COMBINATION THERAPIES TO TREAT VIRAL INFECTIONS

Field of the Invention

The invention relates generally to methods of treating viral infections using combination therapies that include an inhibitor of de novo pyrimidine synthesis and an inhibitor of the pyrimidine salvage pathway.

Background

Many of the deadliest wars in human history have been fought not between different peoples but between humans and viruses. For example, the influenza virus was responsible for 25-50 million deaths duringthe 1918 influenza pandemic; about 36 million people had diedfrom human immunodeficiency virus (HIV) by 2020; and the global death toll from SARS-CoV-2, the coronavirus that causes CO VID-19, was over 4 million by the summer of 2021.

Several elements of virus biology make viral infections challenging to treat. First, because viruses have small genomes that contain relatively few genes, they rely on cellular enzymes and other machinery to reproduce. Consequently, for any given virus, the number of virus-specific gene products that can be targeted for therapeutic intervention is limited. Another obstacle to treating viral infections is the ability of viruses to reproduce quickly under appropriate conditions. One consequence of the short reproduction cycle is a rapid expansion in the number of infectious particles. For example, a single host cell infected with SARS-CoV-2 can produce up to 1000 virion particles over the course of an infection. The short reproduction cycle also allows viruses to evolve through mutations that occur naturally during replication. Evolution can lead to the development and propagation of virus strains with deleterious characteristics, such as increased infectivity and the ability to evade the host immune system.

Summary

One therapeutic strategy for treatment of viral infections is to deplete the nucleotide supply of infected cells. Copious nucleotide pools are needed to support synthesis of viral nucleic acids, whereas non -proliferating cells can survive temporary periods of nucleotide starvation. The invention recognizes thatbrequinar (an inhibitor of dihydrooro tate dehydrogenase (DHODH), an enzyme required fort/c novo synthesis of pyrimidine-based nucleotides) may be used to interfere with nucleic acid synthesis by limiting the availability of nucleotides.

The invention further recognizes that pyrimidines required to support viral replication may be produced either by de novo synthesis or by salvaging pyrimidines generated from the breakdown of other cellular macromolecules. Inhibitors of de novo pyrimidine synthesis may not block viral replication if an adequate supply of pyrimidines is generated via the salvage pathway.

Therefore, the invention provides compositions and methods for treating viral infections using combination therapies that include a de novo pyrimidine synthesis inhibitor, such as brequinar, and an inhibitor of the pyrimidine salvage pathway, such as dipyridamole. By inhibiting both pathways, intracellular pyrimidine pools can be depleted to a degree that prevents the production of new virus particles. The dual inhibition is sustained for a period long enough to hinder viral replication but short enough to avoid damage to host cells. Thus, the combination therapies of the invention are useful for treating a wide variety of viral infections.

In an aspect, the invention provides pharmaceutical compositions that contain a polymorphic form C of brequinar sodium salt and an inhibitor of the pyrimidine salvage pathway.

The composition may contain a defined amount of the brequinar sodium salt in polymorphic form C. The composition may contain at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the brequinar sodium salt in polymorphic form C.

The composition may contain a defined amount of brequinar sodium salt. The composition may contain from about 1 mg to about 100 mg, from about 1 mg to about 200 mg, from about 1 mg to about 300 mg, from about 1 mg to about 400 mg, from about 1 mg to about 500 mg, from about 1 mg to about 600 mg, from about 1 mg to about 800 mg, from about 1 mg to about 1000 mg, from about 1 mgto about 1200 mg, from about 1 mgto about 1500 mg, from about 5 mgto about 100 mg, from about 5 mgto about 200 mg, from about 5 mgto about 300 mg, from about 5 mgto about 400 mg, from about 5 mgto about 500 mg, from about 5 mgto about 600 mg, from about 5 mgto about 800 mg, from about 5 mgto about 1000 mg, from about 5 mg to about 1200 mg, from about 5 mgto about 1500 mg, from about 10 mgto about 100 mg, from about 10 mgto about 200 mg, from about 10 mgto about 300 mg, from about 10 mgto about 400 mg, from about 10 mgto about 500 mg, from about 10 mgto about 600 mg, from about 10 mgto about 800 mg, from about 10 mgto about 1000 mg, from about 10 mgto about 1200 mg, from about 10 mgto about 1500 mg, from about 25 mgto about 100 mg, from about 25 mg to about 200 mg, from about 25 mgto about 300 mg, from about 25 mgto about 400 mg, from about25 mgto about 500 mg, from about25 mgto about 600 mg, fromabout25 mgto about 800 mg, from about 25 mgto about 1000 mg, from about 25 mgto about 1200 mg, from about25 mgto about 1500 mg, from about 50 mgto about lOO mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mgto about 400 mg, from about 50 mg to about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about lOOOmg, from about 50 mgto about 1200 mg, orfrom about 50 mg to about 1500 mg of brequinar sodium salt.

The inhibitor of the pyrimidine salvage pathway may inhibit a nucleoside transporter. The inhibitor of the pyrimidine salvage pathway may inhibit synthesis of both ribonucleotide triphosphates (rNTPs) and deoxyribonucleotide triphosphates (dNTPs). The inhibitor of the pyrimidine salvage pathway may inhibit synthesis of rNTPs but not dNTPs. The inhibitor of the pyrimidine salvage pathway may inhibit synthesis of dNTPs but not rNTPs. The inhibitor of the pyrimidine salvage pathway may be capecitabine, cladribine, clofarabine, cyclopentenyl cytosine (CPE-C), diazo-5-oxo-L-norleucine (DON), dilazep, dipyridamole, floxuridine, fludarabine, fluorouracil (5-FU), gemcitabine, TAS-114, and trifluridine.

The composition may contain a defined amount of the inhibitor of the pyrimidine salvage pathway. The composition may contain the inhibitor of the pyrimidine salvage pathway at from about 1 mgto about 100 mg, from about 1 mgto about 200 mg, from about 1 mgto about 300 mg, from about 1 mgto about 400 mg, from about 1 mgto about 500 mg, from about 1 mgto about 600 mg, from about 1 mgto about 800 mg, from about 1 mgto about 1000 mg, from about 1 mg to about 1200 mg, from about 1 mgto about 1500 mg, from about 5 mgto about 100 mg, from about 5 mgto about 200 mg, from about 5 mgto about 300 mg, from about 5 mgto about 400 mg, from about 5 mgto about 500 mg, from about 5 mgto about 600 mg, from about 5 mg to about 800 mg, from about 5 mgto about 1000 mg, from about 5 mgto about 1200 mg, from about 5 mgto about 1500 mg, from about 10 mgto about 100 mg, from about 10 mgto about 200 mg, from about 10 mgto about 300 mg, from about 10 mgto about400 mg, from about 10 mg to about 500 mg, from about 10 mgto about 600 mg, from about 10 mgto about 800 mg, from about 10 mgto about 1000 mg, from about 10 mgto about 1200 mg, from about 10 mgto about 1500 mg, from about25 mgto about 100 mg, from about25 mgto about200mg, from about25 mgto about300 mg, from about25 mgto about400 mg, fromabout25 mgto about 500 mg, from about25 mgto about 600 mg, from about25 mgto about 800 mg, fromabout25 mg to about 1000 mg, from about25 mgto about 1200 mg, from about25 mgto about 1500 mg, from about 50 mgto about 100 mg, from about 50 mgto about200 mg, from about 50 mgto about 300 mg, from about 50 mgto about400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about 1000 mg, from about 50 mgto about 1200 mg, or from about 50 mgto about 1500 mg.

The composition may contain brequinar and dipyridamole at fixed doses in a single dosage unit. The composition may contain about 10 mg brequinar and about 10 mg dipyridamole, about 25 mg brequinar and about 10 mg dipyridamole, about 50 mg brequinar and about 10 mg dipyridamole, about 75 mg brequinar and about 10 mg dipyridamole, about 100 mg brequinar and about 10 mg dipyridamole, about 125 mg brequinar and about 10 mg dipyridamole, about 150 mg brequinar and about 10 mg dipyridamole, about 175 mg brequinar and about 10 mg dipyridamole, about 200 mg brequinar and about 10 mg dipyridamole, about 250 mg brequinar and about 10 mg dipyridamole, about 300 mg brequinar and about 10 mg dipyridamole, about 10 mg brequinar and about 25 mg dipyridamole, about 25 mg brequinar and about 25 mg dipyridamole, about 50 mg brequinar and about 25 mg dipyridamole, about 75 mg brequinar and about 25 mg dipyridamole, about 100 mg brequinar and about 25 mg dipyridamole, about 125 mg brequinar and about 25 mg dipyridamole, about 150 mg brequinar and about 25 mg dipyridamole, about 175 mg brequinar and about 25 mg dipyridamole, about 200 mg brequinar and about 25 mg dipyridamole, about 250 mg brequinar and about 25 mg dipyridamole, about 300 mg brequinar and about 25 mg dipyridamole, about 10 mg brequinar and about 50 mg dipyridamole, about 25 mgbrequinar and about 50 mg dipyridamole, about 50 mg brequinar and about 50 mg dipyridamole, about 75 mgbrequinar and about 50 mg dipyridamole, about 100 mg brequinar and about 50 mg dipyridamole, about 125 mgbrequinar and about 50 mg dipyridamole, about 150 mg brequinar and about 50 mg dipyridamole, about 175 mgbrequinar and about 50 mg dipyridamole, about 200 mgbrequinar and about 50 mg dipyridamole, about 250 mg brequinar and about 50 mg dipyridamole, about 300 mg brequinar and about 50 mg dipyridamole, about 10 mgbrequinar and about 75 mg dipyridamole, about 25 mg brequinar and about 75 mg dipyridamole, about 50 mgbrequinar and about 75 mg dipyridamole, about 75 mgbrequinar and about 75 mg dipyridamole, about 100 mgbrequinar and about75 mg dipyridamole, about 125 mgbrequinar and about75 mg dipyridamole, about 150 mgbrequinar and about 75 mg dipyridamole, about 175 mgbrequinar and about 75 mg dipyridamole, about 200 mgbrequinar and about 75 mg dipyridamole, about 250 mg brequinar and about75 mg dipyridamole, about 300 mg brequinar and about75 mg dipyridamole, about 10 mg brequinar and about 100 mg dipyridamole, about 25 mg brequinar and about 100 mg dipyridamole, about 50 mg brequinar and about 100 mg dipyridamole, about 75 mgbrequinar and about 100 mg dipyridamole, about 100 mgbrequinar and about 100 mg dipyridamole, about 125 mgbrequinar and about 100 mg dipyridamole, about 150 mg brequinar and about 100 mg dipyridamole, about 175 mgbrequinar and about 100 mg dipyridamole, about200 mgbrequinar and about 100 mg dipyridamole, about 250 mgbrequinar and about 100 mg dipyridamole, about 300 mgbrequinar and about 100 mg dipyridamole, about 10 mgbrequinar and about 125 mg dipyridamole, about 25 mgbrequinar and about 125 mg dipyridamole, about 50 mgbrequinar and about 125 mg dipyridamole, about 75 mgbrequinar and about 125 mg dipyridamole, about 100 mgbrequinar and about 125 mg dipyridamole, about 125 mgbrequinar and about 125 mg dipyridamole, about 150 mgbrequinar and about 125 mg dipyridamole, about 175 mgbrequinar and about 125 mg dipyridamole, about 200 mgbrequinar and about 125 mg dipyridamole, about 250 mgbrequinar and about 125 mg dipyridamole, about 300 mgbrequinar and about 125 mg dipyridamole, about 10 mg brequinar and about 150 mg dipyridamole, about 25 mgbrequinar and about 150 mg dipyridamole, about 50 mg brequinar and about 150 mg dipyridamole, about 75 mg brequinar and about 150 mg dipyridamole, about 100 mgbrequinar and about 150 mg dipyridamole, about 125 mgbrequinar and about 150 mg dipyridamole, about 150 mgbrequinar and about 150 mg dipyridamole, about 175 mgbrequinar and about 150 mg dipyridamole, about 200 mgbrequinar and about 150 mg dipyridamole, about 250 mg brequinar and about 150 mg dipyridamole, about 300 mgbrequinar and about 150 mg dipyridamole, about 10 mg brequinar and about 175 mg dipyridamole, about 25 mgbrequinar and about 175 mg dipyridamole, about 50 mg brequinar and about 175 mg dipyridamole, about 75 mgbrequinar and about 175 mg dipyridamole, about 100 mgbrequinar and about 175 mg dipyridamole, about 125 mgbrequinar and about 175 mg dipyridamole, about 150 mgbrequinar and about 175 mg dipyridamole, about 175 mgbrequinar and about 175 mg dipyridamole, about 200 mgbrequinar and about 175 mg dipyridamole, about 250 mgbrequinar and about 175 mg dipyridamole, about 300 mgbrequinar and about 175 mg dipyridamole, about 10 mgbrequinar and about 200 mg dipyridamole, about 25 mg brequinar and about200 mg dipyridamole, about 50 mgbrequinar and about200 mg dipyridamole, about 75 mg brequinar and about 200 mg dipyridamole, about 100 mg brequinar and about 200 mg dipyridamole, about 125 mg brequinar and about 200 mg dipyridamole, about 150 mgbrequinar and about 200 mg dipyridamole, about 175 mgbrequinar and about200 mg dipyridamole, about 200 mgbrequinar and about 200 mg dipyridamole, about 250 mgbrequinar and about 200 mg dipyridamole, about 300 mgbrequinar and about 200 mg dipyridamole, about 10 mg brequinar and about 250 mg dipyridamole, about 25 mgbrequinar and about 250 mg dipyridamole, about 50 mg brequinar and about 250 mg dipyridamole, about 75 mgbrequinar and about250 mg dipyridamole, about 100 mgbrequinar andabout250 mg dipyridamole, about 125 mgbrequinar and about 250 mg dipyridamole, about 150 mgbrequinar and about 250 mg dipyridamole, about 175 mgbrequinar and about 250 mg dipyridamole, about 200 mgbrequinar and about 250 mg dipyridamole, about 250 mgbrequinar and about 250 mg dipyridamole, about 300 mgbrequinar and about 250 mg dipyridamole, about 10 mg brequinar andabout 300mg dipyridamole, about 25 mg brequinar and about 300 mg dipyridamole, about 50 mgbrequinar and about 300 mg dipyridamole, about 75 mgbrequinar and about 300 mg dipyridamole, about 100 mgbrequinar and about 300 mg dipyridamole, about 125 mgbrequinar and about 300 mg dipyridamole, about 150 mgbrequinar and about 300 mg dipyridamole, about 175 mgbrequinar and about 300 mg dipyridamole, about 200 mgbrequinar and about 300 mg dipyridamole, about 250 mgbrequinar and about 300 mg dipyridamole, or about 300 mgbrequinar and about 300 mg dipyridamole.

The composition may contain an excipient suitable for oral administration. The excipient may be a binder, coating, coloring agent, disintegrant, flavoring agent, preservative, sorbent, or sweetener.

In another aspect, the invention provides combination therapies for treatment of a viral infection in a subject. The combination therapies include a daily dosage of brequinar of from about 10 mg to about 1000 mg and a daily dosage of dipyridamole of from about 20 mg to about 2000 mg. The total daily dosage of brequinar may be from about 10 mg to about 100 mg, from about 10 mg to about 200 mg, from about 10 mg to about 300 mg, from about 10 mg to about 400 mg, from about 10 mg to about 500 mg, from about 10 mg to about 600 mg, from about 10 mg to about 800 mg, from about 10 mg to about 1000 mg, from about 25 mg to about 100 mg, from about 25 mg to about 200 mg, from about 25 mg to about 300 mg, from about 25 mg to about 400 mg, from about25 mgto about 500 mg, from about25 mgto about 600 mg, from about 25 mgto about 800 mg, from about 25 mgto about 1000 mg, from about 50 mgto about 100 mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mg to about400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about lOOO mg, from about lOOmgto about 200 mg, from about lOOmgto about 300 mg, from about lOOmgto about 400 mg, from about 100 mgto about 500 mg, from about lOOmgto about 600 mg, from about 100 mgto about 800 mg, or from about 100 mgto about 1000 mg.

The total daily dosage of dipyridamole maybe from about 20 mgto about 100 mg, from about 20 mgto about 200 mg, from about 20 mgto about 300 mg, from about 20 mgto about 400 mg, from about 20 mgto about 500 mg, from about 20 mgto about 600 mg, from about 20 mg to about 800 mg, from about 20 mgto about lOOOmg, from about 20 mgto about 1200 mg, from about 20 mgto about 1500 mg, from about 20 mgto about 2000 mg, from about 25 mgto about 100 mg, from about 25 mgto about 200 mg, from about 25 mgto about 300 mg, from about25 mgto about 400 mg, from about25 mgto about 500 mg, from about25 mgto about 600 mg, from about 25 mgto about 800 mg, from about 25 mgto about lOOOmg, from about 25 mg to about 1200 mg, from about25 mgto about 1500 mg, from about25 mgto about2000 mg, from about 50 mgto about 100 mg, from about 50 mgto about200 mg, from about 50 mgto about 300 mg, from about 50 mgto about400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about 1000 mg, from about 50 mgto about 1200 mg, from about 50 mgto about 1500mg, from about 50 mg to about 2000 mg, from about lOOmgto about 200 mg, from about 100 mgto about 300 mg, from about lOOmgto about 400 mg, from about 100 mgto about 500 mg, from about 100 mg to about 600 mg, from about 100 mgto about 800 mg, or from about 100 mgto about 1000 mg, from about lOO mgto about 1200 mg, from about lOO mgto about 1500 mg, from about 100 mg to about 2000 mg, from about 200 mgto about 300 mg, from about 200 mgto about 400 mg, from about200 mgto about 500 mg, from about 200 mgto about 600 mg, from about200 mgto about 800 mg, or from about 200 mgto about 1000 mg, from about 200 mgto about 1200 mg, from about200 mgto about 1500 mg, or from about 200 mgto about2000 mg.

The brequinar in the combination therapy may include polymorphic form C of brequinar sodium salt. A defined amount of the brequinar sodium salt may be in polymorphic form C. In the combination therapy, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the brequinar sodium salt may be in polymorphic form C.

Brequinar and dipyridamole may each independently be provided to a subject having a viral infection by a particular route or mode of administration. Each may independently be provided orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, subcutaneously, nasally, pulmonarily, or with or on an implantable medical device (e.g., stent or drug-eluting stent or balloon equivalents).

The daily dosage of brequinar and dipyridamole may each independently be provided in a single unit dose. The daily dosage of brequinar, dipyridamole, or both may be provided in multiple units that are administered simultaneously. The total daily dosage of brequinar, dipyridamole, or both may be provided in two, three, four, or more units that are administered simultaneously. The daily dosage of brequinar, dipyridamole, or both may be provided in multiple doses that are administered at separate times. The daily dosage of brequinar, dipyridamole, or both may be provided in one dose, two doses, three doses, four doses, five doses, or more. The multiple daily doses may be separated by defined intervals, such as about 12 hours, about 8 hours, or about 6 hours.

In another aspect, the invention provides methods of treating a viral infection in a subject by providing the following in a 24-hour period to a subject having a viral infection: at a first time point, a first dose of an inhibitor of de novo pyrimidine synthesis and a first dose of an inhibitor of the pyrimidine salvage pathway; and at a second time point, a second dose of the inhibitor of the pyrimidine salvage pathway and no dose of the inhibitor of de novo pyrimidine synthesis. Administration of the inhibitor of de novo pyrimidine synthesis and/or administration of the inhibitor of the pyrimidine salvage pathway may be administered with a loading dose, for example as the first dose at the first point in time. A loading dose is an initial higher dose of the agent that is given at the beginning of a course of treatment before dropping down to a lower maintenance dose. For example, the dosing regimen may include multiple consecutive dosages at multiple points in time, in which the first one, two, three, or four dosages are higher than subsequent dosages.

The method may include repeating the providing steps over consecutive 24-hour periods. The method may include repeating the providing steps for at least two, at least three, at least four, at least five, at least six, at least seven, at least ten, or at least fourteen consecutive 24-hour periods.

At the first time point, the first dose of the inhibitor of de novo pyrimidine synthesis and the first dose of the inhibitor of the pyrimidine salvage pathway may be provided in a single pharmaceutical composition. At the first time point, the first dose of the inhibitor of de novo pyrimidine synthesis and the first dose of the inhibitor of the pyrimidine salvage pathway may be provided in separate pharmaceutical compositions.

Each dose of the inhibitor of de novo pyrimidine synthesis and each dose of the inhibitor of the pyrimidine salvage pathway may independently be provided to the subject by a particular route or mode of administration, such as any of those described above.

The method may include providing a third dose of the inhibitor of the pyrimidine salvage pathway at a third time point in the 24-hour period. At the third time point, the third dose of the inhibitor of the pyrimidine salvage pathway maybe provided with no dose of the inhibitor of de novo pyrimidine synthesis. At the third time point, the third dose of the inhibitor of the pyrimidine salvage pathway may be provided with a dose of the inhibitor of de novo pyrimidine synthesis.

The inhibitor of de novo pyrimidine synthesis may inhibit dihydroorotate dehydrogenase (DHODH). The inhibitor of de novo pyrimidine synthesis may be brequinar. Brequinar may be provided as polymorphic form C of brequinar sodium salt. A defined amount of the brequinar may be provided as polymorphic form C of brequinar sodium salt, such as any of the percentage described above.

The inhibitor of the pyrimidine salvage pathway may inhibit a nucleoside transporter. The inhibitor of the pyrimidine salvage pathway maybe dipyridamole. In another aspect, the invention provides kits that include the following: at least one single unit dose of a first pharmaceutical composition that comprises a therapeutically effective amount of polymorphic form C of brequinar sodium salt and a therapeutically effective amount of an inhibitor of the pyrimidine salvage pathway; at least one single unit dose of a second pharmaceutical composition that comprises a therapeutically effective amount of the inhibitor of the pyrimidine salvage pathway and is substantially free of the polymorphic form C of brequinar sodium salt; and instructions for administration of the first pharmaceutical composition and the second pharmaceutic composition in a combination therapeutic regimen.

The kit may include a packaging that contains an exact number of single unit doses of the first pharmaceutical composition to be administered in one day and an exactnumberof single unit doses of the second pharmaceutical composition to be administered in one day. The exact number of single unit doses of the first pharmaceutical composition to be administered in one day may be one, two, three, or more. The exact number of single unit doses of the second pharmaceutical composition to be administered in one day may be one, two, three, or more.

The single unit dose of the first pharmaceutical composition may differ from single unit dose of the second pharmaceutical composition by one or more properties. The single unit dose of the first pharmaceutical composition may differs from single unit dose of the second pharmaceutical composition by one or more of size, shape, color, texture, mass, and surface printing.

The therapeutically effective amount of polymorphic form C of brequinar sodium salt may be from about 10 mg to about 100 mg, from about 10 mg to about 200 mg, from about 10 mg to about 300 mg, from about 10 mg to about 400 mg, from about 10 mg to about 500 mg, from about 10 mg to about 600 mg, from about 10 mg to about 800 mg, from about 10 mg to about 1000 mg, from about 25 mg to about 100 mg, from about 25 mg to about 200 mg, from about 25 mgto about 300 mg, from about25 mgto about400 mg, from about25 mgto about 500 mg, from about 25 mgto about 600 mg, from about 25 mgto about 800 mg, from about 25 mg to about 1000 mg, from about 50 mgto about 100 mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mgto about 400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about 1000 mg, from about 100 mg to about 200 mg, from about 100 mgto about 300 mg, from about lOOmgto about400 mg, from about 100 mgto about 500 mg, from about 100 mgto about 600 mg, from about lOOmgto about 800 mg, or from about 100 mgto about 1000 mg.

The inhibitor of the pyrimidine salvage pathway may inhibit a nucleoside transporter. The inhibitor of the pyrimidine salvage pathway maybe dipyridamole.

The therapeutically effective amount of the inhibitor of the pyrimidine salvage pathway may be from about 1 mgto about 100 mg, from about 1 mgto about 200 mg, from about 1 mgto about 300 mg, from about 1 mgto about 400 mg, from about 1 mgto about 500 mg, from about 1 mg to about 600 mg, from about 1 mgto about 800 mg, from about 1 mgto about 1000 mg, from about 1 mgto about 1200 mg, from about 1 mgto about 1500 mg, from about 5 mgto about 100 mg, from about 5 mgto about 200 mg, from about 5 mgto about 300 mg, from about 5 mg to about400 mg, from about 5 mgto about 500 mg, from about 5 mgto about 600 mg, from about 5 mgto about 800 mg, from about 5 mgto about 1000 mg, from about 5 mgto about 1200 mg, from about 5 mgto about 1500 mg, from about lOmgto about 100 mg, from about 10 mg to about 200 mg, from about 10 mgto about 300 mg, from about 10 mgto about 400 mg, from about 10 mgto about 500 mg, from about 10 mgto about 600 mg, from about 10 mgto about 800 mg, from about 10 mgto about lOOOmg, from about 10 mgto about 1200 mg, from about 10 mgto about 1500 mg, from about25 mgto about lOO mg, from about25 mgto about 200 mg, from about25 mgto about 300 mg, from about25 mgto about 400 mg, fromabout25 mg to about 500 mg, from about 25 mgto about 600 mg, from about 25 mgto about 800 mg, from about 25 mgto about lOOOmg, from about 25 mgto about 1200 mg, from about 25 mgto about 1500 mg, from about 50 mg to about 100 mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mgto about 400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mg to about 1000 mg, from about 50 mgto about 1200 mg, or from about 50 mgto about 1500 mg.

In another aspect, the invention provides combination therapies for treatment of a viral infection in a subject. The combination therapies include an inhibitor of de novo pyrimidine synthesis, an inhibitor of the pyrimidine salvage pathway, and a nucleoside analog. The inhibitor of de novo pyrimidine synthesis may inhibit dihydroorotate dehydrogenase (DHODH). The inhibitor of de novo pyrimidine synthesis may be brequinar. Brequinar may be provided as polymorphic form C of brequinar sodium salt. A defined amount of the brequinar may be provided as polymorphic form C of brequinar sodium salt, such as any of the percentage described above.

Each of the inhibitor of de novo pyrimidine synthesis, the inhibitor of the pyrimidine salvage pathway, and the nucleoside analog may independently be provided to a subject having a viral infection by a particular route or mode of administration, such as any of those described above.

The inhibitor of de novo pyrimidine synthesis may be provided to a subject having a viral infection in a pharmaceutical composition that includes the inhibitor of the pyrimidine salvage pathway.

The inhibitor of de novo pyrimidine synthesis may be provided to a subject having a viral infection in a single daily dose, such as any of the daily dosages described above.

The inhibitor of the pyrimidine salvage pathway may inhibit a nucleoside transporter. The inhibitor of the pyrimidine salvage pathway maybe dipyridamole.

The inhibitor of the pyrimidine salvage pathway maybe provided to a subject having a viral infection at a first time point in a pharmaceutical composition that includes the inhibitor of de novo pyrimidine synthesis. The inhibitor of the pyrimidine salvage pathway may be provided to a subject having a viral infection at a second time point in a pharmaceutical composition that is substantially free of the inhibitor of de novo pyrimidine synthesis.

Each of the inhibitor of de novo pyrimidine synthesis, the inhibitor of the pyrimidine salvage pathway, and the nucleoside analog may independently be provided in a single unit dose. Each of the inhibitor of de novo pyrimidine synthesis, the inhibitor of the pyrimidine salvage pathway, and the nucleoside analog may independently be provided in multiple units that are administered simultaneously. Each of the inhibitor of de novo pyrimidine synthesis, the inhibitor of the pyrimidine salvage pathway, and the nucleoside analog may independently be provided in two, three, four, or more units that are administered simultaneously. Each of the inhibitor of de novo pyrimidine synthesis, the inhibitor of the pyrimidine salvage pathway, and the nucleoside analog may independently be provided in multiple doses that are administered at separate times. The daily dosage of each of the inhibitor of de novo pyrimidine synthesis, the inhibitor of the pyrimidine salvage pathway, and the nucleoside analog may independently be provided in one dose, two doses, three doses, four doses, five doses, or more. The multiple daily doses may be separated by defined intervals, such as about 12 hours, about 8 hours, or about 6 hours.

The inhibitor of pyrimidine salvage pathway may be provided to a subject having a viral infection in a single daily dose, such as any of the daily dosages described above.

The nucleoside analog may be abacavir, acyclovir, allopurinol, azacitidine, azathioprine, cladribine, cytarabine, decitabine, didanosine, emtri citab in e, entecavir, favipiravir, floxuridine, fludarabine, fluorouracil, galidesivir, gemcitabine, gemcitabine, idoxuridine, lamivudine, mercaptopurine, molnupiravir, nelarabine, remdesivir, ribavirin, stavudine, telbivudine, trifluridine, trimethoprim, vidarabine, zalcitabine, or zidovudine.

The nucleoside analog may be provided to a subject having a viral infection at a first dose on a first day and at a second dose on subsequent days. The second dose may be lower than the first dose, higher than the first dose, or the same as the first dose. The second dose may be about 25%, about 33%, about 50%, about 67%, or about 75% of the first dose.

Each dose of the nucleoside analog may independently be from about 1 mg to about 100 mg, from about 1 mg to about 200 mg, from about 1 mg to about 300 mg, from about 1 mg to about 400 mg, from about 1 mg to about 500 mg, from about 1 mg to about 600 mg, from about 1 mg to about 800 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 1200 mg, from about 1 mg to about 1500 mg, from about 5 mg to about 100 mg, from about 5 mg to about 200 mg, from about 5 mg to about 300 mg, from about 5 mg to about 400 mg, from about 5 mg to about 500 mg, from about 5 mg to about 600 mg, from about 5 mg to about 800 mg, from about 5 mgto about 1000 mg, from about 5 mgto about 1200 mg, from about 5 mgto about 1500 mg, from about 10 mgto about 100 mg, from about 10 mg to about 200 mg, from about 10 mg to about 300 mg, from about 10 mgto about 400 mg, from about 10 mgto about 500 mg, from about 10 mgto about 600 mg, from about 10 mgto about 800 mg, from about 10 mgto about 1000 mg, from about 10 mg to about 1200 mg, from about 10 mgto about 1500 mg, from about 25 mgto about 100 mg, from about 25 mgto about 200 mg, from about 25 mgto about 300 mg, from about 25 mgto about 400 mg, from about 25 mgto about 500 mg, from about 25 mg to about 600 mg, from about 25 mg to about 800 mg, from about 25 mg to about 1000 mg, from about25 mgto about 1200 mg, from about 25 mgto about 1500 mg, from about 50 mgto about 100 mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mgto about 400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about 1000 mg, from about 50 mg to about 1200 mg, or from about 50 mgto about 1500 mg.

In another aspect, the invention provides methods of treating a viral infection in a subject. The methods include providing to a subject having a viral infection a therapeutically effective amount of brequinar to achieve a brequinar concentration of at least 1 μM in plasma of the subject for a period of at least 24 hours and a therapeutically effective amount of dipyridamole, for example in multiple doses, to achieve a dipyridamole concentration of at least 2 μM in plasma of the subject for a period of at least 24 hours.

The brequinar may be provided as polymorphic form C of brequinar sodium salt. A defined amount of the brequinar sodium salt may be in polymorphic form C. In the combination therapy, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the brequinar sodium salt may be in polymorphic form C.

Each of the brequinar and the dipyridamole may independently be provided by a particular route or mode of administration, such as any of those described above.

Brequinar and dipyridamole may each independently be provided in a single unit dose. Brequinar, dipyridamole, or both maybe provided in multiple units that are administered simultaneously. Brequinar, dipyridamole, or both may be provided in two, three, four, or more units that are administered simultaneously. Brequinar, dipyridamole, or both may be provided in multiple doses that are administered at separate times. Brequinar, dipyridamole, or both may be provided in one dose, two doses, three doses, four doses, five doses, or more per day. The multiple daily doses may be separated by defined intervals, such as about 12 hours, about 8 hours, or about 6 hours.

Brequinar and dipyridamole may each independently be provided at a defined dose, such as any of the doses described above. Brief Description of the Drawings

FIG. 1 is a graph showing the concentration of dipyridamole in the plasma following administration of a 50 mg dose three times per day in a simulated experiment.

FIG. 2 is a graph showing the concentration of dipyridamole in the plasma following administration of a 75 mg dose three times per day or two times per day in a simulated experiment.

FIG. 3 is a graph showing the concentration of dipyridamole in the plasma following administration of a 100 mg dose three times per day, two times per day, or four times per day in a simulated experiment.

FIG. 4 is a graph showing the concentration of dipyridamole in the plasma following administration of a 100 mg dose three times per day or two times per day in a simulated experiment.

FIG. 5 is a graph showing the concentration of dipyridamole in the plasma following administration of a 100 mg dose three times per day in a simulated experiment.

FIG. 6 is a graph showing the concentrations of brequinar and dipyridamole in the plasma following administration according to various regimens in a simulated experiment.

FIG. 7 is a graph showing the concentrations of brequinar and dipyridamole in the plasma following administration according to various regimens in a simulated experiment.

FIG. 8 is a graph showing the concentrations of brequinar and dipyridamole in the plasma following administration of a 100 mg dose of brequinar once per day and a 75 mg dose of dipyridamole three times per day in a simulated experiment.

FIG. 9 is a graph showing the concentrations of brequinar and dipyridamole in the plasma following administration of a 100 mg dose of brequinar once per day and a 100 mg dose of dipyridamole three times per day in a simulated experiment.

FIG. 10 is a graph showing the effects of various agents (brequinar, dipyridamole, brequinar + dipyridamole, and remdesivir) on replication of SARS-CoV-2. FIG. 11 is a three-dimensional graph showing the synergy of brequinar and dipyridamole at inhibiting viral replication.

FIG. 12 is a two-dimensional graph showing the synergy ofbrequinar and dipyridamole at inhibiting viral replication.

FIG. 13 is a graph showingthe effects of various agents (DMSO, brequinar, dipyridamole, brequinar + dipyridamole in presence or absence of uridine) on levels of intracellular ribonucleotide triphosphates (rNTPs) in HEK293T cells (nmoles).

FIG. 14 is a graph showingthe effects of various agents (DMSO, brequinar, dipyridamole, brequinar + dipyridamole in presence or absence of uridine) on levels of intracellular ribonucleotide triphosphates (rNTPs) in HEK293T cells (%baseline).

FIG. 15 is a graph showingthe effects of various agents (DMSO, brequinar, dipyridamole, brequinar + dipyridamole in presence or absence of uridine) on levels of intracellular ribonucleotide triphosphates (rNTPs) in A549 cells (nmoles).

FIG. 16 is a graph showingthe effects of various agents (DMSO, brequinar, dipyridamole, brequinar + dipyridamole in presence or absence of uridine) on levels of intracellular ribonucleotide triphosphates (rNTPs) in A549 cells (%baseline).

FIG. 17 is a graph of results from single agent administration against RSV.

FIG. 18 is a graph of results of dipyridamole and brequinar combination against RSV.

FIG. 19 is a summary of antiviral activity ofBRQ +DPY against influenza.

FIG. 20 is a graph of virus titer reductions following BRQ and DPY administration against influenza.

Detailed Description

When a virus infects a host organism, the virus co-opts cellular enzymes and metabolites, such as pyrimidines, to produce new virus particles. Viral replication depends on synthesis if the nucleic acids that make up the viral genome, and pyrimidine-based nucleotides are key building blocks of nucleic acids. Consequently, ample intracellular pyrimidine pools are necessary to allow viruses to reproduce within infected cells. Cells of host organisms, including humans, use two biochemical pathways to make pyrimidines. The de novo pyrimidine synthesis pathway includes a suite of enzymes that convert glutamine to uridine monophosphate (UMP) in six enzymatic steps. A key step in de novo pyrimidine synthesis is the conversion of dihydroorotate (DHO) to orotate by dihydroorotate dehydrogenase (DHODH). An alternative mechanism for cells to make pyrimidines is to salvage them from the products of nucleic acid degradation. Because nucleosides and nucleobases generated by breakdown of nucleic acid circulate in the bloodstream, one set of proteins required for salvaging pyrimidines include transporters that allow the products to enter cells. Also required for the pyrimidine salvage pathway are enzymes that convert the nucleosides and nucleobases into the nucleotides.

The invention is based on the recognition that viral replication can be blocked by simultaneous inhibition of the de novo and salvage pathways for pyrimidine biosynthesis. By inhibiting both pathways for synthesis of pyrimidines, pyrimidine pools can be depleted. Temporary depletion of pyrimidine pools thwarts viral replication but does not harm healthy cells, which can tolerate brief periods of nucleotide starvation. Consequently, the combination therapies of the invention are effective at treating viral infections without harming host cells.

Inhibitors of de novo pyrimidine synthesis

Combination therapies of the invention include an inhibitor of de novo pyrimidine synthesis. Pyrimidine biosynthesis involves a sequence of six enzymatic reactions that result in the conversion of glutamine to uridine monophosphate as shown below:

The first three steps in de novo pyrimidine synthesis are performed sequentially by a trifunctional, multi-domain enzyme, CAD, that has carbamoyl phosphate synthase, aspartate transcarbamoylase (also called aspartate transcarbamoylase or ATCase), and dihydroorotase activities. In the fourth step, which is the first committed step in de novo pyrimidine biosynthesis, dihydroorotate dehydrogenase (DHODH) catalyzes conversion of dihydrooro tate (DHO) to orotate. The last two steps are catalyzed by another multifunctional enzyme, uridine monophosphate synthetase (UMPS). In the fifth step, orotate is converted to orotidine 5'- monophosphate (OMP) by the orotate phosphoribosyltransferase activity of UMPS. In the sixth step, OMP is converted to uridine monophosphate (UMP) by the OMP decarboxylase (OMPD) activity of UMPS.

Subsequent steps in the synthesis of NTP pyrimidines from UMP are shared by the de novo and salvage pathways. UMP serves as a precursor for rNTPs required for RNA synthesis and dNTPs required for DNA synthesis. To make rNTPs, UMP is sequentially converted to uridine diphosphate (UDP) and uridine triphosphate (UTP) by cytidine monophosphate kinase (CMPK) and nucleoside-diphosphate kinase (NDPK), respectively. UTP is converted into cytidine triphosphate (CTP) by CTP synthetase (CTPS). To make dNTPs required for DNA synthesis, UDP and CDP are deoxygenated into deoxy-UDP (dUDP) and dCDP, respectively, by ribonucleotide reductase (RNR). dUTP is dephosphorylated by dUTPase to produce dUMP. dUMP can be converted by thymidylate synthase to produce deoxy-TMP (dTMP), which is then phosphorylated into dTTP.

In compositions and methods of the invention, a de novo pyrimidine synthesis inhibitor may target any of the reactions involved in de novo pyrimidine biosynthesis. Inhibitors of ATCase, DHODH, or OMPD that may be used in embodiments of the invention are provided below. However, other inhibitors of those enzymes and inhibitors of other enzymes involved in de novo pyrimidine biosynthesis may also be usedin embodiments of the invention.

Several DHODH inhibitors are known in the art. For example, and without limitation, inhibitors of DHODH include brequinar, leflunomide, teriflunomide, GSK983, GSK984, MEDS433, 6Br-oTol, and 6Br-pF. Brequinar, whichhas the systematic name 6-fluoro-2-(2’- fluoro-1, 1’ biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid, has the following structure:

Brequinar and related compounds are describedin, for example, U.S. PatentNos. 4,680,299 and 5,523,408, the contents of which are incorporated hereinby reference. Leflunomide, N-(4'-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide (I), is described in, for example, U.S. PatentNo. 4,284,786, the contents of which are incorporated herein by reference. The use ofMEDS433 to inhibit in vitro replication of SARS-CoV2 is describedin, for example, Calistri, A., et al., The New Generation h DHODH Inhibitor MED S433 Hinders the In Vitro Replication of SARS-CoV-2 and Other Human Coronaviruses, Microorganisms, 2021 Aug 14;9(8): 1731 , doi: 10.3390/microorganisms9081731. Teriflunomide, 2-cyano-3-hydroxy-N- [4-(trifluoromethyl)phenyl]-2-butenamide, is describedin, for example, U.S. PatentNo. 5,679,709, the contents of which are incorporated herein by reference. GSK983, GSK984, 6Br- oTol, and 6Br-pF are described in, for example, U.S. Patent No. 10,736,911 and Deans, etal., Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification, NatChem Biol. 2016 May; 12(5): 361-366. doi:10.1038/nchembio.2050, the contents of which are incorporated herein by reference.

One OMP decarboxylase inhibitor known in the art is pyrazofurin, which has the systematic name 5-[(2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl] -4-hydroxy- lH-pyrazole-3-carboxamide and the following structure:

Pyrazofurin andrelated compounds are described in, for example, U.S. PatentNos. 3,674,774 and 3,802,999, the contents of which are incorporated herein by reference.

One ATCase inhibitor known in the art is N-(phosphonacetyl)-L-aspartate (PALA). PALA is described in, for example, Swyryd et al, N-(Phosphonacetyl)-L- Aspartate, a Potent Transition State Analog Inhibitor of Aspartate Transcarb amylase, Blocks Proliferation of Mammalian Cells in Culture, J. Biol. Chem. Vol. 249, No. 21, Issue ofNovember 10, pp. 6945- 6950, 1974.

The de novo pyrimidine synthesis inhibitor may be provided as a prodrug, analog, derivative, or salt. The DHODH inhibitor may be provided in a micellar formulation. For example, brequinar may be provided as a brequinar analog, a brequinar derivative, a brequinar prodrug, a micellar formulation of brequinar, a brequinar hydrate, or a brequinar salt. The brequinar salt may be a sodium salt. Brequinar sodium salt may be provided in a crystalline form, as described in more detail in the section on pharmaceutical compositions. Inhibitors of the pyrimidine salvage pathway

Combination therapies of the invention include an inhibitor of the pyrimidine salvage pathway. The salvage of pyrimidines involves two sets of gene products: nucleoside transporters and enzymes.

Due to their hydrophilic nature, nucleosides require the action of nucleoside transporters (NTs) to enter cells from the extracellular environment. NTs are divided into two families: the concentrative nucleoside transporters (CNTs) and the equilibrative nucleoside transporters (ENTs), which are both primordial for the salvage of pyrimidine ribonucleotides from their nucleosides. CNTs are transport proteins that allow nucleosides to cross the plasma membrane against a chemical gradient, in a strict Na(+)-dependent manner. Humans have three CNTs, CNT1, CNT2, and CNT3, which are describedin, for example, Gray, et al., The concentrative nucleoside transporter family, SLC28, Eur. J. Physiol. 2004 February; 447(5):728-34, doi: 10. 1007/s00424-003-1107 -y, the contents of which are incorporated for reference. ENTs are passive transport proteins that allow nucleosides to cross the plasma membranes of cells down a chemical gradient. Humans have four ENTs, ENT1, ENT2, ENT3, and ENT4, which are described in, for example, Baldwin, et al., The equilibrative nucleoside transporter family, SLC29, Pflugers Arch. 2004 February; 447(5):735-43, doi: 10.1007/s00424-003-l 103-2, and Okesli, et al., Human Pyrimidine Nucleotide Biosynthesis as a Target for Antiviral Chemotherapy, Curr Opin Biotechnol. 2017 December; 48: 127-134, doi : 10. 1016/j. copbio.2017.03.010, the contents of which are incorporated by reference. When cells degrade nucleic acids, they generate large quantities of nucleosides, and NTs allow the nucleosides to exit the cells and enter the bloodstream. Conversely, when cells synthesize nucleic acids, such as when they harbor replicating viruses, the consumption of nucleosides leads to low levels of intracellular nucleosides, andNTs allow nucleosides from extracellular milieu to enter the cells. Similarly, when nucleoside pools are depleted due to inhibition of the de novo pyrimidine synthesis pathway, NTs permit the influx of nucleosides from the extracellular environmentto support pyrimidine synthesis via the salvage pathway. Because NTPs can be produced either from imported nucleosides or from UMP produced by the de novo pyrimidine synthesis pathway, NTs and the enzymes of the de novo pyrimidine synthesis pathway are independent of each other. A second set of gene products involved in the pyrimidine salvage pathway includes the enzymes that convert the nucleosides and nucleobases into nucleotide triphosph osphates (NTPs) to support nucleic acid synthesis. Examples of such enzymes include CTP synthase, nucleoside triphosphate phosphatase, nucleoside diphosphate kinase, apyrase, nucleoside diphosphate phosphatase, uridylate-cytidylate kinase, pyrimidine-specific 5’ -nucleotidase, uridine-cyti dine kinase (UCK), deoxy cytidine kinase (DCK), cytidine deaminase, uridine nucleosidase, uridine phosphorylase, and uracil phosphoribosyltransferase. Some of these enzymes, such as UCK and cytidine deaminase (CD A), are involved in converting uridine and cytidine to UMP and CMP, and thus are independent of the enzymes of the de novo pyrimidine synthesis pathway. As noted above, however, certain enzymes involved in the conversion UMP into rNTPs or dNTPs required for both synthesis of pyrimidines by both the de novo and salvage pathways.

Compositions and methods of the invention may include any inhibitor of the de novo pyrimidine synthesis pathway. The inhibitor may inhibit a nucleoside transporter (NT), such as one or more CNTs and/or ENTs. The inhibitor may inhibit an enzyme, such as any of those described above. The inhibitor may inhibit an enzyme that functions independently of the de novo pyrimidine synthesis pathway or an enzyme that is also involved in the de novo pathway. Examples of inhibitors of the pyrimidine salvage pathway include capecitabine, cladribine, clofarabine, cyclopentenyl cytosine (CPE-C), diazo-5-oxo-L-norleucine (DON), dilazep, dipyridamole, floxuridine, fludarabine, fluorouracil (5-FU), gemcitabine, TAS-114, and trifluridine. Inhibitors of the pyrimidine salvage pathway are described in more detail in, for example, Okesli, et al., Human Pyrimidine Nucleotide Biosynthesis as a Target for Antiviral Chemotherapy, Curr Opin Biotechnol. 2017 December; 48: 127-134. doi: 10.1016/j . copbio.2017.03.010, the contents of which are incorporated by reference.

Combination therapies that include an inhibitor of de novo pyrimidine synthesis and an inhibitor of the pyrimidine salvage pathway have previously been used to treat cancer. See, for example, Gaidano V., et al., The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia, Cancers (Basel) 2021 Feb 28;13(5): 1003, doi: 10.3390/cancersl3051003, the contents ofwhich are incorporated herein by reference. Nucleoside analogs

Composition and methods of the invention may include one or more nucleoside analogs. Non-limiting examples of nucleoside analogs that may be used in embodiments of the invention include abacavir, acyclovir, allopurinol, azacitidine, azathioprine, cladribine, cytarabine, decitabine, didanosine, emtricitabine, entecavir, favipiravir, floxuridine, fludarabine, fluorouracil, galidesivir, gemcitabine, gemcitabine, idoxuridine, lamivudine, mercaptopurine, molnupiravir, nelarabine, remdesivir, ribavirin, stavudine, telbivudine, trifluridine, trimethoprim, vidarabine, zalcitabine, and zidovudine.

The nucleoside analog may function as a DNA hypomethylating agent. Non-limiting examples of nucleoside analogs that function as hypomethylating agents include azacitidine and decitabine.

Azacitidine is a chemical analog of the nucleoside cytidine that is converted to a nucleotide analog that becomes incorporated into both DNA andRNA. When azacitidine is incorporated into DNA, it forms a covalent bond with DNA methyltransferase to inactivate the enzyme. When azacitidine is incorporated into DNA, it leads to disassembly of polyribosomes, defective methylation and acceptor function of transfer RNA, and inhibition of the production of proteins. Conversion of azacitidine to a nucleotide analog that becomes incorporated into nucleic acids requires phosphorylation of azacitidine by uridine-cytidine kinase (UCK). The mechanism of action of azacitidine is known in the art and described in more detail in, for example, Martens UM, ed. (2010) “11 5 -Azacytidine/ Azacitidine”. Small molecules in oncology. Recent Results in Cancer Research. 184. Heidelberg: Springer, pp. 159-170, doi:l 0.1007/978-3-642-01222-8; Dapp MJ, Clouser CL, Patterson S, Mansky LM (November 2009) “5 -Azacytidine can induce lethal mutagenesis in human immunodeficiency virus type 1”. Journal of Virology. 83 (22): 11950-8, doi: 10.1128/JVI.01406-09; and Diamantopoulos PT, Michael M, Benopoulou O, Bazanis E, Tzeletas G, Meletis J, Vayopoulos G, ViniouNA (January 2012) “Antiretroviral activity of 5-azacytidine during treatment of a HTLV-1 positive myelodysplastic syndrome with autoimmune manifestations”. Virology Journal. 9: 1, doi:10.1186/1743 -422X-9-1, the contents of which are incorporated herein by reference.

Decitabine is also a cytidine analog that is metabolized in vivo into a nucleotide analog that becomes incorporated into DNA. The incorporated form of decitabine forms a covalent bond with DNA methyltransferase and triggers proteasomal degradation of the enzyme, but formation of the covalent bond is not required for degradation. Decitabine is converted to a nucleotide analog via phosphorylation by deoxycytidine kinase. Unlike azacitidine, decitabine does not become incorporated into RNA. The mechanism of action of decitabine is known in the art and described in more detail in, for example, Datta, et al., Novel Insights into the Molecular Mechanism of Action ofDNA Hypomethylating Agents, Genes Cancer. 2012 Jan; 3(1): 71-81, doi: 10. 1177/1947601912452665; Kantarjian H, Issa JP, Rosenfeld CS, et al. (April 2006) “Decitabine improves patient outcomes in myelodysplastic syndromes: results of a phase III randomized study”. Cancer. 106 (8): 1794-1803, doi:10.1002/cncr.21792; and Kantarjian HM, O'Brien S, Cortes J, et al. (August 2003) “Results of decitabine (5 -aza-2'deoxy cytidine) therapy in 130 patients with chronic myelogenous leukemia”. Cancer. 98 (3): 522-528, doi : 10. 1002/cncr.11543, the contents of each of which are incorporated herein by reference.

Providing an agent to a subject

Methods of the invention include providing one or more agents, such as those described above, to a subject. Each of the agents may independently be provided to a subject by any suitable route of administration. For example and without limitation, an agent may be provided orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, subcutaneously, nasally, pulmonarily, or with or on an implantable medical device.

Each agent in a combination therapy may independently be provided to a subject according to a dosing regimen. In combination therapies, the different agents may be provided according to the same dosing regimen. Alternatively, the different agents may be provided according to different dosing regimens. The dosing regimens described belowmay be used for any agent provided in methods of the invention.

A dosing regimen may include a dosage and a schedule of administration. A dosage of an agent includes an amount of the agent. The amount of agent may be expressed in absolute terms, e.g., mass of the agent. The amount of agent may be expressed in terms relating the amount to the subject, e.g., agent mass per subject mass, or agent mass per subject volume. The amount of agent may be expressed in terms that indicate an effect of the agent, e.g., amount of agent that achieves a target concentration in a tissue or sample from the subject. A dosage may include a period of time over which the amount is to be administered to the subject. Thus, the dosage may include an amount of agent per unit of time. The dosage may include a single dose, i.e., the entire amount may be provided at once. Alternatively, the dosage may include multiple, e.g., 2, 3, 4, 6, or 8, doses that collectively achieve the entire amount of the dosage. A schedule of administration may be described by the interval between doses, e.g., every 24 hours, every 48 hours, etc., or by the number doses administered during a given period, e.g., once per week, twice per week, etc.

In methods of the invention, a dosage may include one dose per day, two doses per day, three doses per day, four doses per day, five doses per day, six doses per day, one dose per 36 hours, one dose per 48 hours, one dose per 60 hours, or one dose per 72 hours. Individual doses may be separated by about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours.

In combination therapies of the invention, different agents, such as those described above, may be provided according to the same dosing regimen. Alternatively, different agents may be provided according to different dosing regimens. Dosing regimens for combination therapies may include one or more time points at which multiple agents are provided to the subject. Dosing regimens for combination therapies may include one or more time points at which one or more agents are provided to the subject and one or more agents are withheld from the subject. For example, dosing of one or more drugs in a combination therapy may be administered with a loading dose. A loading dose is an initial higher dose of the agent that is given at the beginning of a course of treatment before dropping down to a lower maintenance dose. For example, the dosing regimen may include multiple consecutive dosages in which the first one, two, three, or four dosages are higher than subsequent dosages.

A dose may be provided to a subject as a single unit, such as in a single pill, tablet, capsule, etc. Alternatively, a dose may be provided to a subject in multiple units, i.e., in a divided dose. A divided dose may contain two, three, four, five, six, or more units, such as pills, tablets, capsules, etc.

In some methods and compositions of the invention, an agent, such as an inhibitor of de novo pyrimidine synthesis, an inhibitor of the pyrimidine salvage pathway, or a nucleoside analog, may be provided in a therapeutically effective amount. A “therapeutically effective amount” is a quantity of an agent that, when administered to a subject, produces a measurable effect in the subject. For example and without limitation, the measured effect may be physiological, biochemical, anatomical, medical, or psychological.

In compositions and methods of the invention, the daily dosage, single dose, or therapeutically effective amount of brequinar maybe from about 10 mg to about 180 mg, from about 26 mgto about 180 mg, from about 51 mgto about 180 mg, from about 76 mgto about 180 mg, from about 101 mgto about 180 mg, from about 126 mgto about 180 mg, from about 151 mgto about 180 mg, from about 10 mgto about 150 mg, from about 26 mgto about 150 mg, from about 51 mgto about 150 mg, from about 76 mgto about 150 mg, from about 101 mgto about 150 mg, from about 126 mgto about 150 mg, from about 10 mgto about 125 mg, from about 26 mgto about 125 mg, from about 51 mgto about 125 mg, from about 76 mgto about 125 mg, from about 101 mgto about 125 mg, from about 10 mg to about 100 mg, from about 26 mg to about 100 mg, from about 51 mgto about 100 mg, from about 76 mgto about 100 mg, from about 10 mgto about 75 mg, from about 26 mgto about 75 mg, from about 51 mgto about 75 mg, from about 10 mgto about 50 mg, from about 26 mgto 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 180 mg.

In compositions and methods of the invention, the daily dosage, single dose, or therapeutically effective amount of brequinar maybe from about 10 mgto about 4000 mg, from about 26 mgto about4000 mg, from about 51 mgto about4000 mg, from about 76 mgto about 4000 mg, from about 101 mgto about 4000 mg, from about 151 mgto about4000 mg, from about 201 mgto about 4000 mg, from about 10 mgto about 2000 mg, from about 26 mgto about 2000 mg, from about 51 mgto about 2000 mg, from about 76 mgto about 2000 mg, from about 101 mgto about 2000 mg, from about 151 mgto about 2000 mg, from about 201 mgto about 2000 mg, from about 10 mgto about 1000 mg, from about 26 mgto about 1000 mg, from about 51 mg to about 1000 mg, from about 76 mgto about 1000 mg, from about 101 mgto about 1000 mg, from about 151 mgto about 1000 mg, from about 201 mgto about 1000 mg, from about 10 mg to about 500 mg, from about 26 mgto about 500 mg, from about 51 mgto about 500 mg, from about 76 mgto about 500 mg, from about 101 mgto about 500 mg, from about 151 mgto about 500 mg, from about 201 mgto about 500 mg, from about 10 mgto about 300 mg, from about 26 mgto about 300 mg, from about 51 mgto about 300 mg, from about 76 mgto about 300 mg, from about 101 mgto about 300 mg, from about 151 mgto about 300 mg, from about 201 mg to about 300 mg, from about 10 mg to about 200 mg, from about 26 mg to about 200 mg from about 51 mgto about200 mg, from about 76 mgto about200 mg, from about 101 mgto about 200 mg, from about 151 mgto about200 mg, from about 10 mgto about 150 mg, from about 26 mgto about 150 mg, from about 51 mgto about 150 mg, from about 76 mgto about 150 mg, from about 101 mgto about 150 mg, from about 10 mg to about 100 mg, from about 26 mg to about 100 mg, from about 51 mgto about 100 mg, from about 76 mgto about 100 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about250 mg, about 300 mg, about 400 mg, about 500 mg, about 1000 mg, about 2000 mg, or about 4000 mg.

In compositions and methods of the invention, the daily dosage, single dose, or therapeutically effective amount of brequinar maybe from about 1 mgto about 100 mg, from about 1 mgto about 200 mg, from about 1 mgto about 300 mg, from about 1 mgto about 400 mg, from about 1 mgto about 500 mg, from about 1 mgto about 600 mg, from about 1 mgto about 800 mg, from about 1 mgto about 1000 mg, from about 1 mgto about 1200 mg, from about 1 mgto about 1500 mg, from about 5 mgto about 100 mg, from about 5 mgto about 200 mg, from about 5 mgto about 300 mg, from about 5 mgto about 400 mg, from about 5 mgto about 500 mg, from about 5 mgto about 600 mg, from about 5 mgto about 800 mg, from about 5 mg to about 1000 mg, from about 5 mgto about 1200 mg, from about 5 mgto about 1500 mg, from about 10 mg to about 100 mg, from about 10 mg to about 200 mg, from about 10 mg to about 300 mg, from about 10 mgto about400 mg, from about 10 mgto about 500 mg, from about 10 mgto about 600 mg, from about 10 mgto about 800 mg, from about 10 mgto about 1000 mg, from about 10 mgto about 1200 mg, from about 10 mgto about 1500 mg, from about 25 mg to about 100 mg, from about 25 mgto about 200 mg, from about 25 mgto about 300 mg, from about 25 mgto about 400 mg, from about 25 mgto about 500 mg, from about 25 mgto about 600 mg, from about 25 mgto about 800 mg, from about 25 mgto about 1000 mg, from about 25 mgto about 1200 mg, from about 25 mgto about 1500 mg, from about 50 mgto about 100 mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mg to about400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about lOOO mg, from about 50 mgto about 1200 mg, orfrom about 50 mgto about 1500 mg.

In compositions and methods of the invention, the daily dosage, single dose, or therapeutically effective amount of brequinar maybe from about 10 mgto about 100 mg, from about 10 mg to about 200 mg, from about 10 mg to about 300 mg, from about 10 mg to about 400 mg, from about 10 mg to about 500 mg, from about 10 mg to about 600 mg, from about 10 mg to about 800 mg, from about 10 mg to about 1000 mg, from about 25 mg to about 100 mg, from about 25 mg to about 200 mg, from about 25 mg to about 300 mg, from about 25 mg to about 400 mg, from about25 mgto about 500 mg, from about25 mgto about 600 mg, from about 25 mgto about 800 mg, from about 25 mgto about 1000 mg, from about 50 mgto about 100 mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mg to about400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about lOOOmg, from about lOO mgto about 200 mg, from about lOOmgto about 300 mg, from about lOOmgto about 400 mg, from about 100 mgto about 500 mg, from about lOOmgto about 600 mg, from about 100 mgto about 800 mg, or from about 100 mgto about 1000 mg.

In compositions and methods of the invention, the daily dosage, single dose, or therapeutically effective amount of an inhibitor of the pyrimidine salvage pathway, such as dipyridamole, maybe from about 1 mgto about 100 mg, from about 1 mgto about 200 mg, from about 1 mgto about 300 mg, from about 1 mgto about 400 mg, from about 1 mgto about 500 mg, from about 1 mgto about 600 mg, from about 1 mgto about 800 mg, from about 1 mgto about 1000 mg, from about 1 mgto about 1200 mg, from about 1 mgto about 1500 mg, from about 5 mgto about 100 mg, from about 5 mgto about200 mg, from about 5 mgto about 300 mg, from about 5 mgto about400 mg, from about 5 mgto about 500 mg, from about 5 mgto about 600 mg, from about 5 mgto about 800 mg, from about 5 mgto about 1000 mg, from about 5 mg to about 1200 mg, from about 5 mgto about 1500 mg, from about 10 mgto about 100 mg, from about 10 mgto about 200 mg, from about 10 mgto about 300 mg, from about 10 mgto about 400 mg, from about 10 mgto about 500 mg, from about 10 mgto about 600 mg, from about 10 mgto about 800 mg, from about 10 mgto about lOOO mg, from about 10 mgto about 1200 mg, from about 10 mgto about 1500 mg, from about 25 mgto about 100 mg, from about 25 mg to about 200 mg, from about 25 mgto about 300 mg, from about 25 mgto about 400 mg, from about25 mgto about 500 mg, from about25 mgto about 600 mg, from about25 mgto about 800 mg, from about25 mgto about lOOOmg, from about25 mgto about 1200 mg, from about 25 mgto about 1500 mg, from about 50 mgto about 100 mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mgto about 400 mg, from about 50 mg to about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about 1000 mg, from about 50 mgto about 1200 mg, or from about 50 mg to about 1500 mg.

In compositions and methods of the invention, the daily dosage, single dose, or therapeutically effective amount of an inhibitor of the pyrimidine salvage pathway, such as dipyridamole, maybe from about 20 mgto about 100 mg, from about 20 mgto about 200 mg, from about 20 mgto about 300 mg, from about 20 mgto about 400 mg, from about 20 mgto about 500 mg, from about 20 mgto about 600 mg, from about 20 mgto about 800 mg, from about 20 mgto about 1000 mg, from about 20 mgto about 1200 mg, from about 20 mgto about 1500 mg, from about 20 mgto about 2000 mg, from about 25 mgto about 100 mg, from about 25 mg to about 200 mg, from about 25 mgto about 300 mg, from about 25 mgto about 400 mg, from about25 mgto about 500 mg, from about25 mgto about 600 mg, from about25 mgto about 800 mg, from about 25 mgto about lOOO mg, from about 25 mgto about 1200 mg, from about 25 mgto about 1500 mg, from about25 mgto about2000 mg, from about 50 mgto about 100 mg, from about 50 mgto about 200 mg, from about 50 mgto about 300 mg, from about 50 mg to about400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about lOOO mg, from about 50 mgto about 1200 mg, from about 50 mg to about 1500 mg, from about 50 mgto about 2000 mg, from about 100 mgto about 200 mg, from about 100 mg to about 300 mg, from about 100 mgto about 400 mg, from about lOO mgto about 500 mg, from about 100 mgto about 600 mg, from about 100 mgto about 800 mg, or from about lOO mgto about lOOO mg, from about lOO mgto about 1200 mg, from about 100 mgto about 1500 mg, from about 100 mgto about 2000 mg, from about 200 mgto about 300 mg, from about 200 mg to about 400 mg, from about 200 mgto about 500 mg, from about 200 mg to about 600 mg, from about 200 mgto about 800 mg, or from about 200 mgto about 1000 mg, from about 200 mgto about 1200 mg, from about200 mgto about 1500 mg, or from about 200 mg to about 2000 mg.

In compositions and methods of the invention, the daily dosage, single dose, or therapeutically effective amount of a nucleoside analog, such as remdesivir, may be from about 1 mg to about 100 mg, from about 1 mgto about200 mg, from about 1 mgto about 300 mg, from about 1 mgto about400 mg, from about 1 mgto about 500 mg, from about 1 mgto about 600 mg, from about 1 mgto about 800 mg, from about 1 mgto about 1000 mg, from about 1 mgto about 1200 mg, from about 1 mgto about 1500 mg, from about 5 mgto about 100 mg, from about 5 mgto about 200 mg, from about 5 mgto about 300 mg, from about 5 mgto about 400 mg, from about 5 mgto about 500 mg, from about 5 mgto about 600 mg, from about 5 mgto about 800 mg, from about 5 mgto about 1000 mg, from about 5 mgto about 1200 mg, from about 5 mg to about 1500 mg, from about 10 mg to about 100 mg, from about 10 mg to about 200 mg, from about 10 mgto about 300 mg, from about 10 mgto about 400 mg, from about 10 mg to about 500 mg, from about 10 mgto about 600 mg, from about 10 mgto about 800 mg, from about 10 mgto about 1000 mg, from about 10 mgto about 1200 mg, from about 10 mgto about 1500 mg, from about 25 mgto about 100 mg, from about 25 mgto about 200 mg, from about 25 mgto about 300 mg, from about25 mgto about400 mg, from about25 mgto about 500 mg, from about25 mgto about 600 mg, from about 25 mgto about 800 mg, from about 25 mg to about 1000 mg, from about25 mgto about 1200 mg, from about 25 mgto about 1500 mg, from about 50 mgto about 100 mg, from about 50 mgto about200 mg, from about 50 mgto about 300 mg, from about 50 mgto about400 mg, from about 50 mgto about 500 mg, from about 50 mgto about 600 mg, from about 50 mgto about 800 mg, from about 50 mgto about 1000 mg, from about 50 mgto about 1200 mg, or from about 50 mgto about 1500 mg.

Methods of the invention may include providing an agent, such as brequinar or dipyridamole, to a subject to achieve a defined concentration of the agent in a body fluid of the subject for a defined period. Methods may include providing multiple agents to a subject to achieve defined concentrations of each agent in plasma of the subject for a defined period.

The defined concentration may be a threshold concentration or minimum concentration. The agent, such as such as brequinar or dipyridamole, may be provided to the subject to achieve a body fluid concentration of at least 1 nM, at least 3 nM, at least 10 nM, at least 30 nM, at least 100 nM, at least 300 nM, at least 500 nM, at least 750 nM, at least 1 μM, at least 1.5 μM, at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 6 μM, at least 8 μM, or at least 10 μM for a defined period.

The defined period may be a minimum period. The agent, such as such as brequinar or dipyridamole, maybe provided to the subject to achieve a defined body fluid concentration for at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours. The body fluid may be amniotic fluid, blood, cerebrospinal fluid, lymphatic fluid, phlegm, plasma, pus, saliva, semen, serum, sputum, sweat, synovial fluid, or urine.

A dosing regimen may contain a single dosage. Alternatively, a dosing regimen may contain multiple dosages. For example, a dosing regimen may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more dosages. A dosing regimen may include multiple dosages provided consecutively. For example, a dosage may include a defined amount of agent provided over a defined period, e.g., one day or 24 hours, andthe dosing regimen may include dosages provided in two or more consecutive periods, e.g., daysor 24-hour periods.

In dosing regimens that include multiple dosages, each dosage may be the same. For example, each dosage may include the same amount of an agent, such as any of those described above. Alternatively, dosing regimens may include dosages that are not all the same. In some embodiments, the dosing regimen includes multiple consecutive dosages in which the first one, two, three, or four dosages are higher than subsequent dosages. In some embodiments, the dosing regimen includes multiple consecutive dosages in which the first the first one, two, three, or four dosages are lower than subsequent dosages. In the aforementioned embodiments, the subsequent dosages may all be the same, or they may differ from each other as well. A variety of other dosage variations are possible within the scope of the invention. For example and without limitation, the dosing regimen may include any of the following sequences of dosages: alternation between high and low dosages; stepwise decreases or increases in individual dosages; stepwise decreases or increases in which one or more steps include two or more dosages that are the same; and patterns in which one or more of the aforementioned sequences is repeated or interspersed another aforementioned sequence. Each dosage may independently be selected from any of the dosages described above. For example, a first dosage may include a defined amount of an agent, and a subsequent dosage may include 200%, 150%, 125%, 100%, 75%, 50%, or 25% of the define amount.

The dosing regimen may include a dosage-free period in which the subject does not receive an agent, such as brequinar or a pharmaceutically acceptable salt thereof. The dosage- free period may be 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 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about

5 days, about ? days, about 10 days, or about 14 days.

The dosage-free period may follow a dosage. The dosage-free period may follow multiple dosages provided over consecutive 24-hour periods. The dosage-free period may follow multiple dosages provided over 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive 24-hour periods.

In some embodiments, an agent, such as brequinar or a pharmaceutically acceptable salt thereof, is not administered during a second phase. In some embodiments, a second phase involves administration of uridine rescue therapy.

In some embodiments, the first phase and the second phase each comprise administering an agent, such as brequinar or a pharmaceutically acceptable salt thereof. In some embodiments, the first phase and the second phase are at different times. In some embodiments, the first phase and the second phase are on different days. In some embodiments, the first phase lasts for a period of time that is less than four days. In some embodiments, the first phase comprises administering an agent, such as brequinar, followed by a period of time in which no agent is administered. In some embodiments, the period of time in which no agent is administered is 3 to 7 days after the dose during the first phase. In some embodiments, the first phase comprises administering more than one dose.

In some embodiments, an agent, such as brequinar or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof according to a multi-phase protocol comprising a first phase in which at least one dose of the agent is administered to the subject and a second phase in which at least one dose of the agent is administered to the subject, wherein one or more doses administered in the second phase differs in amount and/or timing relative to other doses in its phase as compared with the dose(s) administered in the first phase.

In some embodiments, the level of a metabolite, e.g. DHO, is determinedin 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 or differs from that of at least one dose administered before the metabolite level was determined. In some embodiments, the amount of an agent, such as brequinar or a pharmaceutically acceptable salt thereof, that is administered to the patient is adjusted in view of the metabolite level in the subject’ s plasma. For example, in some embodiments, a first dose is administered in the first phase. In some embodiments, metabolite level is determined at a period of time after administration of the first dose.

In some embodiments, if the metabolite level is below a pre-determined level, the amount of agent, such as brequinar, administered in a second or subsequent dose is increased and/or the interval between doses is reduced. For example, in some embodiments, the amount of brequinar administered may be increasedby 5 mg/m ² , 10 mg/m ² , 20 mg/m ² , 25 mg/m ² , 40 mg/m ² , 50 mg/m ² , 60 mg/m ² , 75 mg/m ² , or 100 mg/m ² . In some embodiments, the amount of brequinar administered in a second or subsequent dose is increased by 5 mg/m ² , 10 mg/m ² , 20 mg/m ² , 25 mg/m ² , 40 mg/m ² , 50 mg/m ² , 60 mg/m ² , 75 mg/m ² , 100 mg/m ² , 125 mg/m ² , 150 mg/m ² , 175 mg/m ² , or 200 mg/m ² . In some embodiments, the amount of agent, such as brequinar, administered may be increased by an adjustment amount determined based on change in metabolite levels observed between prior doses of different amounts administered to the subject. The dose may be increasedby an absolute amount. For example and without limitation, the amount of brequinar administered maybe increased by 5 mg, 10 mg, 20 mg, 25 mg, 40 mg, 50 mg, 60 mg, 75 mg, or 100 mg.

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

In some embodiments, if the metabolite level is above a pre-determined level, the amount of agent, such as brequinar, in a second or subsequent dose is decreased and/or the interval between doses is increased. For example, the amount of brequinar administered may be decreased by 5 mg/m ² , 10 mg/m ² , 20 mg/m ² , 25 mg/m ² , 40 mg/m ² , 50 mg/m ² , 60 mg/m ² , 75 mg/m ² , or 100 mg/m ² . In some embodiments, if the metabolite level is above a pre-determined level, the amount of brequinar in a second or subsequent dose is decreased by 5 mg/m ² , 10 mg/m ² , 20 mg/m ² , 25 mg/m ² , 40 mg/m ² , 50 mg/m ² , 60 mg/m ² , 75 mg/m ² , 100 mg/m ² , 125 mg/m ² , 150 mg/m ² , 175 mg/m ² , or 200 mg/m ² . In some embodiments, the amount of brequinar administered may be decreased by an adjustment amount determined based on change in metabolite levels observed between prior doses of different amounts administered to the subject. The dose may be decreased by an absolute amount. For example and without limitation, the amount of brequinar administered maybe decreasedby 5 mg, 10 mg, 20 mg, 25 mg, 40 mg, 50 mg, 60 mg, 75 mg, or 100 mg.

In some embodiments, the methods include administering a later dose of an agent, such as brequinar, to a patient who has previously received an earlier dose of the agent, wherein the patient has had a level of metabolite assessed subsequent to administration of the earlier dose, and wherein the later dose is different from the earlier dose. The later dose may be different from the earlier dose in amount of agent included in the dose, time interval relative to an immediately prior or immediately subsequent dose, or combinations thereof. The amount of agent in the later dose may be less than that in the earlier dose.

The method may include administering multiple doses of an agent, such as brequinar or a pharmaceutically acceptable salt thereof, in which doses are separated from one another by a time period that is longer than 2 days and shorter than 8 days. For example, the time period may be about 3 days, about 4 days, about 5 days, about 6 days or about ? days.

In some embodiments, the metabolite level is determined in a sample from the subject before each dose is administered, and dosing is delayed or skipped if the determined metabolite level is above a pre-determined threshold. For example, the metabolite level may be determined about 12 hours, about24 hours, about 36 hours, about48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours after administration of an agent, such as brequinar.

The method may include administering an agent, such as brequinar or a pharmaceutically acceptable salt thereof, according to a regimen approved in a trial in which a level of metabolite was measured in a patient between doses of the agent. The regimen may include multiple doses whose amount and timing were determined in the trial to maintain the metabolite level within a range determined to indicate a degree of target enzyme inhibition below a toxic threshold and above a minimum threshold. The regimen may include determining the metabolite level in the subject after administration of one or more doses of an agent, such as brequinar.

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

The subject may be a human. The subject may be a patient in a particular category. For example, the patient may be pediatric, newborn, neonates, infants, children, adolescent, pre- teens, teenagers, adults, or elderly. The patient may be in critical care, intensive care, neonatal intensive care, pediatric intensive care, coronary care, cardiothoracic care, surgical intensive care, medical intensive care, long-term intensive care, an operating room, an ambulance, a field hospital, or an out-of -hospital field setting.

Pharmaceutical compositions

In compositions and methods of the invention, one or more agents, such as any of those described above, maybe provided as pharmaceutical compositions. Pharmaceutical compositions of the invention may contain two or more agents. For example and without limitation, compositions may contain an inhibitor of de novo pyrimidine synthesis, such as brequinar, and an inhibitor of the pyrimidine salvage pathway, such as dipyridamole. Each agent may be present in a defined amount such as any of the amounts described above in relation to providing an agent to a subject. If brequinar is included in a pharmaceutical composition, it may be present in a specific crystal form, such as polymorphic form C. The pharmaceutical composition may be suitable for oral administration.

The invention includes compositions that contain combinations of an inhibitor of the de novo pyrimidine synthesis pathway, such as brequinar, and in inhibitor of the pyrimidine salvage pathway, such as dipyridamole, at fixed doses in a single dosage unit, such as a pill, tablet, capsule, etc. For example and without limitation, the single dosage unit may contain about 10 mg brequinar and about 10 mg dipyridamole, about 25 mg brequinar and about 10 mg dipyridamole, about 50 mg brequinar and about 10 mg dipyridamole, about 75 mg brequinar and about 10 mg dipyridamole, about 100 mgbrequinar and about 10 mg dipyridamole, about 125 mg brequinar and about 10 mg dipyridamole, about 150 mg brequinar and about 10 mg dipyridamole, about 175 mgbrequinar and about 10 mg dipyridamole, about 200 mg brequinar and about 10 mg dipyridamole, about 250 mg brequinar and about 10 mg dipyridamole, about 300 mgbrequinar and about 10 mg dipyridamole, about 10 mgbrequinar and about 25 mg dipyridamole, about 25 mgbrequinar and about 25 mg dipyridamole, about 50 mg brequinar and about 25 mg dipyridamole, about 75 mgbrequinar and about 25 mg dipyridamole, about 100 mg brequinar and about 25 mg dipyridamole, about 125 mgbrequinar and about 25 mg dipyridamole, about 150 mgbrequinar and about 25 mg dipyridamole, about 175 mgbrequinar and about 25 mg dipyridamole, about 200 mg brequinar and about 25 mg dipyridamole, about 250 mgbrequinar and about 25 mg dipyridamole, about 300 mgbrequinar and about 25 mg dipyridamole, about 10 mgbrequinar and about 50 mg dipyridamole, about 25 mgbrequinar and about 50 mg dipyridamole, about 50 mgbrequinar and about 50 mg dipyridamole, about 75 mg brequinar and about 50 mg dipyridamole, about 100 mgbrequinar and about 50 mg dipyridamole, about 125 mgbrequinar and about 50 mg dipyridamole, about 150 mg brequinar and about 50 mg dipyridamole, about 175 mgbrequinar and about 50 mg dipyridamole, about 200 mgbrequinar and about 50 mg dipyridamole, about 250 mgbrequinar and about 50 mg dipyridamole, about 300 mgbrequinar and about 50 mg dipyridamole, about 10 mgbrequinar and about 75 mg dipyridamole, about 25 mgbrequinar and about 75 mg dipyridamole, about 50 mg brequinar and about 75 mg dipyridamole, about 75 mgbrequinar and about 75 mg dipyridamole, about 100 mg brequinar and about 75 mg dipyridamole, about 125 mgbrequinar and about75 mg dipyridamole, about 150 mg brequinar and about75 mg dipyridamole, about 175 mgbrequinar and about 75 mg dipyridamole, about 200 mgbrequinar and about 75 mg dipyridamole, about 250 mgbrequinar and about 75 mg dipyridamole, about 300 mg brequinar and about 75 mg dipyridamole, about 10 mgbrequinar and about 100 mg dipyridamole, about 25 mg brequinar and about 100 mg dipyridamole, about 50 mgbrequinar and about 100 mg dipyridamole, about 75 mg brequinar and about 100 mg dipyridamole, about 100 mg brequinar and about 100 mg dipyridamole, about 125 mg brequinar and about 100 mg dipyridamole, about 150 mgbrequinar and about 100 mg dipyridamole, about 175 mgbrequinar and about 100 mg dipyridamole, about 200 mgbrequinar and about 100 mg dipyridamole, about 250 mgbrequinar and about 100 mg dipyridamole, about 300 mgbrequinar and about 100 mg dipyridamole, about 10 mg brequinar and about 125 mg dipyridamole, about 25 mgbrequinar and about 125 mg dipyridamole, about 50 mg brequinar and about 125 mg dipyridamole, about 75 mgbrequinar and about 125 mg dipyridamole, about 100 mgbrequinar andabout 125 mg dipyridamole, about 125 mgbrequinar and about 125 mg dipyridamole, about 150 mgbrequinar and about 125 mg dipyridamole, about 175 mgbrequinar and about 125 mg dipyridamole, about200 mgbrequinar and about 125 mg dipyridamole, about 250 mgbrequinar andabout 125 mg dipyridamole, about 300 mgbrequinar and about 125 mg dipyridamole, about 10 mgbrequinar andabout 150mg dipyridamole, about 25 mgbrequinar and about 150 mg dipyridamole, about 50 mgbrequinar and about 150 mg dipyridamole, about 75 mgbrequinar and about 150 mg dipyridamole, about 100 mgbrequinar and about 150 mg dipyridamole, about 125 mgbrequinar and about 150 mg dipyridamole, about 150 mgbrequinar and about 150 mg dipyridamole, about 175 mgbrequinar and about 150 mg dipyridamole, about 200 mgbrequinar andabout 150 mg dipyridamole, about 250 mgbrequinar and about 150 mg dipyridamole, about 300 mgbrequinar and about 150 mg dipyridamole, about 10 mgbrequinar and about 175 mg dipyridamole, about 25 mgbrequinar and about 175 mg dipyridamole, about 50 mgbrequinar and about 175 mg dipyridamole, about 75 mg brequinar and about 175 mg dipyridamole, about 100 mgbrequinar andabout 175 mg dipyridamole, about 125 mgbrequinar and about 175 mg dipyridamole, about 150 mgbrequinar and about 175 mg dipyridamole, about 175 mgbrequinar andabout 175 mg dipyridamole, about 200 mgbrequinar and about 175 mg dipyridamole, about 250 mg brequinar and about 175 mg dipyridamole, about 300 mgbrequinar and about 175 mg dipyridamole, about 10 mg brequinar and about 200 mg dipyridamole, about 25 mg brequinar and about 200 mg dipyridamole, about 50 mg brequinar and about 200 mg dipyridamole, about 75 mg brequinar and about 200 mg dipyridamole, about 100 mg brequinar and about 200 mg dipyridamole, about 125 mgbrequinar and about 200 mg dipyridamole, about 150 mgbrequinar and about 200 mg dipyridamole, about 175 mgbrequinar and about 200 mg dipyridamole, about 200 mg brequinar and about200 mg dipyridamole, about 250 mgbrequinar and about 200 mg dipyridamole, about 300 mgbrequinar and about 200 mg dipyridamole, about 10 mg brequinar and about 250 mg dipyridamole, about 25 mg brequinar and about 250 mg dipyridamole, about 50 mgbrequinar and about 250 mg dipyridamole, about 75 mg brequinar and about 250 mg dipyridamole, about 100 mg brequinar and about 250 mg dipyridamole, about 125 mgbrequinar and about 250 mg dipyridamole, about 150 mgbrequinar and about 250 mg dipyridamole, about 175 mgbrequinar and about250 mg dipyridamole, about 200 mgbrequinar and about 250 mg dipyridamole, about 250 mgbrequinar and about 250 mg dipyridamole, about 300 mgbrequinar and about 250 mg dipyridamole, about 10 mg brequinar and about 300 mg dipyridamole, about 25 mgbrequinar and about 300 mg dipyridamole, about 50 mg brequinar and about 300 mg dipyridamole, about 75 mgbrequinar and about 300 mg dipyridamole, about 100 mgbrequinar and about 300 mg dipyridamole, about 125 mgbrequinar and about 300 mg dipyridamole, about 150 mgbrequinar and about 300 mg dipyridamole, about 175 mgbrequinar and about 300 mg dipyridamole, about200 mgbrequinar and about 300 mg dipyridamole, about 250 mgbrequinar and about 300 mg dipyridamole, or about 300 mg brequinar and about 300 mg dipyridamole.

A pharmaceutical composition may be in a form suitable for oral use, for example, as tablets, troches, lozenges, fast-melts, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the compounds in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. Examples of excipients suitable for oral formulations include binders, coatings, coloring agents, disintegrants, flavoring agents, preservatives, sorbents, and sweeteners. Examples of specific excipients include inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.

The tablets may be uncoated, or they may be coated by known techniques to delay disintegration in the stomach and absorption lower down in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques describedin U.S. Patents 4,256,108, 4,166,452 and 4,265,874, to form osmotic therapeutic tablets for control release. Preparation and administration of compounds is discussed in U.S. Pat. No. 6,214,841 and U.S. Pub. No. 2003/0232877, the contents of each of which are incorporated by reference herein.

Formulations for oral use may also be presented as hard gelatin capsules in which the compounds are mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the compounds are mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. An alternative oral formulation, where control of gastrointestinal tract hydrolysis of the compound is sought, can be achieved using a controlled-release formulation, where a compound of the invention is encapsulated in an enteric coating.

Aqueous suspensions may contain the compounds in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example, polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxy cetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such a polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the compounds in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compounds in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified, for example sweetening, flavoring, and coloring agents, may also be present.

The pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally- occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soyabean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and agents for flavoring and/or coloring.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Pharmaceutical compositions may include other pharmaceutically acceptable carriers, such as sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin (glycerol), erythritol, xylitol, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employedin pharmaceutical formulations. The pharmaceutically acceptable carrier may be an encapsulation coating. For example, the encapsulation coating may contain carrageenan, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate trimellitate, collagen, gelatin, hydroxypropyl methyl cellulose acetate, a methyl acrylate-methacrylic acid copolymer, polyvinyl acetate phthalate shellac, sodium alginate, starch, or zein.

The agents, including prodrugs, analogs, derivatives, and salts thereof, may be provided as pharmaceutically acceptable salts, such as nontoxic acid addition salts, which are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Other pharmaceutically acceptable salts may be found in, for example, Remington, The Science and Practice of Pharmacy (20th ed. 2000). Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a pharmaceutically acceptable salt is an alkali salt. In some embodiments, a pharmaceutically acceptable salt is a sodium salt. In some embodiments, a pharmaceutically acceptable salt is an alkaline earth metal salt. In some embodiments, pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

Compositions that contain brequinar or a brequinar salt may contain a solid form of the drug. Compositions may contain crystals of brequinar or a brequinar salt, such as brequinar sodium. Crystals of brequinar sodium exist in at east least ten different polymorphic forms, labeled A-J. Compositions may contain a specific polymorphic form of brequinar sodium. For example, compositions may contain polymorphic form A, polymorphic form B, polymorphic form C, polymorphic form D, polymorphic form E, polymorphic form F, polymorphic form G, polymorphic form H, polymorphic form I, or polymorphic form J of brequinar sodium. Compositions may contain all or nearly all of the brequinar sodium salt in polymorphic form C. For example and without limitation, the composition may contain a brequinar sodium salt in which atleast 50%, atleast 60%, atleast 70%, at least 75%, 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 atleast 99.5% of the brequinar sodium salt in polymorphic form C. Crystal forms of brequinar sodium are described in co-owned, co-pending U.S. Application Nos. 63/043382 and 63/043384, the contents of each of which are incorporated herein by reference.

Monitoring metabolite levels

Methods of treating a condition in a subject may include monitoring the level of a metabolite, such as dihydroorotate (DHO), in a sample obtained from the subject. Monitoring the level of a metabolite may include receiving information about the measured level of the metabolite. Monitoring the level of a metabolite may include measuring the metabolite.

In some embodiments, the metabolite is measured by mass spectrometry, optionally in combination with liquid chromatography. Molecules may be ionized for mass spectrometry by any method known in the art, such as ambient ionization, chemical ionization (CI), desorption electrospray ionization (DESI), electron impact (El), electrospray ionization (ESI), fast-atom bombardment (FAB), field ionization, laser ionization (LIMS), matrix-assisted laser desorption ionization (MALDI), paper spray ionization, plasma and glow discharge, plasma-desorption ionization (PD), resonance ionization (RIMS), secondary ionization (SIMS), spark source, or thermal ionization (TIMS). Methods of mass spectrometry are known in the art and described in, for example, U.S. PatentNo. 8,895,918; U.S. Patent No. 9,546,979; U.S. Patent No. 9,761,426; Hoffman and Stroobant, Mass Spectrometry: Principles and Applications (2nd ed.). John Wiley and Sons (2001), ISBN 0-471-48566-7; Dass, Principles and practice of biological mass spectrometry, New York: John Wiley (2001) ISBN 0-471-33053-1; andLee, ed., Mass Spectrometry Handbook, John Wiley and Sons, (2012) ISBN: 978-0-470-53673-5, the contents of each of which are incorporated herein by reference. In certain embodiments, a sample can be directly ionized without the need for use of a separation system. In other embodiments, mass spectrometry is performed in conjunction with a method for resolving and identifying ionic species. Suitable methods include chromatography, capillary electrophoresis-mass spectrometry, and ion mobility. Chromatographic methods include gas chromatography, liquid chromatography (LC), high-pressure liquid chromatography (HPLC), hydrophilic interaction chromatography (HILIC), ultra-performance liquid chromatography (UPLC), and reversed-phase liquid chromatography (RPLC). In a preferred embodiment, liquid chromatography -mass spectrometry (LC-MS) is used. Methods of coupling chromatography and mass spectrometry are known in the art and described in, for example, Holcapek and Brydwell, eds. Handbook of Advanced Chromatography/Mass Spectrometry Techniques, AcademicPress and AOCS Press (2017), ISBN 9780128117323; Pitt, Principles and Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry, The Clinical Biochemist Reviews. 30(1): 19-34 (2017) ISSN 0159-8090; Niessen, Liquid Chromatography -Mass Spectrometry, Third Edition. Boca Raton: CRC Taylor & Francis, pp. 50-90. (2006) ISBN 9780824740825; Ohnesorge et al., Quantitation in capillary electrophoresismass 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, the contents of which are incorporated herein by reference.

A sample may be obtained from any organ or tissue in the individual to be tested, provided that the sample is obtained in a liquid form or can be pre-treated to take a liquid form. For example and without limitation, the sample may be a blood sample, a urine sample, a serum sample, a semen sample, a sputum sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a plasma sample, a pus sample, an amniotic fluid sample, a bodily fluid sample, a stool sample, a biopsy sample, a needle aspiration biopsy sample, a swab sample, a mouthwash sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a synovial fluid sample, a phlegm sample, a saliva sample, a sweat sample, or a combination of such samples. The sample may also be a solid or semi-solid sample, such as a tissue sample, feces sample, or stool sample, that has been treated to take a liquid form by, for example, homogenization, sonication, pipette trituration, cell lysis etc. The sample may be kept in a temperature-controlled environment to preserve the stability of the metabolite. For example, DHO is more stable at lower temperatures, and the increased stability facilitates analysis of this metabolite from samples. Thus, samples may be stored at 4 °C, -20 °C, or -80 °C.

In some embodiments, a sample is treated to remove cells or other biological particulates. Methods for removing cells from a blood or other sample are well known in the art and may include e.g., centrifugation, sedimentation, ultrafiltration, immune selection, etc.

The sample may be obtained from an individual before or after administration to the subject of an agent that alters activity of a metabolic pathway, such as inhibitor of an enzymein the pathway. For example, the sample may be obtained 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days ormorebefore administration of an agent, or it may be obtained 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more after administration of an agent.

The dosing regimen may be adjusted by comparing a measured level of a metabolite, e.g, DHO, in a sample obtained from a subject to a reference that provides an association between the measured level and a recommended dosage adjustment of a DHODH inhibitor, such as brequinar. For example, the reference may provide a relationship between administration of the DHODH inhibitor and levels of the metabolite in the subject. The relationship can be empirically determined from a known dose and time of administration of the DHODH inhibitor and measured levels of the metabolite at one or more subsequent time points. The reference may include a relationship between measured levels of the DHODH inhibitor or a metabolic product of the DHODH inhibitor and measured levels of the metabolite. Methods of dosing the DHODH inhibitor brequinar based on measured metabolite levels are known in the art and described in, for example, International Patent Publication Nos. WO 2019/191030 and WO 2019/191032, the contents of which are incorporated herein by reference.

From the comparison between the measured level of the metabolite and the reference, a dosing regimen may then be adjusted. For example, one or more of the dosages, intervals between doses, and dosage-free periods maybe adjusted. The dosing regimen may ensure that levels of a metabolite, such as DHO, are raised or maintained at a minimum threshold required to achieve a certain effect. For example, the dosing regimen may raise or maintain levels of the metabolite above a threshold level in the subject for a certain time period. The time period may include a minimum, a maximum, or both. For example, the dosing regimen may raise or maintain levels of the metabolite above the threshold level for at least 6 hours, 12, hours, 24 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, or more. The dosing regimen may raise or maintain levels of the metabolite above the threshold level for not more than 24 hours, not more than 36 hours, not more than 48 hours, not more than 60 hours, not more than 72 hours, not more than 84 hours, not more than 96 hours, not more than 5 days, not more than 6 days, not more than 7 days, not more than 10 days, or not more than 2 weeks. The dosing regimen may raise or maintain levels of the metabolite above the threshold level for at least 72 hours but not more than 96 hours, for at least 72 hours but not more than 5 days, for at least 72 hours but not more than 6 days, for at least 72 hours but not more than 7 days, for at least 96 hours but not more than 7 days.

The dosing regimen may ensure that levels of a metabolite, such as DHO, do not exceed or are maintained below a maximum threshold that is associated with toxicity. Levels of the metabolite above a maximum threshold may indicate thatDHODH inhibitor, such as brequinar, is causing or is likely to cause an adverse event in the subject. For example and without limitation, adverse events include abdominal pain, anemia, anorexia, blood disorders, constipation, diarrhea, dyspepsia, fatigue, fever, granulocytopenia, headache, infection, leukopenia, mucositis, nausea, pain at the injection site, phlebitis, photosensitivity, rash, somnolence, stomatitis, thrombocytopenia, and vomiting.

The dosing regimen may include a time point for administration of one or more subsequent doses to raise or maintain levels of the metabolite, such as DHO, above a threshold level for a certain time period. The time point for administration of a subsequent dose may be relative to an earlier time point. For example, the time point for administration of a subsequent dose may be relative to a time point when a previous dose was administered or a time point when a sample was obtained from a subject. Minimum and maximum threshold levels of a metabolite depend on a variety of factors, such as the metabolites and type of sample. Minimum and maximum threshold levels may be expressed in absolute terms, e.g., in units of concentration, or in relative terms, e.g., in ratios relative to a baseline or reference value. For example, the minimum threshold (below which a patient may receive a dose increase or additional dose) could also be calculated in terms of increase from a pre-treatmentDHO level or baseline level.

Minimum threshold levels of DHO or orotate in a human plasma sample may be about 0 ng/ml, about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 100 ng/mL, about 150 ng/mL, about200 ng/mL, about 250 ng/mL, about 300 ng/mL, about 350 ng/mL, about400 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 1250ng/ml, about 1500 ng/ml, about 1750 ng/ml, about2000 ng/ml, about 2500 ng/ml, about3000 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, about30,000 ng/ml, about40,000 ng/ml, about 50,000 ng/ml, about75,000 ng/ml, about 100,000 ng/ml, about 150,000 ng/ml, about 200,000 ng/ml, about 300,000 ng/ml, or about 400,000 ng/ml. The minimum threshold may include any value that falls between the values recited above. Thus, the minimum threshold may include any value between 0 ng/ml and 400,000 ng/ml.

Maximum threshold levels of DHO or orotate in a human plasma sample may be about 50 ng/mL, about 100 ng/mL, about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, about300 ng/mL, about350 ng/mL, about400 ng/mL, about450 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, about950 ng/mL, about lOOOng/mL, about 1250 ng/ml, about 1500 ng/ml, about 1750 ng/ml, about 2000 ng/ml, about2500 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, about25,000 ng/ml, about 30,000 ng/ml, about 40,000 ng/ml, about 50,000 ng/ml, about 75,000 ng/ml, about 100,000 ng/ml, about 150,000 ng/ml, about 200,000 ng/ml, about 300,000 ng/ml, about 400,000 ng/ml, or about 500,000 ng/ml. The maximum threshold may include any value that falls between the values recited above. Thus, the maximum threshold may include any value between 50 ng/ml and 500,000 ng/ml.

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

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

Dosage of a DHODH inhibitor, such as brequinar, also depends on factors such as the type of subject and route of administration. The dosage may fall within a range for a given type of subject and route of administration, or the dosage may be adjusted by a specified amount for a given type of subject and route of administration. For example, dosage of brequinar for oral or intravenous administration to a subject, such as human or mouse, may be about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, or about 100 mg/kg. Dosage ofbrequinar for oral or intravenous administration to a subject, such as a human or mouse, maybe adjusted by about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about25 mg/kg, or about 50 mg/kg. Dosage of brequinar for oral or intravenous administration to an animal subject, such as a human or mouse, may be about 50 mg/m ² , about 100 mg/m ² , about200 mg/m ² , about 300 mg/m ² , about 350 mg/m ² , about400 mg/m ² , about 500 mg/m ² , about 600 mg/m ² , about 700 mg/m ² , about 750 mg/m ² , about 800 mg/m ² , or about 1000 mg/m ² . Dosage of brequinar for oral or intravenous administration to an animal subject, such as a human or mouse, may be adjustedby 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 , or about 400 mg/m ² .

Methods may include determining whether the level of the metabolite is within a threshold range (e.g., above a minimal threshold and/or below a potential toxicity threshold) that warrants dosing, and/or that warrants dosing at a particular level or in a particular amount. Methods of determining the level of a metabolite and adjusting a dosing regimen of a DHODH inhibitor, such as brequinar, based on the determined levels of the metabolite are known in the art and describedin, for example, International Patent Publication Nos. WO 2019/191030 and WO 2019/191032, the contents of which are incorporated herein by reference.

The methods may include providing at least one dose of a DHODH inhibitor, such as brequinar, to a subject whose plasma metabolite level has been determined and is below a predetermined threshold (e.g., a pre-determined potential toxicity threshold and/or a pre-determined potential efficacy threshold). The predetermined threshold reflects percent inhibition of DHODH in the subject relative to a baseline determined for the subject. The baseline may be determined by an assay.

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

The method may comprise a step of re-determining the subject’ s plasma metabolite level after administration of at least one dose. In some embodiments, the subject’s plasma metabolite level is re-determined after each dose. The method may comprise administering at least one further dose of a DHODH inhibitor, such as brequinar, after the subject’s plasma metabolite level has been determined again (e.g., after administering a first or previous dose) to be below the predetermined threshold. If the subject’s plasma metabolite level is determined to be above a predetermined threshold, dosing can be discontinued. Thus, no further dose of the DHODH inhibitor is administered until the subject’s plasma metabolite level has been determined to again be below a pre-determined threshold.

Treating viral infections

The compositions and methods of the invention are useful for treating viral infections in a subject. The infection may be due to any type of virus or class of viruses. For example and without limitation, the infection may be from a virus or group of viruses that have a particular type of genome. For example and without limitation, the virus may be a DNA virus, doublestranded DNA virus, single-stranded DNA virus, double-stranded DNA virus with an RNA intermediate, RNA virus, double-stranded RNA virus, single-stranded RNA virus, positive- stranded single-stranded RNA virus, negative-stranded single-stranded RNA virus, or singlestranded RNA virus with a DNA intermediate. The virus may be from a particular family. For example and without limitation, the virus may be an adenovirus, arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, enterovirus, flavivirus, hepadnavirus, hepatitis virus, herpesvirus, human metapneumovirus, human parainfluenza virus, human respiratory syncytial virus, influenza virus orthomyxovirus, papillomavirus, paramyxovirus, parvovirus, picomavirus, polyomavirus poxvirus, reovirus, retrovirus, rhabdovirus, rhinovirus or togavirus. The coronavirus may be Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-Co V), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The influenza virus may be influenza A, influenza B, influenza C, or influenza D. The influenza A virus may be a HINl, H3N2, N9N2, orH5Nl strain.

The infection may affect a particular tissue, organ, or system. For example and without limitation, the viral infection may affect the respiratory system. Respiratory viral infections include any viral infection of any tissue or cell type within the respiratory system. The infection may occur in the upper respiratory system, lower respiratory system, or both. For example and without limitation, the infection may affect or occur in the alveoli, bronchi, bronchioles, larynx, lungs, nasal cavities, nose, pharynx, respiratory system, sinuses, and trachea. Kits

The invention also provides kits that contain pharmaceutical compositions, such as those described above. The kits may also include instructions on use of the pharmaceutical compositions contained therein. The instructions may include text, images, or both.

Kits may contain individual doses of one or more pharmaceutical compositions. Kits may include packaging that partitions the exact number of doses of each pharmaceutical composition to be administered to a subject in a particular period, such as one day. A kit may provide a defined number of doses of one or more pharmaceutical compositions to be administered to a subject over an extended period, such as one or more weeks, and the kit may contain packaging that partitions a defined number of doses of the one or more pharmaceutical compositions to be administered to a subject over a sub-division of the extended period, such as one day.

In kits that contain doses of multiple pharmaceutical compositions, units of the different pharmaceutical compositions may have properties that allow them to be readily distinguished from each other by a subject, healthcare provider, or other individual. For example and without limitation, units of different pharmaceutical compositions may differ in one or more of size, shape, color, texture, mass, and surface printing.

Examples

Example 1: Pharmacokinetics of brequinar and dipyridamole

To model the efficacy of combination therapies in certain embodiments of the invention, simulations were performed to determine levels of brequinar and dipyridamole in the plasma following at various time points following administration.

FIG. 1 is a graph showing the concentration of dipyridamole in the plasma following administration of a 50 mg dose three times per day in a simulated experiment. Orange dashed line represents a dipyridamole concentration of 2.5 μM.

FIG. 2 is a graph showing the concentration of dipyridamole in the plasma following administration of a 75 mg dose three times per day (blue line) or two times per day (gold line) in a simulated experiment. Orange dashed line represents a dipyridamole concentration of 2.5 μM, and green dashed line represents a dipyridamole concentration of 5 μM.

FIG. 3 is a graph showing the concentration of dipyridamole in the plasma following administration of a 100 mg dose three times per day (blue line), two times per day (gold line), or four times per day (mauve line) in a simulated experiment. Orange dashed line represents a dipyridamole concentration of 2.5 μM, and green dashed line represents a dipyridamole concentration of 5 μM.

FIG. 4 is a graph showing the concentration of dipyridamole in the plasma following administration of a 100 mg dose three times per day (blue line) or two times per day (gold line) in a simulated experiment. Orange dashed line represents a dipyridamole concentration of 2.5 μM, and green dashed line represents a dipyridamole concentration of 5 μM.

FIG. 5 is a graph showing the concentration of dipyridamole in the plasma following administration of a 100 mg dose three times per day (blue line) in a simulated experiment. Orange dashed line represents a dipyridamole concentration of 2.5 μM, and green dashed line represents a dipyridamole concentration of 5 μM.

FIG. 6 is a graph showing the concentrations of brequinar and dipyridamole in the plasma following administration according to the following regimens in a simulated experiment: brequinar at a 300 mg dose once per day for two days followed by a 100 mg dose once per day for three days (purple line); brequinar at a 400 mg dose once per day for one day followed by a 100 mg dose once per day for four days (gold line); and dipyridamole at a 100 mg dose three times per day for five days (blue line) in a simulated experiment. Orange dashed line represents a dipyridamole concentration of 2.5 μM, and green dashed line represents a dipyridamole concentration of 5 μM.

FIG. 7 is a graph showing the concentrations of brequinar and dipyridamole in the plasma following administration according to the following regimens in a simulated experiment: brequinar at a 300 mg dose once per day for two days followed by a 100 mg dose once per day for three days (purple line); brequinar at a 400 mg dose once per day for one day followed by a 100 mg dose once per day for four days (gold line); and dipyridamole at a 100 mg dose two times per day for five days (grey line) in a simulated experiment. Orange dashed line represents a dipyridamole concentration of 2.5 μM, and green dashed line represents a dipyridamole concentration of 5 μM.

FIG. 8 is a graph showing the concentrations of brequinar and dipyridamole in the plasma following administration of a 100 mg dose of brequinar once per day (orange line) and a 75 mg dose of dipyridamole three times per day (blueline) in a simulated experiment.

FIG. 9 is a graph showing the concentrations of brequinar and dipyridamole in the plasma following administration of a 100 mg dose of brequinar once per day (orange line) and a 100 mg dose of dipyridamole three times per day (blueline) in a simulated experiment. Orange dashed line represents a dipyridamole concentration of 2.5 μM, and green dashed line represents a dipyridamole concentration of 5 μM.

Example 2: In Vitro evaluation of antiviral activity of brequinar and dipyridamole against SARS-CoV-2 in Vero Cells

To test whether a DHODH inhibitor and inhibitor of the pyrimidine salvage pathway, either alone or in combination, inhibit viral replication, the DHODH inhibitor brequinar and dipyridamole, an inhibitor of the pyrimidine salvage pathway, were tested for the ability to stop replication of SARS-CoV-2 in vitro.

Methodology

Vero cells were seeded at a density of 1.2 x 10 ⁴ cells per well, 25 pl per well, in black 384-well, in plates sold under the trade name pClear plates (as sold by Greiner Bio-One) and incubated at 37°C and 5% CO2 overnight. On the next day, serially diluted compounds (BRQ and DPY) orDMSO was added into test wells by liquid handler sold under the trade name CyBi- HummingWell as indicated in the following table:

Table 1. Test Concentrations of Compounds

• The “no drug” row provided the 7-point dilution curve with the other drug. Cells were then infected with SARS-CoV-2 PCOV/KOR/KCDC03/2020 strain ata multiplicity of infection (MOI) of 0.0125. The final volume of the cell culture was 50 pl per well. The final concentration of DMSO in the cell culture was 0.5%. The cell control (without virus infection and compounds treatment) and virus control (with virus infection, without compounds treatment) were tested in parallel.

Following 24 hours of incubation at 37°C and 5% CO ₂ , cultures were fixed with 4% paraformaldehyde and then permeabilized with 0.25% TritonX-100. The virus was detected with Anti- SARS-CoV-2 N protein antibody (1 :3000 dilution in 5% normal goat serum in phosphate buffered saline (PBS)) at 37°C for 1 hour, and then stained with Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1 :2000 in 5% normal goat serum in PBS) and 2.5 μg/ml (1 :4000 dilution) of Hoechst 33342. After each step, the plateswere washed with PBS (as sold under the trade name Delbecco’s by Thermo Fisher) twice.

Images were acquired by using Operetta high content imaging system (Equipment setting: 488/405 emission, 20 X Objective, 5 images/well,). The acquired images were analyzed using Columbus software to quantify cell numbers (Hoechst-stained cells) and infected cell numbers (Alexa Fluor 488 stained cells). The infection ratios and the cell numbers were used for analysis of combination effect and cytotoxicity.

Cytotoxicity of the compound combinations was expressed as % Viability, and calculated with the formula below:

Viability (%) = Cell number of test well / Average cell numbers of virus control x 100

The combination indices were calculated using software sold under the trade name MacSynergy II (See Prichard and Shipman 1990). A positive combination index value indicates synergism, and a negative combination index value indicates antagonism.

The antiviral activity of brequinar (BRQ) in combination with dipyridamole (DPY) was evaluated in an immunofluorescence assay (IF A) using Vero cells infected with SARS-CoV-2 pCoV/KOR/KCDC03/2020 (NCCP43326). Results

The combination of BRQ and DPY showed synergistic antiviral activity against SARS- CoV-2. The synergy volume of +404.09 and antagonism volume of -5.93 equates to an absolute combination index of +398.16; this being greater than 100 indicates strong synergism.

Table 2. Combination indices of the 2 -drug combinations against SARS-CoV-2

An absolute combination index value of 25-50 indicates minor synergism or antagonism.

An absolute combination index value of 50-100 indicates moderate synergism or antagonism.

An absolute combination index value of >100 indicates strong synergism or antagonism.

FIG. 10 is a graph showing the effects of various agents on replication of SARS-CoV-2 in vitro. Various concentration of brequinar alone (darkblue circles connected by darkblue line), dipyridamole alone (light blue squares connected by light blue line), brequinar and dipyridamole (red diamonds connected by red line), and remdesivir (grey triangles connected by grey line) were tested on Vero E6 cells infected with SARS-CoV-2.

FIG. 11 . is a MacSynergy graph of dipyridamole and brequinar combination against SARS-CoV-2, showing synergism when the compounds are combined. The graph is a three- dimensional graph showing the synergy of brequinar and dipyridamole at inhibiting viral replication. Positive values indicate synergy volumes at 95% confidence level, and negative values indicate antagonism volumes at 95% confidence level. Values of less than 25 at 95% confidence level indicate insignificant synergism or antagonism; values of 25-50 at 95% confidence level indicate minor but significant synergism or antagonism; values of 50-100 at 95% confidence level indicate moderate synergism or antagonism that may be important in vivo; values of greater than 100 at 95% confidence level indicate strong synergism or antagonism that is likely important in vivo. FIG. 12 is a two-dimensional graph showing the synergy ofbrequinar and dipyridamole at inhibiting viral replication. Positive values indicate synergy volumes at 95% confidence level, and negative values indicate antagonism volumes at 95% confidence level. Relevance of values to significance of synergism or antagonism is as described above. The results in FIGS. 10-12 show that either brequinar or dipyridamole alone inhibits viral replication at high concentrations but that the combination ofbrequinar and dipyridamole inhibits viral replication at much lower concentrations of each agent.

The antiviral activity exhibited by the combination of BRQ+DPY was not associated with overt cytotoxicity on Vero cells at the concentrations tested, as shown in the table below.

Table 3. Percentage Cell Viability

Discussion

The combination ofbrequinar plus dipyridamole exhibited strong synergistic antiviral activity against SARS-CoV-2 at 24 hours in an immunofluorescence assay using Vero cells. The antiviral effect was not driven by overt cytotoxicity of either agent alone or in combination. Example 3: In Vitro evaluation of intracellular ribonucleotide triphosphates (rNTPs) in uninfected cells HEK293T and A549 Cells

FIG. 13 is a graph showing the effects of various agents on levels of intracellular ribonucleotide triphosphates (rNTPs). HEK293 T cells were treated with the following agents: DMSO (blue bars), DMSO + uridine (red bars); 1 μM brequinar (green bars); brequinar + uridine (purple bars); 1 μM dipyridamole (orange bars); dipyridamole + uridine (black bars); brequinar + dipyridamole (brown bars); or brequinar + dipyridamole + uridine (navy bars). Results are presented in terms of absolute amounts of rNTPs.

FIG. 14 is a graph showingthe effects ofvarious agents on levels of intracellular ribonucleotide triphosphates (rNTPs). HEK293 T cells were treated with the following agents: DMSO (blue bars), DMSO + uridine (red bars); 1 μM brequinar (green bars); brequinar + uridine (purple bars); 1 μM dipyridamole (orange bars); dipyridamole + uridine (black bars); brequinar + dipyridamole (brown bars); or brequinar + dipyridamole + uridine (navy bars). Results are presented in terms of relative amounts of rNTPs compared to a starting baseline value.

The results in FIGS. 13 and 14 further show that treatment with brequinar and dipyridamole in combination also produces a dramatic reduction in levels of pyrimidine rNTPs, and co-administration of uridine with brequinar and dipyridamole does not restore pyrimidine rNTP levels. Levels of purine rNTPs are not affected by either treatment. These results showthat simultaneous inhibition of the de novo pyrimidine synthesis pathway and the pyrimidine salvage pathway prevents cells from making pyrimidine rNTPs even in the presence of an abundance of a precursor for the pyrimidine salvage pathway.

FIG. 15 is a graph showingthe effects ofvarious agents on levels of intracellular ribonucleotide triphosphates (rNTPs). A549 cells were treated with the following agents: DMSO (blue bars), DMSO + uridine (red bars); 1 μM brequinar (green bars); brequinar + uridine (purple bars); 1 μM dipyridamole (orange bars); dipyridamole + uridine (blackbars); brequinar + dipyridamole (brown bars); or brequinar + dipyridamole + uridine (navy bars). Results are presented in terms of absolute amounts of rNTPs.

FIG. 16 is a graph showingthe effects ofvarious agents on levels of intracellular ribonucleotide triphosphates (rNTPs). A549 cells were treated with the following agents: DMSO (blue bars), DMSO + uridine (red bars); 1 μM brequinar (green bars); brequinar + uridine (purple bars); 1 μM dipyridamole (orange bars); dipyridamole + uridine (blackbars); brequinar + dipyridamole (brown bars); or brequinar + dipyridamole + uridine (navy bars). Results are presented in terms of relative amounts of rNTPs compared to a starting baseline value.

The results in FIGS. 13-16 show that treatment with brequinar alone produces a dramatic reduction in levels of pyrimidine rNTPs, i.e., CTP and UTP, but levels of pyrimidine rNTPs are restored when uridine is co-administered with brequinar. Levels of purine rNTPs are not affected by either treatment. These results indicate that cells can use the pyrimidine salvage pathway to make pyrimidine rNTPs when the de novo pyrimidine synthesis pathway is impaired.

Discussion

The results, as shown in FIGS. 13-16, support the idea that combination therapies that include inhibitors of both the de novo pyrimidine synthesis pathway and the pyrimidine salvage pathway deplete intracellular pools of pyrimidines to an extent that prevents viral replication.

Example 4: In Vitro evaluation of antiviral activity of brequinar and dipyridamole against respiratory syncytial virus in A549 Cells

The in vitro antiviral activity of brequinar (BRQ) alone and in combination with dipyridamole (DPY) was evaluated using A549 cells infected with respiratory syncytial virus RSV/A/Long.

Methodology

A549 cells were grown at 37°C and 5% CO2 in 24-well plates to 90% confluency. After two washes, cells were infected with RSV/A/Long (103 PFU/mL) for 60 minutes. Following incubation with virus, wells were washed twice, test compounds added, and plates incubated for a total of 72 hours. At 24-, 48-, and 72-hours post-infection, aliquots from each treatment were collected and processed for viral quantification using a standard viral plaque assay (4 dilution format) performed with HEp-2 cells as the indicators.

The test articles were evaluated as single agents using concentrations indicated in the table below. Table 4. Test Article Concentrations as Single Agents

The test articles were also evaluated as single agents and as 2-drug combinations using concentrations indicated in the table below.

Table 5. Test Article Concentrations in Combination

Results

As single agents, the positive control PALIVIZUMAB (PAL; 5 ug/mL) exhibited a significant reduction in virus titer relative to the media negative control to below the limit of quantification (<LLOQ; 2.0 logw PFU/mL) at all timepoints tested. BRQ as a single agent at 1 and 4 uM exhibited a significant decrease in virus titer relative to the negative control at 48 and 72 hr post-infection; virus titers at 24 hr post-infection were not different from the negative control. BRQ at 0.25 uM exhibited a significant reduction in virus titer only at 48 hr postinfection butto a lesser degree than BRQ at 1 or 4 uM. BRQ at 0.0625 uM did not significantly reduce virus titer at any timepoint. As expected from other antiviral assay systems, DPY did not exhibit antiviral activity at either 0.5 or 5 uM at any timepoint.

FIG. 17 is a graph of results from single agent administration at 24 hours post-infection, 48 hours post-infection, and 72 hours post-infection. As shown, BRQ exhibits in vitro antiviral activity against RSV/A/Long.

Results are also shown in the table below.

Table 6. ANOVA with Dunnett’s Multiple Comparisons Test vs C-EMEM Control

BOLD indicates significantly less than C-EMEM Control

Underline indicates significantly greater than C-EMEM Control

The data from BRQ and DPY as single agents were used to inform the design of further studies evaluating BRQ+DPY in combination. The monoclonal antibody viral entry inhibitor PAL (5 ug/mL) was a positive control for the assay. PAL exhibited a significant reduction in virus titer relative to the media negative control to below the limit of quantification (<LLOQ; 2.0 logw PFU/mL) at all timepoints tested. All concentrations of BRQ, both as a single agent and in combination with 1 uM DPY, exhibited significant reductions in virus titer relative to the media negative control. As expected from other antiviral assay systems, DPY did not exhibit antiviral activity at either 0.5 or 5 uM at any timepoint.

FIG. 18 is a graph of results from combination compound administration at 24 hours post-infection, 48 hours post-infection, and 72 hours post-infection. As shown, DPY enhances BRQ in vitro antiviral activity against RSV/A/Long

Results are also shown in the table below.

Table 7. ANOVA with Dunnett’s Multiple Comparisons Test vs C-EMEM Control

BOLD indicates significantly less than C-EMEM Control

The magnitude and significance of reduction in virus titer relative to the positive controls and to BRQ alone demonstrated the enhanced antiviral activity of BRQ in combination with DPY. In comparison to PAL 5 ug/mL, the most illustrative example is data with BRQ at 1 uM. Although single agent BRQ 1 uM significantly reduced virus titer relative to the negative control, it was significantly less potent than PAL at all timepoints; however, BRQ 1 uM and 1 uM DPY exhibited reductions in virus titer that were not different than PAL. BRQ 0.25 uM and 1 uM DPY exhibited similar antiviral activity to PAL at 24 hr post-infection but was less active at 48 and 72 hours. Table 8. ANOVA with Dunnett’s Multiple Comparisons Test vs PAL 5 ug/mL

BOLD indicates significantly less than C-EMEM Control

Underline indicates significantly greater than C-EMEM Control The most illustrative example is data with BRQ at 1 uM. At 24 and 48 hr post-infection,

BRQ 1 uM and DPY 1 uM exhibited high reductions in virus titer, at 72 hr post-infection, BRQ 1 uM + DPY 1 uM showed a significant reduction in virus titer.

Results are shown in the table below. Table 9. ANOVA with Dunnett ’s Multiple Comparisons Test

BOLD indicates significantly less than C-EMEM Control

Underline indicates significantly greater than C-EMEM Control

Finally, a comparison of antiviral activity of BRQ+DPY vs BRQ alone at each concentration also confirmed that DPY enhanced the antiviral activity of BRQ against

RSV/A/Long. Again, the most illustrative example is data with BRQ at 1 uM. The combination of BRQ 1 uM + DPY 1 uM exhibited significantly greater reductions in virus titer than BRQ 1 uM as a single agent. In addition, BRQ 0.25 uM + DPY 1 uM exhibited significantly greater reductions in virus titer relative to BRQ 0.25 uM alone.

Results are shown in the table below.

Table 10. ANOVA with Dunnett’s Multiple Comparisons Test vs BRQ Single Agent

BOLD indicates significantly less than C-EMEM Control

Underline indicates significantly greater than C-EMEM Control

In terms of cytotoxicity, cytopathic effect (CPE) is used as the primary visual readout for the assay. With 72-hourRSV in vitro assays, CPE induced by test materials would be evident at 24- and 48- hours post-infection as RSV does not induce syncytia at these timepoints. No CPE was observed in single agent or combination agent experiments.

Discussion

The combination ofbrequinar plus dipyridamole exhibited significantly greater antiviral activity against RSV/A/Long than with either agent alone. In addition, BRQ + DPY at pharmacologically relevant concentrations of 1 uM each exhibited similar antiviral activity to the potent positive control PAL.

The antiviral effect was not driven by overt cytotoxicity of either agent alone or in combination. Example 5: A Phase II, Randomized, Assessor-blind, Multicenter, Multi-dose, Placebo- controlled Study Assessing the Safety and Anti-coronavirus Response of Brequinar Combined With Dipyridamole in Patients With Mild to Moderate SARS-CoV-2 Infection. (CCBCRISIS04)

Overview

The efficacy of a combination therapy that includes brequinar and dipyridamole in treating SARS-CoV-2 infections is tested in human subjects.

Rationale

Brequinar is a potent DHODH inhibitor that has been previously studied in more than 1,000 cancer, psoriasis, and organ transplant patients as well as 71 patients with confirmed SARS-CoV2 infection. Brequinar has potent in vitro antiviral activity against many RNA viruses, including SARS-CoV-2. The antiviral activity of brequinar against SARS-CoV-2 is likely due to DHODH inhibition and shows nanomolar potency and a high selectivity index in inhibiting viral replication in in vitro studies. Results in 15 hospitalized COVID-19 patients and 56 COVID-19 out-patients demonstrated that brequinar lOOmgx 5 days was safe andwell- tolerated in these populations. Brequinar’ s antiviral activity was demonstrated in the out-patient study as shown by decreased viral load at days 15, 22 and 29 and a shorter duration of viral shedding.

Another factor that may blunt brequinar’s antiviral effect may be pyrimidine salvage. Dipyridamole is a platelet inhibitor marketed for its ability to lengthen abnormally shortened platelet survival time. Dipyridamole has been shown in vitro to block pyrimidine salvage and may potentiate the antiviral activities of RNA-dependent RNA polymerase (RdRp) inhibition on viral genome infection (Liu et al., 2020). Blocking pyrimidine salvage may improve the antiviral efficacy of DHODH inhibition and was usedin this study in combination with brequinar to enhance brequinar’s antiviral activity.

The CRISIS04 trial studied non-hospitalized patients who have a positive SARS-CoV-2 test and had mild to moderate signs and/or symptoms associated with CO VID- 19 infection. Subjects were randomized to receive standard of care(SOC) + 5 days of brequinar, or SOC + 5 days of brequinar with dipyridamole, or SOC + 5 days of placebo. The purpose of this study was to determine if the antiviral activity of higher doses of brequinar and/or the brequinar/dipyridamole combination decreases viral load in patients infected with SARS-CoV-2.

Product and dosage

Subjects were randomized to either standard of care (SOC) + 5 days of brequinar X mg, SOC + 5 days of brequinar X mg + 75 mg dipyridamole thrice daily, or SOC + placebo where X was to be 50 mg for the first cohort, 100 mg for the second cohort, 150 mg for the third cohort and 200 mg for the fourth cohort. Safety and tolerability at a given dose were required before the next cohort can be dosed. The subjects self-administered study medication as directed on Study Days 1-5. Treatment assignment was randomized and blinded.

Design

This was a phase 2 randomized, blinded, multi-center study with approximately 16 subjects per arm and was to have included an expansion cohort at the maximally tolerated dose for a total of approximately 112 subjects. All subjects received SOC per relevant guidelines for treatment of patients with CO VID-19 infection.

Subjects had a documented positive SARS-CoV-2 test result and at least one symptom consistent with SARS-CoV-2 infection rated as mild to moderate in the opinion of the investigator. Study Day 1 may have included screening activities as well as randomization. Study Day 1 and first dose of study drug was administered < 5 days from onset of first symptom. Study visits (virtual or in person) took place on Study Days 1-8, 12, 15, 22, and 29. The visits that included bloodwork were conducted at the study site, or arrangements are made for sample collection at the subject’s home or other appropriate location. Other visits/visit activities forthat visit may have been conducted remotely using telephone, telemedicine or other remote technique. Subjects self-assessed their respiratory rate, heart rate, body temperature and SpO2 and self-completed a symptom assessment on Days 1 through 15, 18, 22, 25, and 29. The site also had a telephone call to communicate with the subject on Study Days 2 through 7 for changes in concomitant medications and assessment of adverse events (or use some sort of patient- reported outcome (PRO) device/app). The Day 1 visit was conducted at the site unless the site was unable to accommodate in-person visits. If the site was unable to accommodate in-person visits, a home visit for lab samples was arranged as long as first dose could be achieved within 5 days of symptom onset. Days 8, 15, and 29 required in-person visits for lab draws and were conducted at the site or via home visit. Any in-person visit may also have had telemedicine or telephone components if all study activities were not completed in person. Telemedicine only visits were conducted by site staff on study Days 4, 12, and 22.

Treatment

All subjects received standard of care(SOC) including treatment for CO VID- 19 signs and/or symptoms as required. Subjects were randomly assigned to SOC +brequinar X mg daily for 5 days, SOC + brequinar X mg daily with dipyridamole 75 mg thrice daily for 5 days, or SOC + placebo daily for 5 days, where X was 50 mg for the first cohort, 100 mg for the second cohort, 150 mg for the third cohort and 200 mg for the fourth cohort. Study encounters were conducted remotely or at the study site depending on site facilities and subject and study team preferences.

Statistical analysis

A separate, detailed statistical analysis plan (SAP) was finalized prior to locking the database. All analyses of safety and efficacy for this study were descriptive in nature and presented by treatment group. Subject demographics and baseline characteristics were summarized. Subject data listings were also provided.

Summaries for quantitative variables included the mean, median, quartiles, standard error, minimum, and maximum. For qualitative variables, the summaries included the number and percentage of subjects for each outcome, and the 95% CI, when appropriate. Any statistical testing was considered exploratory and descriptive. All computations were performed using SAS (Version 9.4 or higher). Safety and tolerability were assessed in terms of AEs, SAEs, and safety laboratory data.

Brief Summary

A Phase 2 multi-center, assessor-blind, randomized study was conducted to assess the safety, tolerability, and antiviral activity of brequinar in combination with dipyridamole.

Detailed Description

This was a Phase 2 clinical trial in two parts. The first part of the trial was to have studied up to 64 subjects using a dose escalation approach, with 16 subjects per cohort for up to 4 cohorts. The brequinar dose started at 50 mg per day in Cohort 1, escalating in the next cohort of 16 to 100 mg, and was to have then escalated to 150 mg, and finally to 200 mg if safety parameters were met. The dipyridamole dose was 75 mgthree times a day (TID) for subjects assigned to the combination arm for all cohorts. All subjects also received standard of care (SOC) for treatment of patients with CO VID- 19 infection. After identifying the highest brequinar dose that was safe and well tolerated, 48 subjects were to be treated in an expansion part comparing the chosen brequinar dose in combination with 75 mg dipyridamole TID to the chosen dose of brequinar alone. The combination of brequinar and dipyridamole shows potent in vitro antiviral activity by blocking DHODH and the pyrimidine salvage pathway, respectively, and the purpose of this study was to establish the safety and antiviral effect of the combination.

During the dose escalation part of the study, subjects were confirmed to have mild to moderate COVID-19 and received 5 days of one of the following oral doses: brequinar alone, brequinar in combination with dipyridamole, or placebo. Subjects had a Screening Visit followed as soon as possible with Study Day 1 . Study visits (virtual or in person) took place at Screening and on specified days. The visits included bloodwork that was conducted at the study site or arrangements made for sample collection at the subject's home or other appropriate location. Other visits/visit activities for that visit were conducted remotely using telemedicine or other remote technique. A viral load sample, vital signs (respiratory rate, heart rate, body temperature and SpO2), and a symptom assessment were completed on specified days.

Study Design

Arms and Interventions

Outcome measures

Primary Outcome Measures:

Secondary Outcome Measures:

Eligibility Criteria

Inclusion Criteria:

Exclusion Criteria:

Initial Clinical Safety of the Brequinar-Dipyridamole Combination

The combination of brequinar and dipyridamole (BRQ-DPY) was initially tested in a human clinical trial. The study represented the initial step to evaluate the safety of this combination in CO VID-19 patients (NCT05166876). Qualifying subjects had a positive screening SARS-CoV-2 RT-PCR test, then were treated for 5 days with either the oral BRQ+DPY combination or oral BRQ alone or placebo. A total of 26 patients were treated in this study, 13 with the combination (BRQ 50 mg QD + 75 mg DPY TID, N = 8 and BRQ 100 mg QD + DPY 75 mg TID, N = 5), 7 with BRQ alone (BRQ 50 mg QD, N = 4 and BRQ 100 mgN = 3), and 6 with placebo.

Brequinar and the combination of brequinar-dipyridamole were safe and well -tolerated in this population. Adverse events (AEs) were reported in 7 of the 26 treated subjects (26.9%), 3/13 (23.1%) for the BRQ-DPY combination-treated subjects, 1/7 (14.3%) for brequinar alone-treated subjects, and 3/6 (50.0%) for placebo-treated subjects. There were no deaths and no serious adverse events. All AEs were considered by the investigators to be Grade 1 - Mild. Skin rash in one subject treated with the BRQ 50 mg + DPY 75 mg TID combination was consideredby the investigator to be possibly related to the combination. The skin rash occurred on day 4 of treatment and resolved within one day. Study drug was not discontinued, and the dose was not changed due to this AE. All other AEs were considered Unlikely or NotRelated. No subjects experienced an AE leading to study drug discontinuation, and no subjects discontinued the study due to an AE. See table below.

Table 11. CCB-CRISIS-04 Overall Summary of Treatment Emergent Adverse Events (Safety

Population) Discussion

The combination ofbrequinar and dipyridamole shows potent in vitro antiviral activity by blocking DHODH and the pyrimidine salvage pathway, respectively, and this study establishes the safety of the combination.

Example 6: In Vitro evaluation of antiviral activity of brequinar and dipyridamole against Dengue virus 2 (DENV2)

The in vitro antiviral activity of brequinar (BRQ) in combination with dipyridamole (DPY) against Dengue virus 2 (DENV2) was evaluated using Huh-7 cells by two methodologies as described below.

Methodology

Neutral Red (CPE/Toxicity)

Huh-7 cells were grown at 37°C and 5% CO ₂ in 96-well plates to at/near confluency. After washes, test compounds were added to the cells in 0. ImL volume at 2x concentration. Virus, at a titer that will cause >80% cytopathic effect (CPE) with a multiplicity of infection (MOI) <0.003, is added in O. lmL volume. Plates were incubated at 37°C and 5% CO ₂ until >80% CPE is observed in the virus no-drug control wells. The plates were then stained with 0.011% neutral red for approximately two hours at 37°C in a 5% CO ₂ incubator. The neutral red medium was removed, and the cells were washed with IX with phosphate buffered solution (PBS) to remove residual dye. The PBS was then completely removed, and the incorporated neutral red is eluted with 50% Sorensen’s citrate buffer/50% ethanol for at least 30 minutes.

Since neutral red dye penetrates living cells, the more intense the red color, the larger the number of viable cells present in the wells. The dye content in each well is quantified using a spectrophotometer at 540 nm wavelength and converted to a percentage of dye present in untreated control wells and normalized based on the virus no-drug control. The 50% effective (EC ₅₀ , virus-inhibitory) concentrations and 50% cytotoxic (CC ₅₀ , cell-inhibitory) concentrations are then calculated by regression analysis. The quotient of CC ₅₀ divided by EC ₅₀ gives the selectivity index (SI) value. Compounds showing SI values >10 are considered active. Visual (Virus Yield Reduction (VYR))/ Neutral Red (Toxicity)

The assay setup was similar to that of the Neutral Red (CPE/Toxicity) assay described above. After sufficient evidence of virus replication is noted, generally 3 days post-infection, supernatants were collected from each infected well (replicate wells are pooled) and tested (or stored) for virus titer determination by endpoint dilution. After maximal CPE is observed, the viable plates were stained with neutral red and the amount of incorporated dye was quantified as described above to determine the CC ₅₀ values. For virus titer determination, briefly, serial 10- fold dilutions of supernatant were plated into 4 replicate wells and the plates were then incubated and cells scored for the presence or absence of virus after distinct CPE was observed. The EC90 value was calculated by regression analysis by plotting the logl 0 of the inhibitor concentration versus logl 0 of virus produced at each concentration. For the VYR assay, the SI was determined by dividing the EC90 by the neutral red CC ₅₀ .

Virus

The DENV2 strain New Guinea C was used to assess antiviral activity.

Test Articles

The combination ofBRQ+DPY was evaluated with eight two-fold serial dilutions of BRQ with fixed concentrations of DPY as follows:

• 0.0063-20.0 μMBRQ plus I μM or 6μMDPY

• 0.16-20.0 μMBRQ plus I μM or 6μMDPY

The positive control was NITD008 at 0.032 to 100 μM.

Serial dilutions of BRQ in the presence ofNITD0080.2 μM were also evaluated.

Results

The combination of BRQ with either 1 μM or 6 μM DPY exhibited potent antiviral activity against DENV2; >21 -fold lower EC90 values relative to NITD008 and 1.5- to 2.5-fold lower than BRQ alone in the virus yield reduction assay.

Table 12: Visual (Virus yield reduction)/Neutral Red (Toxicity)

Table 13: Relative Potency Comparisons

Table 14: Neutral Red (Cytopathic effect/Toxicity)

Table 15: Relative Potency Comparisons

Discussion

The combination of brequinar plus dipyridamole exhibited potent antiviral activity against DENV2 strain New Guinea C with greater activity than BRQ alone or the active positive control NITDOO 8. This antiviral effect was more pronounced when measuring the impact on virus titer (virus yield reduction), a direct measure of virus replication.

The antiviral effect was not driven by overt cytotoxicity.

Example 7: In Vitro evaluation of antiviral activity of brequinar and dipyridamole against Influenza A and B

The in vitro antiviral activity of brequinar (BRQ) in combination with dipyridamole (DPY) against Influenza A and B strains was evaluated using MDCK cells by two methodologies as described below.

Methodology

Neutral Red (CPE/Toxicity)

MDCK cells were grown at 37°C and 5% CO2 in 96-well plates to at/near confluency. After washes, test compounds were added to the cells in 0.1 mL volume at 2x concentration. Virus, at a titer that will cause >80% cytopathic effect (CPE) with a multiplicity of infection (MOI) <0.003, was added in 0. ImL volume. Plates were incubated at 37°C and 5% CO2 until >80% CPE as observed in the virus no-drug control wells. The plates were then stained with 0.011% neutral red for approximately two hours at 37°C in a 5% CO ₂ incubator. The neutral red medium was removed, and the cells were washed with IX with phosphate buffered solution (PBS) to remove residual dye. The PBS was then completely removed, and the incorporated neutral red was eluted with 50% Sorensen’s citrate buffer/50% ethanol for at least 30 minutes. Since neutral red dye penetrates living cells, the more intense the red color, the larger the number of viable cells present in the wells. The dye content in each well was quantified using a spectrophotometer at 540 nm wavelength and converted to a percentage of dye present in untreated control wells and normalized based on the virus no-drug control. The 50% effective (EC ₅₀ , virus-inhibitory) concentrations and 50% cytotoxic (CC ₅₀ , cell-inhibitory) concentrations were then calculated by regression analysis. The quotient of CC ₅₀ divided by EC ₅₀ gives the selectivity index (SI) value. Compounds showing SI values >10 were considered active. Visual (Virus Yield Reduction (VYR))/ Neutral Red (Toxicity)

The assay setup is similar to that of the Neutral Red (CPE/Toxicity) assay described above. After sufficient evidence of virus replication is noted, generally 3 days post-infection, supernatants were collected from each infected well (replicate wells are pooled) and tested (or stored) for virus titer determination by endpoint dilution. After maximal CPE is observed, the viable plates were stained with neutral red and the amount of incorporated dye was quantified as described above to determine the CC ₅₀ values. For virus titer determination, briefly, serial 10- fold dilutions of supernatant were plated into 4 replicate wells and the plates then incubated and cells scored for the presence or absence of virus after distinct CPE was observed. The EC90 value was calculated by regression analysis by plotting the loglO of the inhibitor concentration versus logl 0 of virus produced at each concentration. For the VYR assay, the SI was determined by dividing the EC90 by the neutral red CC ₅₀ .

Viruses

Four Influenza A (IF A) and B (IFB) strains were evaluated in this set of experiments:

• IFA H5N1 HPAI Vietnam/1203/2004

• IFA H1N1 California/07/2009

• IFA H3N2 Perth/16/2009

• IFB Brisbane/60/2008]

Test Articles

The combination ofBRQ+DPY was evaluated with eight two-fold serial dilutions of BRQ with fixed concentrations of DPY as follows:

• 0.0063-20.0 μMBRQ plus I μM or 6μMDPY

• 0.16-20.0 μMBRQ plus I μM or 6μMDPY

The positive control was ribavirin at 0.32 to 1,000 μg/mL.

Results

The combination of BRQ with either 1 μM or 6 μM DPY exhibited potent antiviral activity against all four viral strains tested, including the highly pathogenic IFA H5N1 HPAI. FIG. 19 is a summary of antiviral activity ofBRQ +DPY against influenza.

The combination ofBRQ plus either 1 μM or 6 μM DPY exhibited virus titer reductions at/above a 2 logl 0 relative to the no drug control.

FIG. 20 is a graph of virus titer reductions following BRQ and DPY administration.

Discussion

The combination of brequinar plus dipyridamole exhibited >2 logio reduction in virus titers against IF A and IFB strains, including the highly pathogenic IFA H5N1 HP Al. This antiviral effect was observed at concentrations ofBRQ and DPY that are pharmacologically relevant. In addition, BRQ + DPY at pharmacologically relevant concentrations of 1 μM each exhibited greater antiviral activity than the active positive assay control, ribavirin.

The antiviral effect was not driven by overt cytotoxicity as the SI values ranged from >69 to >1300 in the Neutral Red Assay and from >950to 2900 in the Virus Yield Reduction Assay.

Incorporation by Reference

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

Equivalents

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