EP4313150A1

EP4313150A1 - Pyrimidine biosynthesis inhibitor combination for use in treating viral infections

Info

Publication number: EP4313150A1
Application number: EP22718647.5A
Authority: EP
Inventors: Matthias Dobbelstein; Antje DICKMANNS; Kim Maren STEGMANN
Original assignee: Universitaetsmedizin Goettingen Georg August Universitaet
Current assignee: Universitaetsmedizin Goettingen Georg August Universitaet
Priority date: 2021-03-26
Filing date: 2022-03-25
Publication date: 2024-02-07
Also published as: WO2022200615A1

Abstract

The present invention relates to novel treatment strategies comprising compositions, methods, uses, and kits, including a kit of parts, for the treatment and/or prevention of a viral infection in a subject, for example, a coronavirus infection, using a combination drug approach. Specifically, the combination drug therapy comprises a therapeutically effective amount of a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue or its prodrug.

Description

PYRIMIDINE BIOSYNTHESIS INHIBITOR COMBINATION FOR USE IN TREATING VIRAL INFECTIONS

FIELD OF THE INVENTION

The present invention relates to treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue or its prodrug.

BACKGROUND OF THE INVENTION

The COVID-19 pandemic has emerged as the most serious health crisis in modem times. Its causal pathogen, SARS-CoV-2, is a novel coronavirus presenting unique molecular, pathophysiological, and epidemiological characteristics, with a remarkable rate of contagion. This has resulted in an exponential proliferation of COVID-19 across the globe, without regard to gender, age or race, and with over 100 million confirmed cases and over 3,000,000 confirmed deaths worldwide at the time of this writing.

SARS-CoV-2 has been reported to attack a wide variety of organs, for instance, the heart, the brain and nervous system, and especially the respiratory system. Common respiratory symptoms caused by COVID-19 infections include fever, cough, shortness of breath, and dyspnea. In the most severe clinical presentations, a SARS-CoV-2 infected patient experiences pneumonia, acute respiratory syndrome, and even death, frequently due to multiple organ failure. Given the prevalence and severity of SARS-CoV- 2 infections worldwide, the development of effective treatment protocols and preventive measures such as vaccines for the treatment or prevention of COVID-19 has become a most urgent task spanning numerous fields of medical and drug research and development involving a worldwide collaborative effort.

At the time of this application, several vaccines have been approved by various regulatory agencies for the immediate introduction into the relevant population for the immediate treatment and/or prevention of SARS-CoV-2 infections. Vaccination programs are being developed and expanded at a rapid pace, but this approach may be unable to eradicate the disease completely. This reality thus demands alternative therapies for infected or otherwise susceptible individuals. Effective therapeutics can reduce the disease burden, save lives and prevent further spread of the virus.

Health professionals are increasingly considering a “repurposing” of numerous approved and non- approved drugs for treating COVID-19. On the most basic level, an effective therapeutic can meaningfully interfere with SARS-CoV-2 replication at its source, while attenuating the common cytokine storm experienced by numerous subjects receiving the new vaccine regimens. This would be highly advantageous in combatting both the early and late stages of COVID-19 infection. A cytokine storm refers to a severe immune reaction in which the body rapidly releases an excess of cytokines into the blood causing high fever, inflammation, severe fatigue, and nausea.

Nucleoside and nucleotide analogues have been used as antiviral therapeutics to inhibit or prevent viral replication in infected cells and have been tested for their ability to treat and/or prevent symptoms caused by SARS-CoV-2 infections in presenting patients. Nucleoside analogues are also referred to as nucleosides, which contain a nucleobase and a ribose or analogues derived from either moiety, while nucleotide analogues are known as nucleotides, which contain a nucleic acid analogue, a sugar, and a phosphate group containing one to three phosphate moieties.

For example, one nucleoside analogue, the cytidine analogue β-D-N⁴-hydroxycytidine (referred to as NHC, EIDD-1931) and its prodrug β-D-N⁴-hydroxycytidine-5-isopropyl ester (EIDD-2801, also known as Molnupiravir or MK-4482), were found to show activity against coronavirus replication, in addition to exhibiting an anti-replication effect against murine hepatitis virus (MHV), Middle East respiratory syndrome CoV (MERS-CoV), as well as SARS-CoV and SARS-CoV-2, the latter virus being responsible for COVID-19 infections.

EIDD-2801 has also been shown to block SARS-CoV-2 transmission in a ferret model and also in a human lung model. Molnupiravir is currently in Phase II clinical trials (designated as NCT04392219, NCT04405739, NCT04405570) and is undergoing evaluation in patients suffering from COVID-19.

Turning to nucleic acid specificity, ribonucleosides are efficiently removed from eukaryotic cell DNA. Therefore, treating a viral infection with a mutagenic ribonucleoside analogue would be expected to show a selective incorporation into the viral genome. Currently, it is unknown whether a sufficient concentration of NHC can even be accumulated in a subject, considering NHC pharmacokinetics, such that the NHC could effectively interfere with virus replication and achieve any clinical benefit, in particular if NHC is administered as a single dose therapy.

The ribonucleoside analogue, Remdesivir (GS-5374), which is a monophosphoramidate prodrug of an adenosine analogue and a competitive inhibitor of viral RNA-dependent RNA polymerase (RdRP), has been administered to patients under the auspices of a “compassionate medication” in a compassionate drug use principle, even though Remdesivir has not satisfied the conditions gaining regulatory approval. However, Remdesivir has been reported to only mildly reduce the average time of patient hospitalization following SARS-CoV-2 infection in a limited number of studies. In other clinical settings, Remdesivir failed to result in any significant clinical improvement, which was reported using conventional metrics. Another SARS-CoV-2 treatment option, glucocorticoid dexamethasone, has been reported to provide mild benefit in patients by providing anti-inflammatory and immunosuppressant effects, including an avoidance of excessive inflammatory reactions such as the cytokine storm described above. However, glucocorticoid dexamethasone action does not directly involve interference with virus replication.

Among other potential SARS-CoV-2 therapeutic candidates, two groups of therapeutics targeting viruses, specifically coronaviruses, have been recently developed. For instance, pyrimidine analogues and pyrimidine biosynthesis inhibitors.

For instance, pyrimidine biosynthesis inhibitors typically target the cellular de novo pyrimidine nucleotide biosynthesis pathways. Such interference represents yet another potential approach for treating both aspects of COVID-19 with respect to viral replication and over-production of a subset of inflammatory cytokines. The latter are controlled by pyrimidine nucleotide levels within cells that participate in the inflammatory response.

Pyrimidine biosynthesis inhibitors further include inhibitors of dihydroorotate dehydrogenase (DHODH), which is a key mitochondrial enzyme involved in pyrimidine biosynthesis destined for DNA and RNA synthesis. DHODH is located on the inner membrane of mitochondria and catalyzes the dehydrogenation of dihydroorotate (DHO) to orotic acid, ultimately resulting in the production of uridine and cytidine triphosphates (UTP and CTP). DHODH inhibitors were originally developed and used as immunosuppressants. For example, teriflunomide is one representative approved drug used for this clinical purpose (O'Connor et al., 2011).

Newer drugs exhibiting a higher potency and specificity of DHODH inhibition are currently in clinical trials. Recently, the DHODH inhibitor PTC299 was found to counteract SARS-CoV-2 replication (Luban et al., 2021). Similar results were obtained using other DHODH inhibitors, i.e., S416 (Xiong et al., 2020) and IMU-838 (Hahn et al, 2020). IMU-838 was developed by Immunic AG, Grafelfing, Germany, and is currently undergoing clinical trials (designated as NCT04379271). Conversely, one study failed to demonstrate any clinical benefit of the traditional DHODH inhibitor leflunomide in the context of COVID-19 (Wang et al., 2020a). These inconsistent results strongly suggest that more potent drugs are required in order to achieve reliable and meaningful antiviral effects in infected patients.

Several conventional pyrimidine analogues as well as pyrimidine biosynthesis inhibitors might represent promising candidates for treating SARS-CoV-2 infections. However, the potency of these compounds at currently achievable serum concentrations has not yet been shown to effectively halt the spread and infectivity of COVID-19, especially when administered as a single dose. In fact, most of the currently available antiviral therapeutics used for treating SARS-CoV-2 infections offer incomplete protective value and/or cause severe side effects following administration.

The present invention overcomes the shortcomings of known antiviral drug strategies by providing combination therapy approaches comprising one or more pyrimidine analogue and one or more pyrimidine biosynthesis inhibitor useful for the treatment of a viral infection in a subject, which is achieved at a high level of potency at considerably lower dosages than conventional regimens, based on a surprising observation of drug synergy between these two compounds.

SUMMARY OF THE INVENTION

In one aspect the present disclosure provides for a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of one or more pyrimidine biosynthesis inhibitor in combination with one or more antiviral pyrimidine analogue or its prodrug.

In one aspect the present disclosure provides for one or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject.

The viral infection may be caused by, for example, an RNA virus or a DNA virus.

An viral infection caused by a RNA virus may be an infection by a member of the Coronaviridae virus family, an Influenza virus, HIV (Human Immunodeficiency Virus), an Ebolavirus, a member of the Flaviviridae comprising Hepatitis C virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Zika virus, a member of Rhabdoviruses comprising rabies virus, a member of Paramyxoviridae comprising parainfluenza virus, a Venezuelan equine encephalitis virus, an Equine arteritis virus, Rotaviruses, or an Enterovirus comprising Foot-and-mouth disease virus (FMDV).

In a one embodiment, the viral infection is caused by one or more members of the Coronaviridae virus family. Preferred members are Betacoronavirus or Alphacoronavirus members. Even more preferred members are SARS-CoV, MERS-CoV, SARS-CoV-2 or SADS-CoV.

In another embodiment, the viral infection is caused by SARS-CoV-2 or derivatives thereof, wherein the amino acid sequence of virus proteins encoded by the derivative has at least 40% identity to the amino acid sequence of SARS-CoV2. In an even more particular embodiment, the viral infection is caused by SARS-CoV-2 and SARS-CoV-2 derivatives. The latter derivatives can comprise one or more mutations in the viral proteins. In one embodiment, the subject treated with the disclosed combination methods of treating and/or preventing a viral infection in the subject comprising administering to the subject a therapeutically effective amount of one or more pyrimidine biosynthesis inhibitor in combination with one or more antiviral pyrimidine analogue or its prodrug suffers from COVID-19.

The pyrimidine analogue used in the disclosed method can be selected from the pyrimidine analogues listed in Table 3 or their prodrugs.

In a preferred embodiment, the pyrimidine analogue used in the disclosed method is selected from N⁴- hydroxycytidine/EIDD-1931, N⁴-hydroxycytidine-5 ’-isopropyl ester EIDD-2801/mulnupiravir MK- 4482, Sofosbuvir, N⁴-aminocytidine, Cidofovir, Stavudine, AZT/azidothymidine/zidovudine, Didanosine/2 ’ ,3 ’ -dideoxyinosine/ddl, Zalcitabine/2 ’ ,3 ’ -dideoxycytidine/ddC, Lamivudine/(-)-β-L-3 ’ - thia-2 ’ ,3 ’ -dideoxycytidine/3TC, Emtricitabine/(-)-β-L-3 ’ -thia-2 ’ ,3 ’ -dideoxy-5-fluorocytidine/ (-)-FTC, Idoxuridine/5 -iodo-2 ’ -deoxyuridine/IDU, Trifluridine/5 -trifluoro-2 ’ -deoxythymidine/TFT,

Brivudin/ (E)-5-(2-bromovinyl)-2 ’ -deoxyuridine/B VDU, 2 ’ -C-methyl-4-amino-pyrrolo[2,3 - djpyrimidine ribonucleosides.

Preferably, the pyrimidine analogue used in the disclosed method is a cytidine analogue or its prodrug.

In an even more preferred embodiment, the pyrimidine analogue is N⁴-hydroxycytidine/EIDD- 1931 or its prodrug N⁴-hydroxycytidine-5 ’-isopropyl ester/EIDD-2801.

The pyrimidine biosynthesis inhibitor used in the disclosed method can be selected from the DHODH- inhibitors of Table 1, Table 1-1 and/or the inhibitors of Table 2.

In an embodiment, the pyrimidine synthesis inhibitor is a DHODH inhibitor.

In a preferred embodiment, the DHODH inhibitor is selected from IMU-838, IMU-935, PTC299, S312, S416, Leflunomide, Teriflunomide, Brequinar/DD264, BAY 2402234, IPPA17-A04, GSK983/SW835, Cmpl, NITD-982, Compound A3, FK778/Maritimus, DSM265.

In a similar preferred embodiment, the DHODH inhibitor is selected from MEDS433, RP7214, AG- 636, ASLAN003/Farudodstat and PP-001.

In an even more preferred embodiment the DHODH inhibitor is BAY 2402234.

In a similar more preferred embodiment the DHODH inhibitor is vidofludimus. In the most preferred embodiment the DHODH inhibitor is IMU-838.

In another preferred embodiment the DHODH inhibitor is MEDS433.

In another preferred embodiment the DHODH inhibitor is RP7214.

In another preferred embodiment the DHODH inhibitor is AG-636.

In another preferred embodiment the DHODH inhibitor is PP-001.

In another preferred embodiment the DHODH inhibitor is ASLAN003/Farudodstat.

In another preferred embodiment the DHODH inhibitor is PTC299.

In another preferred embodiment the DHODH inhibitor is Brequinar/DD264.

In yet another preferred embodiment the DHODH inhibitor is teriflunomide.

In an embodiment, the pyrimidine synthesis inhibitor is a cytidine triphosphate (CTP) synthetase inhibitor.

The pyrimidine biosynthesis inhibitor used in the disclosed method can be selected from the CTP synthetase inhibitor of Table 2-2.

In a preferred embodiment, the CTP synthetase inhibitor is cyclopentenyl cytosine (CPEC).

In another embodiment, the pyrimidine biosynthesis inhibitor may be selected from PALA (N- phosphonoacetyl-L-aspartate), 6-Azauridine, Azaribine/triacetyl-6-azauridine.

The pyrimidine biosynthesis inhibitor and the pyrimidine analogue or its prodrug can be administered orally or parenterally, preferably orally.

The pyrimidine biosynthesis inhibitor may be administered together with the pyrimidine analogue or its prodrug or the pyrimidine biosynthesis inhibitor is administered prior to the administration of the pyrimidine analogue or its prodrug. Preferably, the pyrimidine biosynthesis inhibitor is administered together with the pyrimidine analogue or its prodrug. If the pyrimidine biosynthesis inhibitor is administered before the pyrimidine analogue or its prodrug, the pyrimidine synthesis inhibitor can administered up to 7 days before the pyrimidine analogue or its prodrug, with the possibility of additional administrations of the pyrimidine biosynthesis inhibitor during the period up to 7 days before the pyrimidine analogue or its prodrug. Optionally, the pyrimidine biosynthesis inhibitor can be administered 7, 6, 5, 4, 3, 2 days, or 1 day, or 24 hours, 18 hours, 16 hours, 10 hours, 8 hours, or 7, 6, 5, 4, 3, 2 hours, or 60 min, 50 min, 40 min, 30 min, 20 min, 10 min before the pyrimidine analogue or its prodrug.

The pyrimidine biosynthesis inhibitor may be administered at a dose of about 1 to about 1000 μmol.

In an embodiment, BAY 2402234 may be administered at a dose of about 50 to about 1000 μmol.

In another embodiment, Teriflunomide may be administered at a dose of about 5 to about 100 μmol. More preferably, Teriflunomide is administered at a daily dose of 7 mg or 14 mg.

In another embodiment, IMU-838 is administered at a dose of about 10 to about 200 μmol. Optionally, one or more of the pyrimidine biosynthesis inhibitors are administered. If more than one inhibitor is administered, the respective dose may be less than the dose defined for each respective inhibitor. The doses of the respective biosynthesis inhibitor may vary depending on the genus of the subject, the body weight and the age of the subject.

In another embodiment, IMU-838 is administered at a daily dose of about 10 mg to about 45 mg. More preferably, IMU-838 is administered at a daily dose of 30 mg or 45 mg.

The pyrimidine analogue may be administered at a dose of about 1 μmol to about 200 mmol.

In an embodiment, N⁴-hydroxycytidine may be administered at a dose of about 10 to about 200 mmol.

In an embodiment, the N⁴-hydroxycytidine-5’ -isopropyl ester is administered at a dose of about 10 mmol to about 150 mmol. Optionally, one or more of the pyrimidine analogues are administered. If more than one analogue is administered, the respective dose may be less than the dose defined for the respective analogue. The doses of the respective analogue may vary depending on the genus of the subject, the body weight and the age of the subject.

In an embodiment, the N⁴-hydroxycytidine-5 ’-isopropyl ester is administered at a dose of about 100 μmol to about 15 mmol. Optionally, one or more of the pyrimidine analogues are administered. If more than one analogue is administered, the respective dose may be less than the dose defined for the respective analogue. The doses of the respective analogue may vary depending on the genus of the subject, the body weight and the age of the subject.

In an embodiment, the N⁴-hydroxycytidine-5’ -isopropyl ester is administered at a daily dose of 200 mg to 800 mg.

In an embodiment, N⁴-hydroxycytidine-5 ’-isopropyl ester is administered at a daily dose of lower than 800 mg.

The effective daily dosing amount may be about 1 μmol to about 1000 μmol of the pyrimidine biosynthesis inhibitor and about 1 μmol to about 200 mmol of the pyrimidine analogue.

After administration of the pyrimidine biosynthesis inhibitor and the antiviral pyrimidine analogue or its prodrug the viral load is reduced.

The subject treated may be mammalian or avian.

Preferably, the subject is a mammal, and more preferably the mammal is a human.

In a preferred embodiment, the pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N⁴- hydroxycytidine, and wherein the viral infection is caused by SARS-CoV-2. Most preferably, the present method of treating and/or preventing a viral infection in a subject comprises administering to the subject a therapeutically effective amount of IMU-838 in combination with N⁴-hydroxycytidine.

In another preferred embodiment, the pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is EIDD- 2801, and wherein the viral infection is caused by SARS-CoV-2. Most preferably, the present method of treating and/or preventing a viral infection in a subject comprises administering to the subject a therapeutically effective amount of IMU-838 in combination with EIDD-2801.

The administration of the therapeutically effective amount of a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue or its prodrug has a synergistic effect.

In one aspect, the present disclosure provides a pharmaceutical composition comprising one or more pyrimidine biosynthesis inhibitor in combination with one or more antiviral pyrimidine analogue disclosed herein or its prodrug and a pharmaceutically acceptable carrier. In another embodiment, a further component can be optionally included, for example, a second pyrimidine biosynthesis inhibitor and/or a second antiviral pyrimidine analogue.

The pharmaceutical composition may comprise about 1 μmol to about 1000 μmol of the pyrimidine biosynthesis inhibitor and about 1 μmol to about 200 mmol of the pyrimidine analogue or its prodrug.

In an embodiment, the pyrimidine biosynthesis inhibitor of the pharmaceutical composition is selected from IMU-838, IMU-935, PTC299, S312, S416, Leflunomide, Teriflunomide, Brequinar/DD264, BAY 2402234, IPPA17-A04, GSK983/SW835, Cmpl, NITD-982, Compound A3, FK778/Maritimus, DSM265, PALA (N-phosphonoacetyl-L-aspartate), 6-Azauridine, Azaribine/triacetyl-6-azauridine and wherein the pyrimidine analogue is selected from N⁴-hydroxycytidine/EIDD- 1931, N⁴-hydroxycytidine- 5’-isopropyl ester /EIDD-2801/mulnupiravir/MK-4482, Sofosbuvir, N⁴-aminocytidine, Cidofovir, Stavudine, AZT/azidothymidine/zidovudine, Didanosine/2’,3’-dideoxyinosine/ddI, Zalcitabine/ 2’, 3’- dideoxycytidine/ddC, Lamivudine/(-)-β-L-3 ’-thia-2’,3 ’-dideoxycytidine/3TC, Emtricitabine/(-)-β-L-3 ’ -thia-2 ’ ,3 ’ -dideoxy-5-fluorocytidine/ (-)-FTC, Idoxuridine/5-iodo-2 ’ -deoxyuridine/IDU,

Trifluridine/5 -trifluoro-2 ’ -deoxythymidine/TFT, Brivudin/(E)-5 -(2-bromovinyl)-2 ’ - deoxyuridine/BVDU, 2’-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides.

In a preferred embodiment, the pyrimidine biosynthesis inhibitor of the pharmaceutical composition is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N⁴-hydroxycytidine.

In another preferred embodiment, the pyrimidine biosynthesis inhibitor of the pharmaceutical composition is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is EIDD-2801.

In another aspect, the present disclosure provides a composition comprising one or more pyrimidine biosynthesis inhibitor and one or more pyrimidine analogue or its prodrug.

In a preferred embodiment, the pyrimidine biosynthesis inhibitor of the composition is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N⁴-hydroxycytidine.

In another preferred embodiment, the pyrimidine biosynthesis inhibitor of the composition is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is EIDD-2801. In one aspect, the present disclosure provides a kit or a kit of parts comprising a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue, provided together or separately, respectively, for treating and/or preventing a viral infection.

In yet another aspect, the present invention discloses a kit of parts comprising the pharmaceutical composition, and optionally technical instructions with information about the administration and dosage of the composition.

In another aspect, the present invention discloses a kit of parts comprising the composition and optionally technical instructions with information about the administration and dosage of the composition.

The present invention offers a number of advantages over conventional antiviral therapeutic approaches.

For instance, a combination therapeutic approach comprising a pyrimidine analogue, e.g., NHC with a pyrimidine biosynthesis inhibitor, e.g., a DHODH inhibitor, for the treatment of viral infections according to the invention has shown to be therapeutically superior compared to a single drug treatment.

The presently disclosed pyrimidine analogues and pyrimidine biosynthesis inhibitors when administered individually can only reach limited blood concentrations for a short period of time, especially when applied orally. Such respective achievable concentrations might not be sufficient to realize a strong inhibition of virus replication, and thus any clinical benefit. Indeed, teriflunomide, a DHODH inhibitor, has already failed in this respect in one clinical study (Wang et al, 2020a). However, the combination of a pyrimidine analogue, for example NHC, and a pyrimidine biosynthesis inhibitor, for example a DHODH inhibitor, is disclosed herein to substantially increase achieving clinical efficacy, through a significantly higher potency of action. The fact that DHODH inhibitors suppress the immune response further alleviates the disease-causing consequences of viral infection in a manner analogous to e.g., dexamethasone.

The combined use of NHC and a DHODH inhibitor (e.g., IMU-838) synergistically suppress SARS- CoV-2 replication. This means that much lower doses can be used compared to a single treatment. For example, for IMU-838, a lower daily dose than 30 to 45 mg (as mentioned in WO2021/214033) may produce a beneficial therapeutic effect and further reduces the risk of hematuria (as mentioned in W02019/101888). In this way, a lower loading dose may be avoided. Similar applies to NHC, the recommended dose of 800 mg twice daily can be reduced to minimize the potential risk of mutagenic effects. In addition, lower doses are more convenient for the patient and economically more favourable. Each of the pyrimidine analogues and pyrimidine biosynthesis inhibitors described herein have been shown to display toxic side effects when administered individually. For instance, teriflunomide was shown to induce severe neutropenia, paresthesia, as well as gastrointestinal dysfunctions. The combination of a pyrimidine analogue, for example NHC, and a pyrimidine biosynthesis inhibitor, for example a DHODH inhibitor, as disclosed herein, was demonstrated to exhibit a lower effective dose, thereby reducing the toxic side effects commonly associated with each compound applied alone. Because the disclosed combination therapy did not reveal apparent cytotoxicity at concentrations shown to antagonize the virus, increased toxicities of the described combination therapeutic approach in subjects is not anticipated and has not been observed to date. That only the virus but not the host cell uses RNA as the genomic material provides further evidence that the disclosed therapeutic combinations are well-tolerated by subjects.

In various embodiments, any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or otherwise herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges are encompassed within the scope of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of several proof of principle studies using the combination treatment strategies according to the present invention. FIG. 1A: qRT-PCR results in terms of virus RNA progeny as a percent of SARS-CoV-2 infected Vero E6 cells that were either left untreated or treated with the DHODH inhibitor BAY 2402234, DHODH inhibitor teriflunomide, and cytidine analogue NHC (EIDD- 1931), either alone or in combination. FIG. IB: qRT-PCR of SARS-CoV-2 infected Vero E6 cells, which were either left untreated or treated with different concentrations of the DHODH inhibitor BAY 2402234, different concentrations of NHC (EIDD-1931), and different concentrations of NHC (EIDD- 1931) in combination with different concentrations ofDHODH inhibitor BAY 2402234. FIG. 1C: qRT- PCR of SARS-CoV-2 infected Calu-3 cells, which were either left untreated or treated with different concentrations of the DHODH inhibitor BAY 2402234, different concentrations of NHC (EIDD-1931), and different concentrations of NHC (EIDD-1931) in combination with different concentrations of DHODH inhibitor BAY 2402234. FIG. ID: qRT-PCR of SARS-CoV-2 infected Vero E6 cells, which were either left untreated or treated with different concentrations of the DHODH inhibitor teriflunomide, different concentrations of NHC (EIDD-1931), and different concentrations of NHC (EIDD-1931) in combination with different concentrations ofDHODH inhibitor teriflunomide. FIG. IE: qRT-PCR of SARS-CoV-2 infected Calu-3 cells, which were either left untreated or treated with different concentrations of the DHODH inhibitor teriflunomide, different concentrations of NHC (EIDD-1931), and different concentrations of NHC (EIDD-1931) in combination with different concentrations of DHODH inhibitor teriflunomide. FIG. IF: Immunoblot analysis of virus proteins (nucleoprotein and spike protein) of SARS-CoV-2 infected Vero E6 cells, which were either left untreated or treated with the DHODH inhibitor BAY 2402234, or NHC (EIDD-1931), or treated with NHC (EIDD-1931) in combination with DHODH inhibitor BAY 2402234. FIG. 1G: Immunoblot analysis of virus proteins (nucleoprotein and spike protein) of SARS-CoV-2 infected Vero E6 cells, which were either left untreated or treated with the DHODH inhibitor teriflunomide, or NHC (EIDD-1931), or treated with NHC (EIDD-1931) in combination with the DHODH inhibitor teriflunomide.

FIG. 2 illustrates experimental results using several combination therapeutic embodiments according to the present invention. FIG. 2A: Combination therapy of IMU-838 and NHC. Cytopathic effect (CPE) 48 hours after viral transfection. Positive control: cells transfected with virus, no treatment. Negative control: cells only. Inoculum: virus only, no cells. Different levels of CPE are depicted with different shading. FIG. 2B: Combination therapy of IMU-838 and NHC. qRT-PCR results, c(t) values. FIG. 2C: Combination therapy of IMU-838 and NHC. qRT-PCR results, virus RNA progeny in %. FIG.2D: Combination therapy of IMU-838 and NHC. qRT-PCR results, virus copies in mL. FIG. 2E: Combination therapy of MEDS433 and NHC. qRT-PCR results, virus copies in mL. FIG. 2F: Combination therapy of PTC299 and NHC. qRT-PCR results, virus copies in mL. FIG. 2G: Combination therapy of RP7214 and NHC. qRT-PCR results, virus copies in mL. FIG. 2H: Combination therapy of AG-636 and NHC. qRT-PCR results, virus copies in mL.

FIG.3 illustrates experimental results using several combination therapeutic embodiments according to the present invention towards influenza A virus. FIG. 3A: Combination therapy of BAY 2402234 and NHC. qRT-PCR results, virus RNA progeny in %. FIG.3B: Combination therapy of teriflunomide and NHC. qRT-PCR results, virus RNA progeny in %. FIG. 3C: Combination therapy of IMU-838 and NHC. qRT-PCR results, virus RNA progeny in %.

FIG. 4 illustrates experimental results using a combination therapeutic embodiment according to the present invention targeting the CTP synthetase (CTPS). FIG. 4A: Combination therapy of CTPS inhibitor cyclopentenyl cytosine (CPEC) and NHC. qRT-PCR results, virus copies in mL. FIG. 4B: Combination therapy of CPEC and NHC. qRT-PCR results, virus RNA progeny in %. FIG. 4C: Combination therapy of CPEC and NHC, also optionally in the presence of uridine or cytidine. qRT- PCR results, virus RNA progeny in %.

DETAILED DESCRIPTION OF THE INVENTION

The present invention overcomes the shortcomings of conventional antiviral drugs by demonstrating a synergistic, or enhanced, therapeutic effect of a combined therapeutic strategy compared to the administration of a single drug alone. For instance, a synergistic therapeutic effect is presently described when a pyrimidine analogue and a pyrimidine biosynthesis inhibitor are combined as a single approach for treating a viral infection in a subject.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The definitions being provided herein are being described in the context of the presently disclosed invention.

As used herein, “synergy” refers to an interaction or cooperation giving rise to a whole that is greater than the simple sum of its parts, namely synergy occurs when the combined action of two or more agents is greater than could have been predicted based on the performance of the agents when used alone. A synergistic effect commonly provides functional or other advantages based on cooperating elements which operate together as a functional unit to achieve an enhanced result compared to either of the components alone. Exemplary synergistic effects in a treatment context can include an increased or otherwise enhanced effect by the cooperative components on the same cellular system, improved bioavailability, increased potency, or an enhanced prevention or delay of a physiological response in a cell, for example, creating a prolonged physiological or therapeutic effect. Several methods have been developed to calculate the action to be expected for a given active ingredient combination, for instance, the Chou-Talalay method (Chou, 2010), or the Colby method, (Colby 1967). If the action actually observed is greater than the expected action, then the action of the combination is super-additive, i.e., there is a synergistic effect.

As disclosed herein, the term “analogue” refers to a structurally related nucleoside, polypeptide, nucleic acid molecule, or other compound or derivative thereof having a similar or identical function of a corresponding reference polypeptide, nucleic acid molecule or compound.

The term “nucleoside” refers to a glycosylamine that resembles a nucleotide without a phosphate group. A nucleoside consists simply of a nucleobase (also known as a nitrogenous base) and a five-carbon sugar, for instance ribose or 2'-deoxyribose. In a nucleoside, the anomeric carbon is linked through a glycosidic bond to the N9 of a purine or the N1 of a pyrimidine. The term “nucleotide” generally refers to a substance comprising a nucleobase, a five-carbon sugar, and one or more phosphate groups.

Accordingly, the respective terms “nucleoside analogue” and “nucleotide analogue” are understood in the context of the present invention. Nucleoside analogues can contain modifications in the nucleobase and/or in the ribose components, for example, substitutions at the carbon atoms comprising removing the 3 ’-OH or adding halogens to the 1' or 2’ carbon atom. A “ribonucleoside” is understood to mean a type of nucleoside that includes ribose as a component. An exemplary ribonucleoside according to the invention is cytidine. Other examples of nucleosides herein can be uridine, adenosine, guanosine, thymidine and inosine.

One example of a ribonucleoside analogue according to the present invention is “EIDD-1931”, which is an orally bioavailable ribonucleoside analogue associated with broad-spectrum antiviral activity against a variety of RNA viruses including, but not limited to, influenza, Ebola, CoV, and Venezuelan equine encephalitis virus (VEEV). Another example of a ribonucleoside analogue according to the present invention is “EIDD-2801”, also known as Molnupiravir or MK-4482, which is an antiviral drug that is typically orally active that was initially developed for treating influenza. EIDD-2801 is a prodrug of the synthetic nucleoside derivative N⁴-hydroxycytidine (NHC, EIDD-1931) and is believed to exert its antiviral action through the introduction of copying errors during viral RNA replication. EIDD-2801 activity has also been demonstrated against coronaviruses including SARS, MERS and SARS-CoV-2.

The term "prodrug" means a derivative that is converted into the pharmacologically active drug (or a precursor of it) by a reaction with an enzyme, gastric acid or the like under a physiological condition in the living body, e.g., by oxidation, reduction, hydrolysis or the like, each of which is carried out enzymatically. Examples of prodrugs that can be used to improve bioavailability include esters, optionally substituted esters, branched esters, optionally substituted branched esters, carbonates, optionally substituted carbonates, carbamates, optionally substituted carbamates, thioesters, optionally substituted thioesters, branched thioesters, optionally substituted branched thioesters, thiocarbonates, optionally substituted thiocarbonates, S-thiocarbonate, optionally substituted S-thiocarbonate, dithiocarbonates, optionally substituted dithiocarbonates, thiocarbamates, optionally substituted thiocarbamates, oxymethoxy carbonyl, optionally substituted oxymethoxycarbonyl, oxymethoxythiocarbonyl, optionally substituted oxymethoxythiocarbonyl, oxymethylcarbonyl, optionally substituted oxymethylcarbonyl, oxymethylthiocarbonyl, optionally substituted oxymethylthiocarbonyl, L-amino acid esters, D-amino acid esters, N- substituted L-amino acid esters, N,N-disubstituted L-amino acid esters, N-substituted D-amino acid esters, N,N-disubstituted D-amino acid esters, sulfenyl, optionally substituted sulfenyl, imidate, optionally substituted imidate, hydrazonate, optionally substituted hydrazonate, oximyl, optionally substituted oximyl, imidinyl, optionally substituted imidinyl, imidyl, optionally substituted imidyl, aminal, optionally substituted aminal, hemiaminal, optionally substituted hemiaminal, acetal, optionally substituted acetal, hemiacetal, optionally substituted hemiacetal, carbonimidate, optionally substituted carbonimidate, thiocarbonimidate, optionally substituted thiocarbonimidate, carbonimidyl, optionally substituted carbonimidyl, carbamimidate, optionally substituted carbamimidate, carbamimidyl, optionally substituted carbamimidyl, thioacetal, optionally substituted thioacetal, S-acyl-2-thioethyl, optionally substituted S-acyl-2-thioethyl, bis-(acyloxybenzyl)esters, optionally substituted bis- (acyloxybenzyl)esters, (acyloxybenzyl)esters, optionally substituted (acyloxybenzyl)esters and BAB- esters or ProTide. The ester can be a carboxylate ester or derived from inorganic acids like phosphoric acid, sulfuric acid, nitric acid, boric acid or carbonic acid. In case of a pyrimidine analogue, a preferred prodrug is the modification of one ore more hydroxyl moieties in the pharmacologically active drug via esterification with isobutyric acid. An even more preferred prodrug can be obtained by esterification of the 5'-OH-moiety in the ribose part of a pyrimidine analogue with isobutyric acid.

The term “pyrimidine” as used herein refers to an aromatic heterocyclic organic compound where one of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring) features a nitrogen atom at positions 1 and 3 in the ring. The other diazines are pyrazine (nitrogen atoms at the 1 and 4 positions) and pyridazine (nitrogen atoms at the 1 and 2 positions). In nucleic acids, three types of nucleobases are pyrimidine derivatives, namely cytosine, thymine and uracil.

A “pyrimidine analogue” according to the present invention may include a structurally related pyrimidine or derivative thereof providing a similar or identical function of a corresponding reference pyrimidine. Pyrimidine analogues can contain modifications in the pyrimidine base and/or in the sugar moiety. Preferred pyrimidine analogues are shown in Table 3.

A preferred pyrimidine analogue is NHC or a prodrug thereof (e.g., molnupiravir). A more referred pyrimidine analogue is molnupiravir.

A “pyrimidine biosynthesis inhibitor” as used herein refers to a compound that effectively inhibits the biosynthesis of a pyrimidine. De novo biosynthesis of a pyrimidine is catalyzed mainly by three different gene products including CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), UMPS (uridine monophosphate synthase, and DHODH (dihydroorotate dehydrogenase). DHODH catalyzes the fourth enzymatic step in the de novo pyrimidine biosynthesis pathway, the ubiquinone-mediated oxidation of dihydroorotate to orotate. The term also includes enzymes that enable the recycling of pyrimidines from the degradation of RNA and DNA, i.e., salvage pathways. Preferred pyrimidine biosynthesis inhibitors target the enzyme DHODH with structures shown in Table 1 and Table 1-1.

Preferred pyrimidine biosynthesis inhibitors are DHODH inhibitors.

An "inhibitor" is a compound, that acts as a substrate and binds to an enzyme or receptor and decreases the enzyme's activity or receptor’s activity in a reversible or irreversible manner. The decrease of activity is 10% or more, preferably 50% or more and more preferably 90% or more compared to the absence of the compound. The half maximal inhibitory concentration (IC50) is usually in the range of nM to mM. For a DHODH inhibitor, the in vitro inhibition can be determined as outlined in J Med. Chem. 2006;49: 1239; in this assay, the IC50 is preferably below 10 mM, more preferably below 1 mM and more preferably below 0.5 mM. The term “inhibit” as used herein refers to the ability of the compounds described herein to completely or substantially eliminate a physiological activity or otherwise reduce such activity when compared to the same activity in the absence of the compound. An inhibitor is a compound that acts to inhibit an activity in the manner described above. "Vidofludimus" as used herein refers to 2-((3-fluoro-3'-methoxy-[l,l'-biphenyl]-4- yl)carbamoyl)cyclopent-l-enecarboxylic acid and shall include its free acid form, and its pharmaceutically acceptable salt forms, such as the calcium, potassium, magnesium, choline or sodium salt. The term shall also include pharmaceutically acceptable solvates, hydrates, solvates of a salt, crystals and polymorphs. Preferred is vidofludimus calcium.

“IMU-838” (also termed “vidofludimus calcium”) is the calcium salt of vidofludimus, including pharmaceutically acceptable solvates, hydrates, crystals and polymorphs. A preferred structure for IMU- 838 is the dihydrate of 1-cyclopentene-l -carboxylic acid, 2-(((3-fluoro-3'-methoxy(l,r-biphenyl)-4- yl)amino)carbonyl)-, calcium salt (2:1) with the structure as follows:

A more preferred crystalline form of IMU-838 is the white crystalline „Polymorph A“ which is characterized as described in WO 2019/175396. In a special embodiment, „Polymorph A“ of IMU-838 is characterized by an X-ray powder diffraction pattern having characteristic peaks at 2 theta (±0.2°) of 5.91°, 9.64°, 16.78°, 17.81°, 19.81° and 25.41°. In a particular special embodiment, „Polymorph A“ of IMU-838 is characterized as outlined in Fig. 1 from WO 2019/175396. In a particular special embodiment, „Polymorph A“ is characterized by an FT Raman absorption spectrum having the following characteristic peaks expressed in cm ¹ at 1664, 1624, 1617, 1532, 1449 and 1338. In a particular special embodiment, „Polymorph A“ is characterized by an IR absorption spectrum having characteristic peaks expressed in cm^-1 at 1980, 1659, 1584, 1335 and 1145. As used herein, the term “cell” has its ordinary and customary meaning as understood by one of ordinary skill in the art in light of this specification. “Cell” can refer to one or more cells. In some embodiments, the cells are normal cells, for example, human cells in different stages of development, or human cells from different organs or tissue types. In some embodiments, the cells are non-human cells, for example, other types of mammalian cells (e.g., mouse, rat, pig, dog, cow, and horse). In some embodiments, the cells are derived from other types of animal or plant cells. In other embodiments, the cells can be any prokaryotic or eukaryotic cells.

In several respective embodiments according to the invention, the pyrimidine analogue NHC (EIDD- 1931) is applied with a DHODH inhibitor, either co-administered simultaneously or sequentially, for instance, the DHODH inhibitor teriflunomide, IMU-838, or BAY 2402234, wherein the administration of each respective NHC-DHODH inhibitor combination produces a significant synergistic effect against the replication of a virus, preferably a SARS-CoV-2 variant. Without being bound to theory, this synergistic result may have occurred in view of the following: DHODH inhibitors reduce the biosynthesis of pyrimidines and may therefore lead to a shortage of pyrimidines following DHODH inhibitor application, for example, a shortage of cytidine triphosphate within a cell. Furthermore, pyrimidine analogues can replace pyrimidines such as cytidine when incorporated into nascent RNA, for instance, the pyrimidine analogue NHC can replace cytidine. In this case, an increase in NHC molecules that may become incorporated into nascent RNA may result when cytidine is scarce or otherwise deficient in an infected cell. The increased incorporation of NHC may then lead to a higher incidence of mutations that preclude a correct synthesis of one or more viral proteins within the infected cell.

Additional mechanisms might further increase the synergy between the two classes of antiviral drugs according to the present invention. For example, the described drug combinations can contribute to a reversal of the host cell shutdown by way of DHODH inhibition. Moreover, in the context of an infected organism, DHODH inhibition might diminish the excessive proliferation and activation of immune cells to thereby diminish excessive inflammation (the above-described “cytokine storm”), whereas DHODH inhibition with antiviral pyrimidine analogues can still counteract virus replication in an infected cell.

The synergistic effect of the pyrimidine analogue NHC (EIDD-1931) and the DHODH inhibitors teriflunomide, IMU-838, and BAY 2402234 against the replication of SARS-CoV-2 is also applicable to the combination of any pyrimidine biosynthesis inhibitor with any pyrimidine analogue since an inhibition of cellular pyrimidine biosynthesis leads to a lower concentration of pyrimidine nucleotides, including their respective triphosphate metabolites, in the cell. On the other hand, pyrimidine nucleoside analogues mostly act in a similar manner as triphosphate metabolites and compete for their natural counterparts. Thus, by decreasing the amount of natural pyrimidine nucleosides in a cell, the likelihood of incorporating the antiviral nucleoside analogues is thereby increased.

The observed synergism between the two classes of drugs described herein, i.e., pyrimidine analogues and pyrimidine biosynthesis inhibitors, came as a surprise to the present inventors. Such strong antiviral synergistic effects exerted by the combinations according to the invention were not expected in view of conventional knowledge regarding each respective compound as described above.

Moreover, viral infections other than a viral infection caused by SARS-CoV-2 can be successfully treated with the presently disclosed combination therapy approaches. Therefore, it is understood that the inventive combination therapy strategies are not only directed to the treatment of SARS-CoV-2 but are also effective for treating any virus defined herein, including Coronaviridae and influenza viruses. The evidence presented herein clearly demonstrates a synergistic effect of pyrimidine analogues and pyrimidine biosynthesis inhibitors advantageously leading to a higher antiviral potency effect with less toxicity compared to conventional treatment paradigms.

Methods of treatment

The present invention relates to methods for treating and/or preventing a viral infection by administering to a subject in need thereof a combination therapy comprising a pyrimidine biosynthesis inhibitor and a pyrimidine analogue. Any viral infection can be effectively treated using this novel combination therapy as defined herein.

As used herein, the term “subject” refers to a mammal (e.g., a human, rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit) or bird (e.g., a chick, goose, turkey), which is in need of prevention and/or treatment of a disease or disorder such as viral infection or cancer. The subject may have a viral infection, e.g., an influenza infection, or be predisposed to developing an infection, or was in contact with another infected individual. Subjects predisposed to developing an infection, or subjects who may be at elevated risk for contracting an infection (e.g., of coronavirus or influenza virus), include subjects with compromised immune systems because of autoimmune disease, subjects receiving immunosuppressive therapy (for example, following organ transplant), subjects afflicted with human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), subjects with forms of anemia that deplete or destroy white blood cells, subjects receiving radiation or chemotherapy, or subjects afflicted with an inflammatory disorder. Additionally, subjects of very young (e.g., 5 years of age or younger) or old age (e.g., 65 years of age or older) are at increased risk. In some embodiments, the subject is preferably a mammal, most preferably a human. In a preferred embodiment, the viral infection is caused or otherwise facilitated by a member of the Coronaviridae virus family or by an influenza virus. The members of the Coronaviridae comprises SARS-CoV, MERS-CoV, SADS-CoV, and SARS-CoV-2. Most preferred is the treatment of a viral infection caused by SARS-CoV-2, wherein the viral infection causes the subject to suffer from the condition of COVID-19.

In a preferred embodiment, the treatment of a viral infection is caused by muted form of SARS-CoV-2, wherein the viral infection causes the subject to suffer from the condition of COVID-19.

The methods of treating and/or preventing a viral infection according to the present invention comprise a step of administering to the subject a therapeutically effective amount of a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue or its prodrug in order to achieve a therapeutically effective outcome.

An “effective amount” or “therapeutically effective amount” as used herein refers to an amount of compound to be delivered or administered that is sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to treat, improve symptoms of, diagnose, prevent, and/or delay the onset or progression of the disease, such as a viral infection, or otherwise reduce the pathological consequences thereof. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and the effective concentration of the compounds that are administered.

As used herein, the term “increase” refers to alter positively by at least about 5%, including, but not limited to, alter positively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

As used herein, the term “reduce” refers to alter negatively by at least about 5% including, but not limited to, alter negatively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

As used herein, the term “therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to a viral infection, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the viral infection. As used herein, the term “prodrug” refers to any substance, molecule or entity which is in a form predicate for that substance, molecule or entity to act as a therapeutic upon chemical or physical alteration. Prodrugs may by covalently bonded or sequestered in some way and which release or are converted into the active drug moiety prior to, upon or after being administered to a subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group respectively.

The pyrimidine biosynthesis inhibitor useful in the disclosed methods is preferably an inhibitor selected from Table 1 or Table 2.

The pyrimidine biosynthesis inhibitor useful in the disclosed methods is preferably an inhibitor selected from Table 1-1 or Table 2.

In a preferred embodiment, the pyrimidine biosynthesis inhibitor used in the inventive antiviral combination treatment is an inhibitor of DHODH (a “DHODH inhibitor”). Exemplary and effective DHODH inhibitors are shown in Table 1. Preferred DHODH inhibitors for use in the present invention include BAY 2402234, teriflunomide, and IMU-838. Most preferred is IMU-838.

In a preferred embodiment, the pyrimidine biosynthesis inhibitor used in the inventive antiviral combination treatment is the DHODH inhibitor Brequinar.

In a preferred embodiment, the pyrimidine biosynthesis inhibitor used in the inventive antiviral combination treatment is an inhibitor of DHODH (a “DHODH inhibitor”). Exemplary and effective DHODH inhibitors are shown in Table 1 - 1. Preferred DHODH inhibitors for use in the present invention include MEDS433, PTC299, RP7214, and AG-636.

The selected pyrimidine biosynthesis inhibitor can be used in combination with one of the antiviral pyrimidine analogues shown in Table 3. In a preferred embodiment, the pyrimidine analogue is a cytidine analogue. In an even more preferred embodiment, the pyrimidine analogue is EIDD-1931 (NHC) or EIDD-2801. In another preferred embodiment, the pyrimidine analogue is a uridine analogue.

Preferably, the method of treating or preventing a viral infection in a subject comprises administering to the subject an effective amount of BAY 2402234, teriflunomide, or IMU-838 in combination with NHC. Most preferably, the method of treating or preventing a viral infection in a subject comprises administering to the subject an effective amount of IMU-838 in combination with NHC.

More preferably, the method of treating or preventing a viral infection in a subject comprises administering to the subject an effective amount of BAY 2402234, teriflunomide, or IMU-838 in combination with Molnupiravir (EIDD-2801). Most preferably, the method of treating or preventing a viral infection in a subject comprises administering to the subject an effective amount of IMU-838 in combination with Molnupiravir (EIDD-2801).

The compounds of the present invention may be administered by any route which results in a therapeutically effective outcome. These routes include, but are not limited to oral, transdermal, intravenous, intramuscular, parenteral, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), and submucosal. In a preferred embodiment, the pyrimidine biosynthesis inhibitor and the pyrimidine analogue or its prodrug are administered orally.

The pyrimidine biosynthesis inhibitor and the pyrimidine analogue or its prodrug can be administered to a subject concurrently, which means simultaneously. Alternatively, the pyrimidine biosynthesis inhibitor can be administered prior to the pyrimidine analogue or its prodrug. The pyrimidine biosynthesis inhibitor may be administered 7 days before the pyrimidine analogue or its prodrug. In some embodiments, the pyrimidine biosynthesis inhibitor may be administered 7, 6, 5, 4, 3, 2, or 1 day before the pyrimidine analogue or its prodrug. In some embodiments, the pyrimidine biosynthesis inhibitor may be administered 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 24 hours before the pyrimidine analogue or its prodrug. In some embodiments, the pyrimidine biosynthesis inhibitor may be administered 10, 20, 30, 40 50 or 60 minutes before the pyrimidine analogue or its prodrug. Preferably, the pyrimidine biosynthesis inhibitor and the pyrimidine analogue or its prodrug are administered concurrently.

The dose to be administered depends on the specific inhibitor used in the combination therapy and the age and body weight of the subject to be treated. The pyrimidine biosynthesis inhibitor may be administered at a dose of about 1 μmol to about 1000 μmol. The combination therapies according to the present invention provide for administering lower doses of each respective drug, namely, at a lower dose of the pyrimidine analogue and/or a lower dose of the pyrimidine biosynthesis inhibitor compared to when any of the two drugs is administered individually independent of a combined approach.

For example, BAY 2402234 may be administered to a subject at a dose of about 1 to about 50 mg/kg body weight, preferably at a dose of about 1 to about 10 mg/kg body weight, more preferably at a dose of about 1 to about 5 mg/kg body weight. Alternatively, BAY 2402234 may be administered at a dose of about 50 to about 1000 μmol, preferably about 200 to about 800 μmol, more preferably about 200 to about 500 μmol.

In another example, teriflunomide may be administered at a dose of about 5 to about 20 mg/day or about 5 to about 100 μmol preferably at a dose of 14 mg/day or 52 μmol, most preferably at a dose of about 5 to about 25 μmol.

In yet another example, IMU-838 may be administered at a dose of about 10-200 μmol, preferably at a dose of up to about 50 mg or at a dose of up to about 140 μmol.

In yet another example, IMU-838 may be administered at a dose of about 10 to 50 mg day, preferably at a dose of 30 mg/day to 45 mg/day.

A pyrimidine analogue or its prodrug as disclosed herein can be administered at a dose of about 1 μmol to about 200 mmol. The dosage depends on the specific analogue used, which would be known to the skilled person.

In one embodiment, N⁴-hydroxycytidine (EIDD-1931) can be administered at a dose of about 10 to about 200 mmol or about 100-1000 mg/kg/day. Preferably, a dosage is administered at about 200 to about 500 mg/kg/day orally or about 50 to about 154 mmol per day.

In one embodiment, N⁴-hydroxycytidine (EIDD-1931) can be administered at a daily dose of about 0.2 to about 10 mmol or about 100-1000 mg/day. Preferably, a dosage is administered at about 200 to about 500 mg/day orally or about 50 to about 154 mmol per day.

In another embodiment, N⁴-hydroxycytidine-5’ -isopropyl ester (EIDD-2801) can be administered at a dose of about 10 to about 150 mmol, preferably about 100 to about 500 mg/kg or about 50 to about 120 mmol.

In one embodiment, Molnupiravir (EIDD-2801) can be administered at a dose of about 0.3 mmol to about 2.5 mmol or about 80-650 mg/day. Preferably, a dosage is administered at about 100 to about 500 mg/day orally.

In one embodiment, Molnupiravir (EIDD-2801) can be administered twice daily at a dose below 800 mg/dose. Preferably, a dosage is administered twice daily at about 50 to about 500 mg/dose. More preferably, a dosage is administered twice daily at about 50 to about 250 mg/dose. The effective daily dosing amount of the disclosed compounds to a subject may be about 1 μmol to about 1000 μmol of the pyrimidine biosynthesis inhibitor and about 1 μmol to 200 mmol of the pyrimidine analogue or its prodrug.

As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, according to the limitations of the applied measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, “about” can mean within an order of magnitude, preferably within 5- fold, and more preferably within 2-fold, of a value.

The dose of the respective pyrimidine analogue or its prodrug and the dose of the respective pyrimidine biosynthesis inhibitor in the compound therapy can be individually lower in amount compared when administered concurrently compared to instances when the respective compounds are administered individually as a single drug dose.

The timing of when to initiate the combination therapy according to the present invention for treating a viral infection in a subject is preferably during the first week when viral symptoms manifest, for example, when pharyngeal shedding is at its highest and the peak virus replication has not yet been reached. Since virus replication and shedding may continue for several weeks, early intervention by administering the present combination therapies disclosed herein provides the maximal clinical benefit.

Combination therapies

DHODH inhibitor in combination with NHC/EIDD-1931

In one aspect, the present invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of a DHODH inhibitor in combination with an antiviral pyrimidine analogue, preferably β-D-N⁴-hydroxycytidine (NHC/ EIDD-1931). The DHODH inhibitor is preferably selected from Table 1. More preferably, the DHODH inhibitor is selected from the group of DHODH inhibitors comprising Teriflunomide, BAY 2402234, IMU-838 and IMU-935, PTC299. In a preferred embodiment, the viral infection is caused by a member of the Coronaviridae virus family or an Influenza virus. The member of the Coronaviridae virus family can be a betacoronavirus or an alphacoronavirus, preferably SARS-CoV, MERS-CoV, SARS-CoV-2, or SADS-CoV, and most preferably SARS-CoV-2. In another aspect, the present invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of a DHODH inhibitor in combination with an antiviral pyrimidine analogue, preferably β-D-N⁴-hydroxycytidine (NHC/ EIDD-1931). The DHODH inhibitor is preferably selected from Table 1-1. More preferably, the DHODH inhibitor is selected from the group of DHODH inhibitors comprising MEDS433, RP7214, and AG-636.

In one embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of BAY 2402234 in combination with an antiviral pyrimidine analogue, preferably β-D-N⁴-hydroxycytidine (NHC/ EIDD- 1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of teriflunomide in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine (NHC/ EIDD-1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In yet another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of IMU- 838 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine (NHC/ EIDD- 1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of IMU-935 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine (NHC/ EIDD-1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of leflunomide in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine (NHC/ EIDD-1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human. In yet another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of PTC299 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine (NHC/ EIDD- 1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

The DHODH inhibitor and the NHC according to the present invention can be administered according to one of the administration routes disclosed herein, preferably orally or parenterally, most preferably orally. The DHODH inhibitor can be administered concurrently with the NHC or before the NHC, preferably concurrently with the NHC.

DHODH inhibitor in combination with EIDD-2801

In another aspect, the present invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of a DHODH inhibitor in combination with an antiviral pyrimidine analogue, preferably β-D-N⁴-hydroxycytidine-5’- isopropyl ester (EIDD-2801). The DHODH inhibitor is preferably selected from Table 1. More preferably, the DHODH inhibitor is selected from the group of DHODH inhibitors comprising teriflunomide, BAY 2402234, IMU-838, IMU-935 and PTC299. In a preferred embodiment, the viral infection is caused by a member of the Coronaviridae virus family or an Influenza vims. The member of the Coronaviridae vims family can be a betacoronavims or an alphacoronavims, preferably SARS- CoV, MERS-CoV, SARS-CoV-2, or SADS-CoV, and most preferably SARS-CoV-2.

In yet another aspect, the present invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of a DHODH inhibitor selected from Table 1-1 in combination with an antiviral pyrimidine analogue, preferably β-D-N⁴-hydroxycytidine-5 ’-isopropyl ester (EIDD-2801). More preferably, the DHODH inhibitor is selected from the group of DHODH inhibitors comprising MEDS433, RP7214, AG-636 and PP-001. In a preferred embodiment, the viral infection is caused by a member of the Coronaviridae vims family or an Influenza vims. The member of the Coronaviridae vims family can be a betacoronavims or an alphacoronavims, preferably SARS-CoV, MERS-CoV, SARS-CoV-2, or SADS- CoV, and most preferably SARS-CoV-2.

In one embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of BAY 2402234 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5 ’-isopropyl ester (EIDD- 2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of teriflunomide in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5’ -isopropyl ester (EIDD-2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of vidofludimus in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5’ -isopropyl ester (EIDD-2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In yet another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of IMU- 838 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5’ -isopropyl ester (EIDD-2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In still another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of IMU- 935 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5’ -isopropyl ester (EIDD-2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of leflunomide in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5’ -isopropyl ester (EIDD-2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of PTC299 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5 ’-isopropyl ester (EIDD- 2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of MEDS433 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5 ’ -isopropyl ester (EIDD- 2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of RP7214 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5 ’ -isopropyl ester (EIDD- 2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of AG-636 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5 ’-isopropyl ester (EIDD- 2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of Brequinar in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5 ’ -isopropyl ester (EIDD- 2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of PP-001 in combination with the antiviral pyrimidine analogue β-D-N⁴-hydroxycytidine-5 ’ -isopropyl ester (EIDD- 2801), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.

In another embodiment, the invention relates to a method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of ASLAN003/Farudodstat in combination with the antiviral pyrimidine analogue b-D-N⁴- hydroxycytidine-5’ -isopropyl ester (EIDD-2801), wherein the viral infection is caused by SARS-CoV- 2 and wherein the subject is a mammal, preferably human.

The DHODH inhibitor and the β-D-N⁴-hydroxycytidine-5’ -isopropyl ester according to the present invention can be administered according to one of the administration routes disclosed herein, preferably orally or parenterally, most preferably orally. The DHODH inhibitor can be administered concurrently with the β-D-N⁴-hydroxycytidine-5 ’ -isopropyl ester or before the β-D-N⁴-hydroxycytidine-5 ’ -isopropyl ester, preferably concurrently with the β-D-N⁴-hydroxycytidine-5 ’ -isopropyl ester. Pyrimidine biosynthesis inhibitors

In addition to the preferred DHODH inhibitors disclosed above, numerous alternative DHODH inhibitors have been developed for treating leukemia and additional purposes (e.g. WO2010115736, WO2020144638, WO2020161663, WO2020212897, W02021038490, W02021070132, WO2021084498, W02021084500, WO2021085582, WO2021156787, WO2021238881,

WO2021240423, WO2021240424, WO2021240429 and WO2022025174). DHODH inhibitors have also been developed for treating malaria and other parasite-induced diseases. Combining the respective DHODH-inhibitors with pyrimidine analogues in the manner of the present invention can also potentiate the therapy of these diseases.

The following DHODH inhibitors may be used for the antiviral combination therapies according to the present invention concurrently with an antiviral pyrimidine analogue or its prodrug:

TABLE 1: Exemplary DHODH Inhibitors

TABLE 1-1: Additional Exemplary DHODH Inhibitors

In other embodiments, the following pyrimidine biosynthesis inhibitors, which are not classified as DHODH inhibitors, may be used in the disclosed antiviral combination therapies: TABLE 2: Exemplary alternative pyrimidine biosynthesis inhibitors

TABLE 2-2: Exemplary CTP synthetase Inhibitors Additional enzymatic events are required for de novo pyrimidine biosynthesis. At present, development of inhibitors towards such enzymatic activities is still in its infancy. Without being bound to a particular theory, according to the mechanisms of action disclosed herein for DHODH, if clinically useful inhibitors are subsequently developed that target these enzymes involved in pyrimidine biosynthesis, such inhibitors would also represent good candidates for combination therapies with antiviral pyrimidine analogues such as EIDD-1931 as disclosed herein. Such enzyme targets can include, but are not limited to, the following: carbamoyl phosphate synthetase II, aspartate carbamoyltransferase (i.e., aspartate transcarbamylase); and dihydroorotase; orotate phosphoribosyltransferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). Additional candidates are enzymes involved in the recycling of pyrimidines from the degradation of RNA (salvage pathway), including but not limited to uridine phosphorylase or pyrimidine-nucleoside phosphorylase or uracil-phosphoribosyltransferase.

The following exemplary pyrimidine analogues and their prodrugs are contemplated for use according to the present invention:

TABLE 3: Pyrimidine analogues

Combinations of inhibitors comprising pyrimidine analogues

The following combinations of DHODH inhibitors and pyrimidine analogues can be effectively used in the presently disclosed antiviral therapeutic strategies.

TABLE 4: Exemplary Combinations of DHODH Inhibitors and pyrimidine analogues

The following combinations of pyrimidine biosynthesis inhibitors, which represent alternatives to a combination of the aforementioned DHODH inhibitors and pyrimidine analogues, may be effectively practiced according to the present invention. TABLE 5: Alternative combinations of pyrimidine biosynthesis inhibitors and pyrimidine analogues

Viruses

The present invention relates to methods for treating or preventing a viral infection in a subject comprising administering a combination therapy as described herein.

The term “virus” includes any virus whose infection in the body of a subject is treatable or preventable by administration of a combination therapy as described herein. The term “virus” also refers to a CoV- dependent respiratory virus which is a virus that infects the respiratory tissue of a subject, for instance, an infection in the upper and/or lower respiratory tract, trachea, bronchi and lungs.

In one embodiment of the invention, the virus can be a DNA virus including the family of Herpesviridae, which comprises the genera of Herpes simplex virus, Varicella Zoster Virus, Cytomegalovirus, Epstein Barr Virus; Hepatitis B virus; Adenoviruses; and Papillomaviruses. As used herein a “DNA virus” is a virus with a genome composed of deoxyribonucleic acid (DNA) that is replicated by a DNA polymerase. A DNA virus can be divided between those that have two strands of DNA in their genome (double-stranded DNA or ‘dsDNA’ viruses), and those that have one strand of DNA in their genome (single-stranded DNA or ‘ssDNA) viruses). In another embodiment of the invention, the virus can be a RNA virus including the family of Coronaviridae, which comprises the genera of coronavirus, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome (MERS) coronavirus), or an RNA RNS virus comprising HIV (Human Immunodeficiency Virus), Influenza virus, Ebolavirus, Flaviviridae comprising Hepatitis C virus, West Nile virus, Dengue virus, Yellow fever virus, Zika virus, Rhabdoviruses comprising rabies virus, Paramyxoviridae comprising parainfluenza virus, Venezuelan equine encephalitis virus, Equine arteritis virus, Rotaviruses, and Enterovirus comprising Foot-and-mouth disease virus (FMDV). Coronaviruses can further include the genera of alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses.

The term “viral infection” refers to the invasion and multiplication of a virus in the body of a subject. The term “coronavirus infection” or “CoV infection,” as used herein, refers to infection involving a coronavirus such as SARS-CoV-2, MERS-CoV, or SARS-CoV. Symptoms of a coronavirus infection can include high fever, dry cough, shortness of breath, pneumonia, gastro-intestinal symptoms such as diarrhea, organ failure (kidney failure and renal dysfunction), septic shock, and death in severe cases.

The inventive combination therapeutic approaches according to the present invention can be used in the treatment of viral infections caused by a wide variety of viruses described herein. The replication of all the viruses contemplated for antiviral action by the instant combination therapies can be inhibited to a significantly greater extent by such combination therapies comprising pyrimidine biosynthesis inhibitors and pyrimidine analogues, compared to administration of these compounds alone. In a preferred embodiment, the viral infection is caused by SARS-CoV-2. A viral infection caused by SARS-CoV-2 further comprises an infection caused by derivatives of SARS-CoV-2, wherein the amino acid sequence of the virus proteins encoded by the derivative has at least 40% identity to the amino acid sequence of the corresponding SARS-CoV2 proteins. More preferably, the amino acid sequence of the derivative has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SARS-CoV2. Derivatives of SARS-CoV-2 can comprise any mutation of the virus, such mutations include those as presented in Wang et al., 2021, Communications Biology vol. 4:228. In addition, it is understood that the present invention is efficient in the treatment of infections caused by viruses, which evolved from SRAS-CoV-2, leading to diseases like COVID-19, for instance, COVID-21, COVID-22, or other COVID diseases.

Pharmaceutical Compositions

In one aspect, the present disclosure provides a pharmaceutical composition comprising a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue as disclosed herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition can comprise a further therapeutic component, for example, a second pyrimidine biosynthesis inhibitor and/or a second antiviral pyrimidine analogue. In some embodiments, the further therapeutic agent is selected from Tables 1-4.

Kit or Kit of Parts

The present invention further provides a kit or a kit of parts comprising one or more components that include, but are not limited to, a therapeutically effective amount of a pyrimidine biosynthesis inhibitor (Tables 1-2 and Tables 4-5) in combination with an antiviral pyrimidine analogue (Tables 3-5) or its prodrug for treating and/or preventing a viral infection in a subject. In another embodiment, each component in the therapeutic combination can be formulated as a single composition or separately in two or more compositions, optionally with a pharmaceutically acceptable carrier, in a pharmaceutical composition. Each component can be provided in a single, common container. Alternatively, each component can be provided in a separate container.

The kit can additionally include a package insert including information concerning the respective components in the combination or the respective pharmaceutical compositions and dosage forms in the respective kit or kit of parts. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.

Embodiments of the present invention are:

1. A method of treating and/or preventing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of one or more pyrimidine biosynthesis inhibitor in combination with one or more antiviral pyrimidine analogue or its prodrug.

2. The method of embodiment 1, wherein the viral infection is caused by an RNA virus or a DNA virus.

3. The method of embodiment 2, wherein the RNA virus is a member of the Coronaviridae virus family, an Influenza virus, HIV (Human Immunodeficiency Virus), Ebolavirus, Flaviviridae comprising Hepatitis C virus, West Nile virus, Dengue virus, Yellow fever virus, Zika virus, Rhabdoviruses comprising rabies virus, Paramyxoviridae comprising parainfluenza virus, Venezuelan equine encephalitis virus, Equine arteritis virus, Rotaviruses, or Enterovirus comprising Foot-and-mouth disease virus (FMDY).

4. The method of emdiment 3, wherein the member of the Coronaviridae virus family is a Betacoronavirus or Alphacoronavirus, preferably a SARS-CoV, MERS-CoV, SARS-CoV-2 or SADS- CoV.

5. The method of embodiment 1, wherein the viral infection is caused by SARS-CoV-2 or derivatives thereof, wherein the amino acid sequence of virus proteins encoded by the derivative has at least 40% identity to the amino acid sequence of SARS-CoV2.

6. The method of embodiment 1 and embodiment 5, wherein the subject suffers from COVID-19.

7. The method of any of embodiment 1 to 6, wherein the pyrimidine analogue is selected from the pyrimidine analogues listed in Table 3 or their prodrugs.

8. The method any of embodiments 1 to 6, wherein the pyrimidine analogue is selected from N⁴- hydroxycytidine/EIDD-1931, N⁴-hydroxycytidine-5 ’-isopropyl ester /EIDD-2801/mulnupiravir/MK- 4482, Sofosbuvir, N⁴-aminocytidine, Cidofovir, Stavudine, AZT/azidothymidine/zidovudine, Didanosine/2 ’ ,3 ’ -dideoxyinosine/ddl, Zalcitabine/2 ’ ,3 ’ -dideoxycytidine/ddC, Lamivudine/(-)-β-L-3 ’ - thia-2 ’ ,3 ’ -dideoxycytidine/3TC, Emtricitabine/(-)-β-L-3 ’ -thia-2 ’ ,3 ’ -dideoxy-5-fluorocytidine/(-)-FTC, Idoxuridine/ 5 -iodo-2 ’ -deoxyuridine/IDU, Trifluridine/5 -trifluoro-2 ’ -deoxythymidine/TFT,

9. The method of any of embodiments 1 to 6, wherein the pyrimidine analogue is a cytidine analogue or its prodrug.

10. The method of any of embodiments 1 to 9, wherein the pyrimidine analogue is N⁴- hydroxycytidine/EIDD-1931 or its prodrug N⁴-hydroxycytidine-5 ’-isopropyl ester/EIDD-2801.

11. The method of any of embodiments 1 to 10, wherein the pyrimidine biosynthesis inhibitor is selected from the DHODH-inhibitors of Table 1 and the inhibitors listed in Table 2.

12. The method of any of embodiments 1 to 10, wherein the pyrimidine biosynthesis inhibitor is selected from the DHODH-inhibitors of Table 1-1 and the inhibitors listed in Table 2. 13. The method of any of embodiments 1 to 12, wherein the pyrimidine synthesis inhibitor is a DHODH inhibitor.

14. The method of embodiment 13, wherein the DHODH inhibitor is selected from IMU-838, IMU-935, PTC299, S312, S416, Leflunomide, Terifhmomide, Brequinar/DD264, BAY 2402234, IPPA17-A04, GSK983/SW835, Cmpl, NITD-982, Compound A3, FK778/Maritimus, DSM265.

15. The method of embodiment 13, wherein the DHODH inhibitor is BAY 2402234.

16. The method of embodiment 13, wherein the DHODH inhibitor is IMU-838.

17. The method of embodiment 13, wherein the DHODH inhibitor is Brequinar/DD264.

18. The method of embodiment 13, wherein the DHODH inhibitor is teriflunomide.

19. The method of any of embodiment 1 to 18, wherein the pyrimidine biosynthesis inhibitor and the pyrimidine analogue or its prodrug are administered orally or parenterally.

20. The method of any of embodiment 1 to 19, wherein the pyrimidine synthesis inhibitor is administered together with the pyrimidine analogue or its prodrug.

21. The method of any of embodiment 1 to 19, wherein the pyrimidine synthesis inhibitor is administered up to 7 days before the pyrimidine analogue or its prodrug, with the possibility of additional administrations of the pyrimidine biosynthesis inhibitor during the period up to 7 days before the pyrimidine analogue or its prodrug.

22. The method of any of embodiment 1 to 21, wherein the pyrimidine biosynthesis inhibitor is administered at a dose of about 1 to about 1000 μmol.

23. The method of embodiment 15, wherein BAY 2402234 is administered at a dose of about 50 to about 1000 μmol.

24. The method of embodiment 18, wherein Teriflunomide is administered at a dose of about 5 to about 100 μmol.

25. The method of embodiment 16, wherein IMU-838 is administered at a dose of about 10 to about 200 μmol. 26. The method of any of embodiment 1 to 25, wherein the pyrimidine analogue is administered at a dose of about 1 mhioΐ to about 200 mmol.

27. The method of embodiment 10, wherein the N⁴-hydroxycytidine is administered at a dose of about 10 to about 200 mmol.

28. The method of embodiment 10, wherein the N⁴-hydroxycytidine-5 ’-isopropyl ester is administered at a dose of about 10 to about 150 mmol.

29. The method of any of embodiment 1-21, wherein the effective daily dosing amount is about 1 μmol to about 1000 μmol of the pyrimidine biosynthesis inhibitor and about 1 μmol to about 200 mmol of the pyrimidine analogue.

30. The method of any of the preceding embodiments, wherein after administration of the pyrimidine biosynthesis inhibitor and the antiviral pyrimidine analogue or its prodrug the viral load is reduced.

31. The method of any of the preceding embodiments, wherein the subject is mammalian or avian.

32. The method of embodiment 31, wherein the mammal is a human.

33. The method of embodiment 1, wherein the pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N⁴-hydroxycytidine, and wherein the viral infection is caused by SARS-CoV-2.

34. A pharmaceutical composition comprising an effective amount of one or more pyrimidine biosynthesis inhibitor and one or more pyrimidine analogue or its prodrug.

35. The pharmaceutical composition of embodiment 34, comprising about 1 μmol to 1000 μmol of the pyrimidine biosynthesis inhibitor and about 1 μmol to 200 mmol of the pyrimidine analogue or its prodrug.

36. The pharmaceutical composition of embodiments 34 or 35, wherein the pyrimidine biosynthesis inhibitor is selected from IMU-838, IMU-935, PTC299, S312, S416, Leflunomide, Teriflunomide, Brequinar/DD264, BAY 2402234, IPPA17-A04, GSK983/SW835, Cmpl, NITD-982, Compound A3, FK778/Maritimus, DSM265, PALA (N-phosphonoacetyl-L-aspartate), 6-Azauridine, Azaribine/triacetyl-6-azauridine and wherein the pyrimidine analogue is selected from N⁴- hydroxycytidine/EIDD-1931, N⁴-hydroxycytidine-5 ’-isopropyl ester /EIDD-2801/mulnupiravir/MK- 4482, Sofosbuvir, N⁴-aminocytidine, Cidofovir, Stavudine, AZT/azidothymidine/zidovudine, Didanosine/2’,3’-dideoxyinosine/ddI, Zalcitabine/ 2’,3’-dideoxycytidine/ddC, Lamivudine/(-)-β-L-3’- thia-2 ’ ,3 ’ -dideoxycytidine/3TC, Emtricitabine/ (-)-β-L-3 ’ -thia-2 ’ ,3 ’ -dideoxy-5-fluorocytidine/(-)-FTC, Idoxuridine/ 5 -iodo-2 ’ -deoxyuridine/IDU, Trifluridine/5 -trifluoro-2 ’ -deoxythymidine/TFT,

Brivudin/ (E)-5-(2-bromovinyl)-2 ’ -deoxyuridine/B VDU, 2 ’ -C-methyl-4-amino-pyrrolo[2,3 - d]pyrimidine ribonucleosides.

37. The pharmaceutical composition of embodiment 36, wherein the pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N⁴-hydroxycytidine.

38. A composition comprising one or more pyrimidine biosynthesis inhibitor and one or more pyrimidine analogue or its prodrug.

39. A pharmaceutical composition of any of embodiments 34 to 37 or the composition of claim 38 comprising a pyrimidine biosynthesis inhibitor and a pyrimidine analogue or its prodrug, wherein the pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N⁴-hydroxycytidine.

40. A kit of parts comprising the pharmaceutical composition of embodiments 34 to 37 or 39, and optionally technical instructions with information on the administration and dosage of the composition and optionally technical instructions with information on the administration and dosage of the composition.

41. A kit of parts comprising the composition of embodiment 38 or 39, and optionally technical instructions with information on the administration and dosage of the composition.

42. The method of any of embodiments 1 to 33, wherein the administration of the therapeutically effective amount of a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue or its prodrug has a synergistic effect.

43. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject.

44. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 43, wherein the viral infection is caused by an RNA virus or a DNA virus.

45. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 43 and 44, wherein the RNA virus is a member of the Coronaviridae virus family, an Influenza virus, HIV (Human Immunodeficiency Virus), Ebolavirus, Flaviviridae comprising Hepatitis C virus, West Nile virus, Dengue virus, Yellow fever virus, Zika virus, Rhabdoviruses comprising rabies virus, Paramyxoviridae comprising parainfluenza virus, Venezuelan equine encephalitis virus, Equine arteritis virus, Rotaviruses, or Enterovirus comprising Foot-and-mouth disease virus (FMDV).

46. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 45, wherein the member of the Coronaviridae virus family is a Betacoronavirus or Alphacoronavirus, preferably a SARS-CoV, MERS- CoV, SARS-CoV-2 or SADS-CoV.

47. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 46, wherein the viral infection is caused by SARS-CoV-2 or derivatives thereof, wherein the amino acid sequence of virus proteins encoded by the derivative has at least 40% identity to the amino acid sequence of SARS-CoV2.

48. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 47, wherein the subject suffers from COVID-19.

49. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 48, wherein the pyrimidine analogue is selected from the pyrimidine analogues listed in Table 3 or their prodrugs.

50. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 48, wherein the pyrimidine analogue is selected from N⁴-hydroxycytidine/EIDD- 1931, N⁴-hydroxycytidine-5 ’ -isopropyl ester/EIDD- 2801/mulnupiravir/MK-4482, Sofosbuvir, N⁴-aminocytidine, Cidofovir, Stavudine, AZT/ azidothymidine/zidovudine, Didanosine/2 ’ ,3 ’ -dideoxyinosine/ddl, Zalcitabine/2 ’ ,3 ’ - dideoxycytidine/ddC, Lamivudine/(-)-β-L-3 ’ -thia-2 ’ ,3 ’ -dideoxycytidine/3 TC, Emtricitabine/ (-)-β-L- 3 ’ -thia-2 ’ ,3 ’ -dideoxy-5-fluorocytidine/(-)-FTC, Idoxuridine/5-iodo-2 ’ -deoxyuridine/IDU,

51. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 48, wherein the pyrimidine analogue is a cytidine analogue or its prodrug.

52. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 51 , wherein the pyrimidine analogue is N⁴- hydroxycytidine/EIDD-1931 or its prodrug N⁴-hydroxycytidine-5 ’ -isopropyl ester/EIDD-2801.

53. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 52, wherein the pyrimidine biosynthesis inhibitor is selected from the DHODH-inhibitors of Table 1 and the inhibitors listed in Table 2.

54. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 52, wherein the pyrimidine biosynthesis inhibitor is selected from the DHODH-inhibitors of Table 1-1 and the inhibitors listed in Table 2.

55. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 54, wherein the pyrimidine synthesis inhibitor is a DHODH inhibitor.

56. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 55, wherein the DHODH inhibitor is selected from IMU- 838, IMU-935, PTC299, S312, S416, Leflunomide, Teriflunomide, Brequinar/DD264, BAY 2402234, IPPA17-A04, GSK983/SW835, Cmpl, NITD-982, Compound A3, FK778/Maritimus, DSM265.

57. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 55, wherein the DHODH inhibitor is BAY 2402234.

58. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 55, wherein the DHODH inhibitor is IMU-838.

59. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 55, wherein the DHODH inhibitor is Brequinar/DD264.

60. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 55, wherein the DHODH inhibitor is teriflunomide.

61. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 52, wherein the pyrimidine biosynthesis inhibitor is a cytidine triphosphate (CTP) synthetase inhibitor.

62. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 52, wherein the pyrimidine biosynthesis inhibitor is selected from the cytidine triphosphate (CTP) synthetase inhibitors of Table 2-2.

63. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 52, wherein the pyrimidine biosynthesis inhibitor is the CTP synthetase inhibitor is cyclopentenyl cytosine (CPEC).

64. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 63, wherein the pyrimidine biosynthesis inhibitor and the pyrimidine analogue or its prodrug are administered orally or parenterally.

65. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 64, wherein the pyrimidine synthesis inhibitor is administered together with the pyrimidine analogue or its prodrug.

66. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 65, wherein the pyrimidine synthesis inhibitor is administered up to 7 days before the pyrimidine analogue or its prodrug, with the possibility of additional administrations of the pyrimidine biosynthesis inhibitor during the period up to 7 days before the pyrimidine analogue or its prodrug.

67. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 66, wherein the pyrimidine biosynthesis inhibitor is administered at a dose of about 1 to about 1000 μmol.

68. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 57, wherein BAY 2402234 is administered at a dose of about 50 to about 1000 μmol.

69. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 60, wherein Teriflunomide is administered at a dose of about 5 to about 100 μmol.

70. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 58, wherein IMU-838 is administered at a dose of about 10 to about 200 μmol.

71. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claims any of embodiments 43 to 67, wherein the pyrimidine analogue is administered at a dose of about 100 μmol to about 20 mmol.

72. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 52, wherein the N⁴-hydroxycytidine is administered at a dose of about 100 μmol to about 10 mmol.

73. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 52, wherein the N⁴-hydroxycytidine-5 ’-isopropyl ester is administered at a dose of about 100 μmol to about 10 mmol.

74. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 63, wherein the effective daily dosing amount is about 1 μmol to about 1000 μmol of the pyrimidine biosynthesis inhibitor and about 1 μmol to about 20 mmol of the pyrimidine analogue.

75. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 74, wherein after administration of the pyrimidine biosynthesis inhibitor and the antiviral pyrimidine analogue or its prodrug the viral load is reduced.

76. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of embodiments 43 to 75, wherein the subject is mammalian or avian.

77. One or more pyrimidine biosynthesis inhibitors) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 76, wherein the mammal is a human.

78. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 43, wherein the pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N⁴-hydroxycytidine, and wherein the viral infection is caused by SARS-CoV-2.

79. One pyrimidine biosynthesis inhibitor(s) in combination with one antiviral pyrimidine analogue or its prodrug for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 43, wherein the pyrimidine biosynthesis inhibitor is IMU-838, wherein the pyrimidine analogue is prodrug N⁴-hydroxycytidine-5’ -isopropyl ester/EIDD-2801, and wherein the viral infection is caused by SARS-CoV-2. 80. One pyrimidine biosynthesis inhibitor(s) in combination with one antiviral pyrimidine analogue or its prodrug for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 43, wherein the pyrimidine biosynthesis inhibitor is Brequinar, wherein the pyrimidine analogue is prodrug N⁴-hydroxycytidine-5’ -isopropyl ester/EIDD-2801, and wherein the viral infection is caused by SARS-CoV-2.

81. One pyrimidine biosynthesis inhibitor(s) in combination with one antiviral pyrimidine analogue or its prodrug for use in a method of treating and/or preventing a viral infection in a subject according to embodiment 43, wherein the pyrimidine biosynthesis inhibitor is BAY 2402234, wherein the pyrimidine analogue is prodrug N⁴-hydroxycytidine-5 ’ -isopropyl ester/EIDD-2801, and wherein the viral infection is caused by SARS-CoV-2.

EXAMPLES

EXAMPLE 1: Analysis of a combination therapy comprising DHODH inhibitors BAY 2402234 or teriflunomide with EIDD-1931

SARS-CoV-2 was isolated from a sample of a subject and established with the following assays in order to observe viral replication, as described previously (Stegmann et al., 2020). The virus was then replicated in Vero E6 and a human lung epithelial cell line Calu-32B4 “Calu-3” cells, followed by qRT- PCR analysis in order to quantify the viral genomes released from the cultivated cells. Calu-3 cells represent a model that reproduces the environment of bronchial epithelia (Kreft et al, 2015).

Specifically, 30,000 Vero E6 cells or 90,000 Calu-3 cells were seeded per well on a 24-well plate and incubated for 8 hours at 37°C. The cells were then treated with the respective drugs at the concentrations indicated in the figures and incubated for additional 24 hours at 37°C. Cells were subsequently infected with 4E+06 SARS-CoV-2 RNA-copies per well and incubated for additional 48 hours at 37°C.

For the qRT-PCR for virus quantification, the virus was inactivated and samples were harvested. RNA was isolated from the samples and virus yield was quantified using a TaqMan probe.

Using this assay, the effect of NHC along with the effect of the DHODH inhibitors BAY 2402234 (Christian et al, 2019) and teriflunomide, a clinically approved immunosuppressive drug (O'Connor et al, 2011), were quantified in terms of virus replication and the synthesis of viral proteins.

In another set of experiments using EIDD-1931 in combination with BAY 2402234, the concentrations of each respective drug individually reduced the virus yield only moderately, still producing more than 20% of the viral genomes. Strikingly, when these two drugs were combined at the concentrations used for each individual drug, the virus yield decreased dramatically to less than 0.2%, which is nominally above the level of the inoculum itself (FIG. 1A).

In another set of experiments using EIDD-1931 in combination with teriflunomide, again, the concentrations of each respective drug individually reduced the virus yield only moderately, still producing about 60% of the viral genomes. Strikingly, when these two drugs were combined at the concentrations used for each individual drug, virus yield decreased dramatically to less than 0.7%, which is nominally above the level of the inoculum itself (FIG. 1A).

EXAMPLE 2: Dose response studies using EIDD-1931 in combination with BAY 2402234

In a second set of experiments, qRT-PCR was performed in the manner described for Experiment 1, using different concentrations of each respective drug. Several different concentrations of EIDD-1931 in combination with several different concentrations of BAY 2402234 revealed a synergistic suppression of viral replication, both in Vero E6 cells (FIG. IB) as well as in Calu-3 cells (FIG. 1C). Respective concentrations are depicted in the Figures.

These results demonstrate that even using very low concentrations of each respective compound in the experiments, namely 100 nM of the NHC and InM of BAY 2402234, exceptional virus clearance could be observed in this combination approach.

EXAMPLE 3: Dose response studies using EIDD-1931 in combination with teriflunomide

In a third set of experiments, qRT-PCR is performed as described for Experiment 1 using different concentrations of each respective drug. Several different concentrations of EIDD-1931 in combination with several different concentrations of teriflunomide revealed a synergistic suppression of viral replication, both in Vero E6 cells (FIG. ID) as well as in human lung epithelial cell line Calu-3 2B4 “Calu-3” cells (FIG. IE) a model that reproduces the environment of bronchial epithelia (Kreft et al., 2015). Respective concentrations are depicted in the Figures.

The results demonstrate that even using very low concentrations of each respective compound in the experiments, namely 100 nM of the NHC and 10 mM teriflunomide, exceptional virus clearance could be observed in this combination approach.

EXAMPLE 4: Immunoblot studies using EIDD-1931 in combination with BAY 2402234

SARS-CoV-2 was isolated from a sample derived from a subject and established using the assays to observe viral replication, as described previously (Stegmann et al., 2020). The virus was replicated in Vero E6 cells; immunoblot analysis of the resulting virus proteins was performed.

Specifically, 30,000 Vero E6 cells were seeded per well on a 24-well plate and incubated for 8 hours at 37°C. The cells were then treated with lOnM BAY 2402234 and lOOnM EIDD-1931 and then incubated for additional 24 hours at 37°C. The cells were subsequently infected with 4E+06 SARS-CoV-2 RNA- copies per well and incubated for an additional 48 hours at 37°C.

For the subsequent immunoblot analysis, wells were washed in PBS, the samples were harvested, lysed and sonicated before performing the blot via SDS-PAGE and transfer onto a nitrocellulose membrane (FIG. IF). The same combinatory antiviral treatment effect was observed when analyzing the levels of the spike and nucleoprotein of SARS-CoV-2. Notably, neither of the drugs was capable of suppressing the synthesis of either of these SARS-CoV-2 proteins individually; only the combination of these two drugs was effective in reducing both viral proteins to below a level of detection. EXAMPLE 5: Immunoblot studies using EIDD-1931 in combination with teriflunomide

SARS-CoV-2 was isolated from a sample of a subject and established with assays in order to observe viral replication, as described previously (Stegmann et al., 2020). The virus was then replicated in Vero E6 cells, followed by immunoblot analysis in order to quantify the viral proteins released from the cultivated cells.

Specifically, 30,000 Vero E6 cells were seeded per well on a 24-well plate and incubated for 8 hours at 37°C. The cells were then treated with 30mM teriflunomide and lOOnM EIDD-1931 and then incubated for additional 24 hours at 37°C. The cells were subsequently infected with 4E+06 SARS-CoV-2 RNA- copies per well and incubated for an additional 48 hours at 37°C.

For the subsequent immunoblot analysis, wells were washed in PBS, the samples were harvested, lysed and sonicated before performing the blot via SDS-PAGE and transfer onto a nitrocellulose membrane (FIG. 1G). The same combinatory antiviral treatment effect was observed when analyzing the levels of the spike and nucleoprotein of SARS-CoV-2. Notably, neither drug was capable of suppressing the synthesis of either of these SARS-CoV-2 proteins individually; only the combination of these two drugs were effective in reducing both viral proteins to below a level of detection.

EXAMPLE 6: Combination studies using the DHODH inhibitor IMU-838 and N⁴- hydroxycytidine (NHC)

Vero E6 cells were treated with the DHOH inhibitor IMU-838 in combination with N⁴-hydroxycytidine (NHC). The positive control used in this study was virus only without any treatment; the negative control was no virus infection. The cytopathic effect was analyzed visually under a microscope (FIG. 2A). qRT- PCR analysis was performed as described herein for Example 1 : c(t) values are shown in FIG. 2B, the virus RNA progeny expressed as a % are shown in FIG. 2C. An overview of the different concentrations of the DHODH inhibitor and different concentrations of NHC used in these experiments are shown in FIG. 2D. The respective concentrations are also depicted in the figures.

All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants to relate to each and every individual publication, patent application, or patent identified even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. EXAMPLE 7: Combination studies using the DHODH inhibitors MEDS433, PTC299, RP7214, AG-636, respectively, and N⁴-hydroxycytidine (NHC)

30,000 Vero E6 cells were seeded into 24-well-plates using DMEM GlutaMAX (Gibco) containing 2% fetal bovine serum and incubated for 8 hrs at 37°C. Cells were treated with NHC and/or various DHODH inhibitors (MEDS433, PTC299, RP7214, AG-636) at the indicated concentrations for 24 hrs before infection. Cells were infected with SARS-CoV-2 strain GOE 001 (30 FFU) and further incubated in the presence of the same drugs for 48 hrs. At 48 hrs post infection (p.i.), the cell culture supernatant was mixed with the Lysis Binding Buffer from the Magnapure LC Kit #03038505001 (Roche, 1:1 ratio) containing > 3 M guanidine thiocyanate (GTC), and the viral RNA was isolated using Trizol LS. Quantitative RT-PCR was performed using a TaqMan probe to detect and quantify SARS-CoV-2 RNA (according to Corman et al., 2020). The amount of SARS-CoV-2 RNA copies per mL are depicted in FIG. 2E to FIG. 2H. Note the logarithmic scale and that the inoculum (6.5E+06 virus RNA copies per mL) was used to infect the cells.

EXAMPLE 8s: Combination studies using the DHODH inhibitors BAY 2402234, teriflunomide, IMU-838, respectively, and N⁴-hydroxycytidine (NHC) with Influenza A virus replication 15,000 Madin-Darby Canine Kidney (MDCK) cells were seeded into 24-well-plates using DMEM(+)including 2% fetal bovine serum and incubated at 37°C overnight. Cells were treated with NHC and/or the DHODH inhibitors BAY 2402234 (Selleckchem, S8847), teriflunomide (Selleckchem, S4169), and IMU-838 (Immunic Therapeutics) at the indicated concentrations for 24 hrs before infection. Cells were infected with Influenza A/WSN/33 (MOI 0.02) and further incubated in the presence of the same drugs for 48 hrs. At 48 hrs post infection (p.i.), the Influenza A virus containing cell culture supernatant was mixed with the Lysis Binding Buffer from the Magnapure LC Kit #03038505001 (Roche, 1:1 ratio) containing > 3 M guanidine thiocyanate (GTC), and the viral RNA was isolated using Trizol LS. Quantitative RT-PCR was performed involving a TaqMan probe to detect and quantify Influenza A virus RNA (according to a WHO protocol, https://www.who.int/csr/resources/publications/swineflu/CDCRealtimeRTPCR_SwineHlAssay- 2009_20090430.pdf). The amount of RNA found upon infection without drug treatment was defined as 100%, and the other RNA quantities were normalized accordingly. RNA was also isolated from the virus inoculum used to infect the cells.

As shown in FIG. 3A to FIG. 3 pyrimidine analogue NHC and the DHODH inhibitors also synergistically interfere with Influenza A virus replication.

Claims

1. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject.

2. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claim 1, wherein the subject suffers from COVID-19.

3. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claim 1 and claim 2, wherein the pyrimidine analogue is N⁴- hydroxycytidine/EIDD-1931 or its prodrug N⁴-hydroxycytidine-5’ -isopropyl ester/EIDD-2801.

4. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claims 1 to 3, wherein the pyrimidine biosynthesis inhibitor is selected from the DHODH-inhibitors of Table 1 and the inhibitors listed in Table 2.

5. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claims 1 to 3, wherein the pyrimidine biosynthesis inhibitor is selected from the DHODH-inhibitors of Table 1-1 and the inhibitors listed in Table 2.

6. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claim 4, wherein the DHODH inhibitor is IMU-838.

7. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claims 1 to 6, wherein the pyrimidine biosynthesis inhibitor and the pyrimidine analogue or its prodrug are administered orally or parenterally.

8. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claims 1 to 7, wherein the pyrimidine synthesis inhibitor is administered together with the pyrimidine analogue or its prodrug.

9. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claim 6, wherein IMU-838 is administered at a dose of about 10 to about 200 μmol.

10. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to claim 3, wherein the N⁴-hydroxycytidine-5 ’-isopropyl ester is administered at a dose of about 100 μmol to about 10 mmol.

11. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of the preceding claims, wherein after administration of the pyrimidine biosynthesis inhibitor and the antiviral pyrimidine analogue or its prodrug the viral load is reduced.

12. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of the preceding claims, wherein the subject is a human.

13. A pharmaceutical composition comprising an effective amount of one or more pyrimidine biosynthesis inhibitor and one or more pyrimidine analogue or its prodrug.

14. A pharmaceutical composition comprising an effective amount of one or more pyrimidine biosynthesis inhibitor and one or more pyrimidine analogue or its prodrug, wherein said one or more pyrimidine biosynthesis inhibitor is a pyrimidine biosynthesis inhibitor as described in any of claims 1 to 13, and wherein said one or more pyrimidine analogue or its prodrug is a pyrimidine analogue or its prodrug as described in any of claims 1 to 13.

15. One or more pyrimidine biosynthesis inhibitor(s) in combination with one or more antiviral pyrimidine analogue(s) or its prodrug(s) for use in a method of treating and/or preventing a viral infection in a subject according to any of the preceding claims, wherein the administration of the therapeutically effective amount of a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue or its prodrug has a synergistic effect in the method of treating/and or preventing said viral infection.