wherein the patient receives A method is provided having metabolite levels evaluated wherein later doses differ from earlier doses. Later doses may differ from earlier doses in the amount of brequinal included in the dose, in the time interval relative to the immediately preceding or following dose, or combinations thereof. The amount of brequinar in the later dose may be less than the amount of brequinar in the earlier dose.

The method can comprise administering multiple doses of brequinar separated from each other by a period of time greater than 2 days and less than 8 days. For example, the period can be about 3 days.

In some embodiments, the metabolite level is determined in a sample from the subject before each dose is administered, and if the determined metabolite level is above a predetermined threshold, dosing is delayed or skipped. be. For example, metabolite levels can 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 brequinar.

The method can comprise administering brequinar according to a regimen approved in clinical trials in which metabolite levels have been measured in patients between doses of brequinar. The regimen consists of multiple doses, the amount and timing of which are determined in clinical trials to maintain metabolite levels within ranges determined to indicate the extent of target enzyme inhibition below the toxicity threshold and above the minimum threshold. can include A regimen can include determining metabolite levels in a subject after administration of one or more doses of brequinar.

In some embodiments, the regimen comprises dosing cycles in which an established pattern of doses is administered over a first period of time. In some embodiments, the regimen includes multiple dosing cycles. In some embodiments, the regimen includes rest periods during which brequinar is not administered.

Combination Therapy A method of treating a condition in a subject may involve the use of a combination therapy comprising a composition containing polymorphic Form C of brequinal sodium salt and a second agent. Combination therapy can be used to treat any of the conditions described above, the choice of the second agent depending on the condition being treated.

For cancer treatment, the second agent can target a signaling pathway. The second agent can target the mitogen-activated protein kinase pathway, the AKT pathway or the phosphoinositide 3-kinase pathway. The second agent can target one or more effectors in the signaling pathway. The second agent is p38, enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), E12/E47, epidermal growth factor receptor (EGFR), FOXC2, Hippo, kallikrein-related peptidase 6 (KLK6), neuropilin -2 (NRP2), polo-like kinase 1 (PLK1), Polycomb group PcG (PRC2), Raf, sex-determining region Y-box 2 (SOX2), SNAI1, SNAI2, transforming growth factor beta (TGF-β), TWIST1 and yes-associated protein 1 (YAP1), ZEB1 and/or ZEB2 pathways can be targeted.

The second agent can be an inhibitor of p38. By way of example and without limitation, p38 inhibitors include AMG-548, asiatic acid, BIRB-796, BMS-582949, doramapimod, LY-2228820, pamapimod, PH- 797804, SB-202190, SB-203580, SB-239063, SCIO-323, SCIO-469, SD-169, SKF-86002, TAK-715, VX-702 or VX-745. Other p38 inhibitors are disclosed, for example, in U.S. Pat. Nos. 5,945,418; 6,093,742; 6,410,540; 6,617,324; 6,632,945; 6,635,644; 7,125,898; 7,135,575; 7,169,779; 7,423,047; 7,425,555; 7,521,447; 7,642,276; 410,160; U.S. Patent Application Publication No. 20020065296; and International Patent Publication No. WO1999/032110.

For treatment of viral infections, the second agent can be an antiviral agent. The second agent can be a direct-acting antiviral agent, such as an agent that interferes with the function of viral proteins or enzymes. By way of example and without limitation, antiviral agents include 3C-like major protease inhibitors, eIF4E inhibitors, helicase inhibitors, inhibitors of viral structural proteins, inhibitors of virulence factors, inosine monophosphate dehydrogenase (IMPDH ) inhibitors, interferons, papain-like proteinase inhibitors, protease inhibitors or RNA-dependent RNA polymerase inhibitors. The eIF4E inhibitor can be ribavirin. IMPDH inhibitors include AS2643361, EICAR, FF-10501, mizoribine, mycophenolic acid, mycophenolate mofetil, ribavirin, selenazofurin, SM-108, taribavirin, tiazofurin, VX-148, VX-497 or It can be VX-944. The interferon can be peginterferon alpha-2a or peginterferon alpha-2b. The protease inhibitor can be lopinavir, ritonavir or a combination thereof. The RNA dependent RNA polymerase inhibitor can be AT-511, AT-527, AT-9010, Beclabuvir, Dasabvir, Deleobvir, Favipiravir, Filibuvir, Radalbuvir, Remdesivir, Setrobuvir or Sofosbuvir . The virulence factor can be Nsp1, Nsp3c or ORF7a.

A second agent can be an anti-inflammatory agent. Anti-inflammatory agents include corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs). By way of example and without limitation, anti-inflammatory agents include acematricin, acetate, aloe vera extract, alpha-methyldexamethasone, amcinafide, arnica flower, aspirin, beclomethasone dipropionate, benorylate, benoxaprofen, betamethasone and its esters, chloroprednisone, chloroprednisone acetate, clescinolone, clidanac, clobetasol valerate, clocortelone, comfrey root, desonide, desoxycorticostero acetate desoxymethasone, dexamethasone, dexamethasone phosphate, dichlorisone, dichlorisone, diclofenac, diflorazone diacetate, diflucortolone and its derivatives, difluprednate, disalacid, enolic acid, Commiphom extract from the genus Commiphora mukul, extract from the genus Rubia cordifolia, fenamic acid derivatives, fenclofenac, fenugreek seeds, fluadrenolone, flurbiprofen ), fluchloronide, flucortin butyl ester, fluchlorone acetonide, flufenamic acid derivatives such as N-(α,α,α-trifluoro-m-tolyl)anthranilic acid), flunisolide, fluocinonide, fluocortolone , fluoromethalone, fluperolone, fluprednidine, fluprednisolone, fluandrenolone, furofenac, halcinonide, hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocol cyclopentylpropionate Tison, Yoshi hydrocortisone herbate, hydroxytriamcilone, indomethacin, isozepac, ketoprofen, matricarria flower, meclofenamic acid derivatives (e.g. sodium meclofenamate), medrysone, mefenamic acid derivatives (e.g. N-(2,3 -xyl-yl)anthranilic acid), meprednisone, methylprednisolone, naproxen, oxicams (e.g., piroxicam and isoxicam), oxyphenbutazone, paramethasone, phenylbutazone, prednisolone, prednisone, ibuprofen, etc. Pyrazolidinediones such as propionate, feprazone, safaprin, salicylic acid derivatives, sulfinpyrazone, sulindac, suprofen, tolmetin, triamcinolone acetonide, trimethasone, trisilate, willow Contains bark, as well as Zomepilac.

Combination therapy can include multiple periods during which different drugs or different combinations of drugs are provided to the subject. The different agents or different combinations of agents can be provided concurrently, sequentially, in overlapping periods, in temporally separated periods, or in any other manner known to those of skill in the art. An exemplary temporal regimen for administering combination therapy is provided below.

By way of example and not limitation, combination therapy can include a first period and a second period following the first period, wherein these periods are as follows: first but no second agent is provided to the subject, a first period; and a second period, wherein the second agent is provided to the subject but not the first agent; A first period during which one agent and a second agent are provided to the subject and a second period during which the second agent is provided to the subject but not the first agent; the first agent but no second agent is provided to the subject, and a second period of time during which the first agent and the second agent are provided to the subject; and a first combination of agents and a second period during which a second, different combination of agents is provided to the subject. In certain embodiments, brequinar or a pharmaceutically acceptable salt thereof is provided to the subject in a first period of time and an anti-inflammatory agent is provided to the subject in a second period of time following the first period of time. Brequinar or a pharmaceutically acceptable salt thereof may be withheld from the subject for a second period of time. An anti-inflammatory drug may or may not be provided in the first period. Brequinar or a pharmaceutically acceptable salt thereof can be provided in the first period according to a dosing regimen such as any of those described above. In combination therapy, different therapeutic agents can be provided in a single formulation. For example, combination therapy can include providing brequinar or a pharmaceutically acceptable salt thereof in the same formulation as a second agent, such as an antiviral or anti-inflammatory agent. In some embodiments, brequinar or a pharmaceutically acceptable salt thereof and dexamethasone are provided in the same formulation.

Alternatively, in combination therapy, different therapeutic agents can be provided in separate formulations. For example, combination therapy can include providing brequinar, or a pharmaceutically acceptable salt thereof, and a second agent, such as an antiviral or anti-inflammatory agent, in separate formulations. In some embodiments, brequinar or a pharmaceutically acceptable salt thereof and dexamethasone are provided in separate formulations.

(Example 1)
Extensive research was conducted to discover a crystalline form of brequinar (BRQ) that would be a suitable pharmaceutical form for commercial oral manufacture. Although earlier methods of producing BRQ provide brequinar as a lyophilized powder suitable for intravenous administration when dissolved in aqueous media, such methods produce a crystalline form suitable for commercial manufacture of capsules or tablets. do not.

The polymorphic behavior of BRQ was investigated to identify the optimal polymorph for further development. A typical manufacturing research batch of brequinar sodium salt designated Pattern A (Research Batch: PS04375-1-E-P4) was used to perform polymorph screening and selection. Equilibration at 25°C and 50°C experiments, equilibration under temperature cycling experiments, crystallization with fast and slow cooling experiments, crystallization with slow evaporation experiments, crystallization with anti-solvent addition experiments, and steam. The polymorphic behavior of this salt was investigated by crystallization using diffusion experiments.

FIG. 1 is an X-ray powder diffraction (XRPD) pattern of brequinal sodium salt from batch PS04375-1-E-P4.
FIG. 2 is a differential scanning calorimetry (DSC) thermogram of brequinal sodium salt from batch PS04375-1-E-P4.
FIG. 3 is a thermogravimetric analysis (TGA) thermogram of brequinal sodium salt from batch PS04375-1-E-P4.

The above results indicate that the study batch brequinal sodium salt, designated pattern A, is polymorphic. Ten hydrates were identified: patterns A, B, C, D, E, F, G, H, I and J.

FIG. 4 is an overlay of XRPD scan patterns AJ of brequinar sodium salt from batch PS04375-1-E-P4.

The polymorphic forms of brequinar are summarized in Table 3.

Of the polymorphic forms of brequinar sodium salt, patterns B, D, E, F, G, I and J are unstable and convert to pattern C under ambient conditions. The starting material, pattern A, is also a metastable form and transforms into different polymorphs that occur in polymorphic screening tests. Pattern C remains the same under vacuum drying conditions at 50° C. for 18 hours. The data provided herein demonstrate that Pattern C is the desired form for commercial development due to its stability in manufacturing and storage.

FIG. 5 is a DSC thermogram of polymorphic Form C of brequinar sodium salt from batch FR00514-2-SL-B9.
FIG. 6 is a TGA thermogram of polymorphic Form C of brequinar sodium salt from batch FR00514-2-SL-B9.
FIG. 7 is a graph of the ¹ H NMR spectrum of polymorphic Form C of brequinal sodium salt from batch FR00514-2-SL-B9.

最も望まれる比が９７／３である、９５／５～９８／２の比のイソプロパノール／水の混合物に６－フルオロ（Ｆｌｏｕｒｏ）－２－（２’－フルオロ－１，１’－ビフェニル－４－イル）－３－メチル－４－キノリンカルボン酸（１．０ｅｑ）を溶解することにより、６－フルオロ－２－（２’－フルオロ－１，１’－ビフェニル－４－イル）－３－メチル－４－キノリンカルボン酸ナトリウム塩の合成を実行した。水酸化ナトリウム（１．０５ｅｑ）を添加し、全て溶解されるまで７０～８０℃の温度に混合物を温めた。溶解のために最も望まれる温度は、７３～７７℃である。混合物を５５～６５℃に冷却し、６－フルオロ－２－（２’－フルオロ－１，１’－ビフェニル－４－イル）－３－メチル－４－キノリンカルボン酸ナトリウム塩の種晶を添加し、２０～３０℃に冷却し、ｎ－ヘプタンを添加した。ｎ－ヘプタンの添加のために最も望まれる温度は、２３～２７℃である。次に、混合物を２０～３０℃に維持し、６～２４時間撹拌した。次に、固体を濾過し、３０～５０℃で乾燥させた。 (Example 2)
A general scheme for the preparation of polymorphic Form C of brequinal sodium salt is illustrated in the following schematic:

6-Fluoro-2-(2′-fluoro-1,1′-biphenyl-4 in a mixture of isopropanol/water in ratios of 95/5 to 98/2, with the most desired ratio being 97/3. 6-fluoro-2-(2′-fluoro-1,1′-biphenyl-4-yl)-3-yl)-3-methyl-4-quinolinecarboxylic acid (1.0 eq) was dissolved in 6-fluoro-2-(2′-fluoro-1,1′-biphenyl-4-yl)-3- Synthesis of methyl-4-quinolinecarboxylic acid sodium salt was carried out. Sodium hydroxide (1.05 eq) was added and the mixture warmed to a temperature of 70-80° C. until all dissolved. The most desirable temperature for dissolution is 73-77°C. The mixture is cooled to 55-65° C. and seeded with 6-fluoro-2-(2′-fluoro-1,1′-biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid sodium salt. and cooled to 20-30° C. and n-heptane was added. The most desirable temperature for the addition of n-heptane is 23-27°C. The mixture was then maintained at 20-30° C. and stirred for 6-24 hours. The solid was then filtered and dried at 30-50°C.

(Example 3)
Polymorphic Form C of brequinar sodium salt was prepared according to the following scheme. 360.15 g (0.96 mol) of 6-fluoro-2-(2′-fluoro-1,1′-biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid was dissolved in 2,541 mL of isopropanol and 79 mL of H _↓2 O (97v:3v). 38.80 g (0.97 mol) of sodium hydroxide was added and warmed to 73-77° C. until completely dissolved. The reaction mixture was filtered, then cooled to 57-61° C. and seeded. The temperature was adjusted to 23-27°C, n-heptane was added and the mixture was stirred at 23-27°C for 24 hours. The solid was then filtered off and dried under vacuum at 40°C.

FIG. 8 is an X-ray powder diffraction (XRPD) pattern of polymorph Form C of brequinal sodium salt prepared according to the present method. The method gave an 88.3% yield of a white crystalline solid. The purity was 99.6% and the water content by KF (w/w) was 5.2%.

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

Equivalents Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, may be incorporated herein by reference to the scientific and patent literature cited herein. It will be apparent to those skilled in the art from the full content of The subject matter herein contains important information, exemplifications and guidance that may be adapted to the practice of the invention in its various embodiments and equivalents thereof.

Claims (38)

A method of making a crystal comprising polymorphic Form C of brequinal sodium salt, comprising:
combining a solid form of brequinaric acid, sodium hydroxide (NaOH) and a solvent to form a solution;
and C. crystallizing brequinal sodium salt from said solution to produce crystals comprising polymorphic C form of brequinal sodium salt. 2. The method of claim 1, wherein the solvent comprises isopropanol and water. 3. The method of claim 2, wherein the isopropanol:water ratio in the solvent is from about 95:5 to about 98:2. 4. The method of claim 3, wherein the ratio of isopropanol:water in the solvent is about 97:3. 2. The method of claim 1, wherein said NaOH is combined with said brequinaric acid in about 0.5 to about 1.6 molar equivalents. 6. The method of claim 5, wherein the NaOH is combined with the brequinaric acid in about 1.0 to about 1.1 molar equivalents. The method of claim 1, wherein said combining step comprises incubating said combined brequinaric acid, NaOH and solvent at about 70°C to about 80°C. 2. The method of claim 1, wherein said combining step comprises removing insoluble materials from said solution. 2. The method of claim 1, wherein said combining step comprises incubating said combined brequinaric acid, NaOH and solvent at about 55°C to about 65°C. The combining step comprises:
incubating the combined brequinaric acid, NaOH and solvent at about 70° C. to about 80° C.;
removing insoluble materials from the combined brequinaric acid, NaOH and solvent;
and incubating the combined brequinaric acid, NaOH and solvent at about 55°C to about 65°C. 2. The method of claim 1, wherein the crystallizing step comprises adding seed crystals of polymorphic Form C of brequinal sodium salt to the solution to produce a seeded mixture. 12. The method of claim 11, wherein the crystallizing step comprises incubating the seeded mixture at about 20°C to about 30°C. 12. The method of claim 11, wherein crystallizing comprises adding an alkane to the solution to produce an alkane-containing mixture. 14. The method of claim 13, wherein said alkane is n-heptane. 12. The method of claim 11, wherein crystallizing comprises incubating the alkane-containing mixture for about 6 hours to about 24 hours. 12. The method of claim 11, wherein the crystallizing step comprises removing the solvent from polymorphic Form C of brequinal sodium salt. The step of crystallizing comprises
adding seed crystals of polymorphic Form C of brequinal sodium salt to said solution to form a seeded mixture;
incubating the seeded mixture at about 20° C. to about 30° C.;
adding an alkane to the seeded mixture to produce an alkane-containing mixture. incubating the alkane-containing mixture for about 6 hours to about 24 hours;
removing said solvent from polymorphic Form C of brequinal sodium salt. Crystals containing polymorphic form C of brequinar sodium salt. 20. The crystal of claim 19, wherein polymorphic form C comprises brequinal sodium salt hydrate. A pharmaceutical composition comprising brequinal sodium salt, wherein at least 75% of said brequinal sodium salt is polymorphic Form C. 22. The pharmaceutical composition of claim 21, wherein at least 95% of said brequinal sodium salt is polymorphic Form C. 22. The pharmaceutical composition of claim 21, wherein polymorphic form C comprises brequinal sodium salt hydrate. 22. The pharmaceutical composition according to claim 21, formulated for oral administration. 22. The pharmaceutical composition according to claim 21, formulated for intravenous or topical administration. 1. A method of treating a condition in a subject comprising providing a subject with the condition a pharmaceutical composition comprising brequinal sodium salt, wherein at least 75% of said brequinal sodium salt is polymorphic Form C. ,Method. 27. The method of claim 26, wherein at least 95% of said brequinal sodium salt is polymorphic Form C. 27. The method of claim 26, wherein polymorphic form C comprises brequinal sodium salt hydrate. 27. The method of claim 26, wherein said composition is provided orally. 27. The method of claim 26, wherein said condition is selected from the group consisting of autoimmune disorders, cancer, metabolic disorders and viral infections. 31. The method of claim 30, wherein said condition is a viral infection. 31. The viral infection comprises a virus selected from the group consisting of adenovirus, coronavirus, enterovirus, human metapneumovirus, human parainfluenza virus, human respiratory syncytial virus, influenza virus and rhinovirus. The method described in . wherein said viral infection is from the group consisting of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) 33. The method of claim 32, comprising the selected coronavirus. 34. The method of claim 33, wherein the coronavirus is SARS-CoV-2. 31. The method of claim 30, wherein said condition is cancer. The cancer is acute lymphoblastic leukemia, acute myeloid leukemia, adult T-cell leukemia/lymphoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, gastric cancer, glioblastoma, glioma , head and neck cancer, renal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, myeloma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer and uterine cancer 36. The method of claim 35, selected from the group consisting of cancer. 31. The method of claim 30, wherein said condition is an autoimmune disorder. said condition is an autoimmune disorder, wherein said autoimmune disorder is selected from the group consisting of arthritis, hepatitis, chronic obstructive pulmonary disease, multiple sclerosis, Crohn's disorder, irritable bowel syndrome (IBS) and tendonitis. 38. The method of claim 37, wherein

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