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EP4313150A1 - Combinaison d'inhibiteur de la biosynthèse de la pyrimidine destinée à être utilisée dans le traitement d'infections virales - Google Patents

Combinaison d'inhibiteur de la biosynthèse de la pyrimidine destinée à être utilisée dans le traitement d'infections virales

Info

Publication number
EP4313150A1
EP4313150A1 EP22718647.5A EP22718647A EP4313150A1 EP 4313150 A1 EP4313150 A1 EP 4313150A1 EP 22718647 A EP22718647 A EP 22718647A EP 4313150 A1 EP4313150 A1 EP 4313150A1
Authority
EP
European Patent Office
Prior art keywords
pyrimidine
prodrug
combination
analogue
viral infection
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22718647.5A
Other languages
German (de)
English (en)
Inventor
Matthias Dobbelstein
Antje DICKMANNS
Kim Maren STEGMANN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaetsmedizin Goettingen Georg August Universitaet
Original Assignee
Universitaetsmedizin Goettingen Georg August Universitaet
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitaetsmedizin Goettingen Georg August Universitaet filed Critical Universitaetsmedizin Goettingen Georg August Universitaet
Publication of EP4313150A1 publication Critical patent/EP4313150A1/fr
Pending legal-status Critical Current

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Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • A61K31/195Carboxylic acids, e.g. valproic acid having an amino group
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses

Definitions

  • 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.
  • COVID-19 pandemic has emerged as the most serious health crisis in modem times.
  • 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.
  • a SARS-CoV-2 infected patient experiences pneumonia, acute respiratory syndrome, and even death, frequently due to multiple organ failure.
  • 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.
  • 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.
  • one nucleoside analogue the cytidine analogue ⁇ -D-N 4 -hydroxycytidine (referred to as NHC, EIDD-1931) and its prodrug ⁇ -D-N 4 -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.
  • MHV murine hepatitis virus
  • MERS-CoV Middle East respiratory syndrome CoV
  • SARS-CoV SARS-CoV
  • SARS-CoV-2 SARS-CoV-2
  • 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.
  • 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.
  • NHC 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.
  • Remdesivir 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.
  • 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.
  • glucocorticoid dexamethasone 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.
  • glucocorticoid dexamethasone action does not directly involve interference with virus replication.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • HIV Human Immunodeficiency Virus
  • Ebolavirus a member of the Flaviviridae comprising Hepatitis C virus, a West Nile virus, a Dengue virus, a Yellow fever virus, a Zika virus
  • 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.
  • 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.
  • 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.
  • 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.
  • the pyrimidine analogue used in the disclosed method is selected from N 4 - hydroxycytidine/EIDD-1931, N 4 -hydroxycytidine-5 ’-isopropyl ester EIDD-2801/mulnupiravir MK- 4482, Sofosbuvir, N 4 -aminocytidine, Cidofovir, Stavudine, AZT/azidothymidine/zidovudine, Didanosine/2 ’ ,3 ’ -dideoxyinosine/ddl, Zalcitabine/2 ’ ,3 ’ -dideoxycytidine/ddC, Lamivudine/(-)- ⁇ -L-3 ’ - thia-2 ’ ,3 ’ -dideoxycytidine/3TC, Emtricitabine/(-)- ⁇ -L-3 ’ -thia-2 ’ ,3 ’ -dideoxy-5-flu
  • the pyrimidine analogue used in the disclosed method is a cytidine analogue or its prodrug.
  • the pyrimidine analogue is N 4 -hydroxycytidine/EIDD- 1931 or its prodrug N 4 -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.
  • the pyrimidine synthesis inhibitor is a DHODH inhibitor.
  • 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.
  • the DHODH inhibitor is selected from MEDS433, RP7214, AG- 636, ASLAN003/Farudodstat and PP-001.
  • the DHODH inhibitor is BAY 2402234.
  • the DHODH inhibitor is vidofludimus. In the most preferred embodiment the DHODH inhibitor is IMU-838.
  • the DHODH inhibitor is MEDS433.
  • the DHODH inhibitor is RP7214.
  • the DHODH inhibitor is AG-636.
  • the DHODH inhibitor is PP-001.
  • the DHODH inhibitor is ASLAN003/Farudodstat.
  • the DHODH inhibitor is PTC299.
  • the DHODH inhibitor is Brequinar/DD264.
  • the DHODH inhibitor is teriflunomide.
  • 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.
  • the CTP synthetase inhibitor is cyclopentenyl cytosine (CPEC).
  • 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.
  • the pyrimidine biosynthesis inhibitor is administered together with 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.
  • 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 ⁇ mol.
  • BAY 2402234 may be administered at a dose of about 50 to about 1000 ⁇ mol.
  • Teriflunomide may be administered at a dose of about 5 to about 100 ⁇ mol. More preferably, Teriflunomide is administered at a daily dose of 7 mg or 14 mg.
  • IMU-838 is administered at a dose of about 10 to about 200 ⁇ mol.
  • 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.
  • 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 ⁇ mol to about 200 mmol.
  • N 4 -hydroxycytidine may be administered at a dose of about 10 to about 200 mmol.
  • the N 4 -hydroxycytidine-5’ -isopropyl ester is administered at a dose of about 10 mmol to about 150 mmol.
  • 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.
  • the N 4 -hydroxycytidine-5 ’-isopropyl ester is administered at a dose of about 100 ⁇ mol to about 15 mmol.
  • 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.
  • the N 4 -hydroxycytidine-5’ -isopropyl ester is administered at a daily dose of 200 mg to 800 mg.
  • N 4 -hydroxycytidine-5 ’-isopropyl ester is administered at a daily dose of lower than 800 mg.
  • the effective daily dosing amount may be about 1 ⁇ mol to about 1000 ⁇ mol of the pyrimidine biosynthesis inhibitor and about 1 ⁇ mol to about 200 mmol of the pyrimidine analogue.
  • the viral load is reduced.
  • the subject treated may be mammalian or avian.
  • the subject is a mammal, and more preferably the mammal is a human.
  • the pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N 4 - hydroxycytidine, and wherein the viral infection is caused by SARS-CoV-2.
  • 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 N 4 -hydroxycytidine.
  • 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.
  • 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.
  • the present disclosure provides a pharmaceutical composition
  • 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.
  • 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 ⁇ mol to about 1000 ⁇ mol of the pyrimidine biosynthesis inhibitor and about 1 ⁇ mol to about 200 mmol of the pyrimidine analogue or its prodrug.
  • 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 N 4 -hydroxycytidine/EIDD- 1931, N 4 -hydroxycytidine- 5’-isopropyl ester /EIDD-2801/mulnupiravir/MK-4482, Sofosbuvir, N 4 -aminocytidine, Cidofovir, Stavudine, AZT/a
  • 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.
  • 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 N 4 -hydroxycytidine.
  • 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.
  • the present disclosure provides a composition comprising one or more pyrimidine biosynthesis inhibitor and one or more pyrimidine analogue or its prodrug.
  • 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 N 4 -hydroxycytidine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a pyrimidine analogue e.g., NHC
  • a pyrimidine biosynthesis inhibitor e.g., a DHODH inhibitor
  • 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.
  • NHC and a DHODH inhibitor synergistically suppress SARS- CoV-2 replication.
  • a DHODH inhibitor e.g., IMU-838
  • a lower daily dose than 30 to 45 mg may produce a beneficial therapeutic effect and further reduces the risk of hematuria (as mentioned in W02019/101888).
  • a lower loading dose may be avoided.
  • the recommended dose of 800 mg twice daily can be reduced to minimize the potential risk of mutagenic effects.
  • lower doses are more convenient for the patient and economically more favourable.
  • 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.
  • 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.
  • 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. 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. 1A qRT-PCR results in terms of virus RNA progeny as a percent of SARS-CoV-2 infected Ver
  • 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. 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),
  • 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 %.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the anomeric carbon is linked through a glycosidic bond to the N9 of a purine or the N1 of a pyrimidine.
  • nucleotide generally refers to a substance comprising a nucleobase, a five-carbon sugar, and one or more phosphate groups.
  • 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.
  • 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).
  • 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 N 4 -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.
  • 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.
  • the ester can be a carboxylate ester or derived from inorganic acids like phosphoric acid, sulfuric acid, nitric acid, boric acid or carbonic acid.
  • 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.
  • pyrimidine 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).
  • 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.
  • 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.
  • inhibitor 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 remindPolymorph A“ which is characterized as described in WO 2019/175396.
  • subjectPolymorph 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°.
  • recipientPolymorph A“ of IMU-838 is characterized as outlined in Fig. 1 from WO 2019/175396.
  • 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.
  • centrePolymorph A“ is characterized by an IR absorption spectrum having characteristic peaks expressed in cm -1 at 1980, 1659, 1584, 1335 and 1145.
  • 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.
  • 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.
  • 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.
  • 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.
  • pyrimidine analogues can replace pyrimidines such as cytidine when incorporated into nascent RNA, for instance, the pyrimidine analogue NHC can replace cytidine.
  • NHC can replace cytidine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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%.
  • 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.
  • 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.
  • the pyrimidine biosynthesis inhibitor used in the inventive antiviral combination treatment is an inhibitor of DHODH (a “DHODH inhibitor”).
  • DHODH inhibitor an inhibitor of DHODH
  • 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.
  • the pyrimidine biosynthesis inhibitor used in the inventive antiviral combination treatment is the DHODH inhibitor Brequinar.
  • the pyrimidine biosynthesis inhibitor used in the inventive antiviral combination treatment is an inhibitor of DHODH (a “DHODH inhibitor”).
  • DHODH inhibitor an inhibitor of DHODH
  • 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.
  • the pyrimidine analogue is a cytidine analogue.
  • the pyrimidine analogue is EIDD-1931 (NHC) or EIDD-2801.
  • the pyrimidine analogue is a uridine analogue.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the pyrimidine biosynthesis inhibitor may be administered 10, 20, 30, 40 50 or 60 minutes before the pyrimidine analogue or its prodrug.
  • 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 ⁇ mol to about 1000 ⁇ mol.
  • 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.
  • 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.
  • BAY 2402234 may be administered at a dose of about 50 to about 1000 ⁇ mol, preferably about 200 to about 800 ⁇ mol, more preferably about 200 to about 500 ⁇ mol.
  • teriflunomide may be administered at a dose of about 5 to about 20 mg/day or about 5 to about 100 ⁇ mol preferably at a dose of 14 mg/day or 52 ⁇ mol, most preferably at a dose of about 5 to about 25 ⁇ mol.
  • IMU-838 may be administered at a dose of about 10-200 ⁇ mol, preferably at a dose of up to about 50 mg or at a dose of up to about 140 ⁇ mol.
  • 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 ⁇ mol to about 200 mmol.
  • the dosage depends on the specific analogue used, which would be known to the skilled person.
  • N 4 -hydroxycytidine (EIDD-1931) can be administered at a dose of about 10 to about 200 mmol or about 100-1000 mg/kg/day.
  • a dosage is administered at about 200 to about 500 mg/kg/day orally or about 50 to about 154 mmol per day.
  • N 4 -hydroxycytidine (EIDD-1931) can be administered at a daily dose of about 0.2 to about 10 mmol or about 100-1000 mg/day.
  • a dosage is administered at about 200 to about 500 mg/day orally or about 50 to about 154 mmol per day.
  • N 4 -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.
  • 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.
  • Molnupiravir (EIDD-2801) can be administered twice daily at a dose below 800 mg/dose.
  • a dosage is administered twice daily at about 50 to about 500 mg/dose.
  • 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 ⁇ mol to about 1000 ⁇ mol of the pyrimidine biosynthesis inhibitor and about 1 ⁇ mol to 200 mmol of the pyrimidine analogue or its prodrug.
  • 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.
  • 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 ⁇ -D-N 4 -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.
  • 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.
  • 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 ⁇ -D-N 4 -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.
  • 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 ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD- 1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.
  • an antiviral pyrimidine analogue preferably ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD- 1931)
  • the invention in another embodiment, 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 ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD-1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.
  • 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 ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD-1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.
  • 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 ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD- 1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.
  • the subject is a mammal, preferably human.
  • the invention in another embodiment, 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 ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD-1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.
  • 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 ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD-1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.
  • the invention in another embodiment, 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 ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD-1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.
  • 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 ⁇ -D-N 4 -hydroxycytidine (NHC/ EIDD- 1931), wherein the viral infection is caused by SARS-CoV-2 and wherein the subject is a mammal, preferably human.
  • 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 ⁇ -D-N 4 -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.
  • 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 ⁇ -D-N 4 -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.
  • 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.
  • 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 ⁇ -D-N 4 -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.
  • 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.
  • 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 ⁇ -D-N 4 -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 invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD-2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5’ -isopropyl ester
  • the invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD-2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5’ -isopropyl ester
  • 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 ⁇ -D-N 4 -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.
  • EIDD-2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5’ -isopropyl ester
  • 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 ⁇ -D-N 4 -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.
  • EIDD-2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5’ -isopropyl ester
  • the invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD-2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5’ -isopropyl ester
  • the invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD- 2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5 ’-isopropyl ester
  • the invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD- 2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5 ’ -isopropyl ester
  • the invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD- 2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5 ’ -isopropyl ester
  • the invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD- 2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5 ’-isopropyl ester
  • the invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD- 2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5 ’ -isopropyl ester
  • the invention in another embodiment, 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 ⁇ -D-N 4 -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.
  • EIDD- 2801 antiviral pyrimidine analogue ⁇ -D-N 4 -hydroxycytidine-5 ’ -isopropyl ester
  • the invention in another embodiment, 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-N 4 - 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.
  • ASLAN003/Farudodstat in combination with the antiviral pyrimidine analogue b-D-N 4 - 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 ⁇ -D-N 4 -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 ⁇ -D-N 4 -hydroxycytidine-5 ’ -isopropyl ester or before the ⁇ -D-N 4 -hydroxycytidine-5 ’ -isopropyl ester, preferably concurrently with the ⁇ -D-N 4 -hydroxycytidine-5 ’ -isopropyl ester.
  • DHODH inhibitors have been developed for treating leukemia and additional purposes (e.g. WO2010115736, WO2020144638, WO2020161663, WO2020212897, W02021038490, W02021070132, WO2021084498, W02021084500, WO2021085582, WO2021156787, WO2021238881,
  • 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.
  • DHODH inhibitors may be used for the antiviral combination therapies according to the present invention concurrently with an antiviral pyrimidine analogue or its prodrug:
  • 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
  • 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 present invention relates to methods for treating or preventing a viral infection in a subject comprising administering a combination therapy as described herein.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • coronavirus infection refers to the invasion and multiplication of a virus in the body of a subject.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pyrimidine biosynthesis inhibitor in combination with an antiviral pyrimidine analogue as disclosed herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can comprise a further therapeutic component, for example, a second pyrimidine biosynthesis inhibitor and/or a second antiviral pyrimidine analogue.
  • the further therapeutic agent is selected from Tables 1-4.
  • kits 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.
  • 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.
  • information concerning the respective components in the combination or the respective pharmaceutical compositions and dosage forms in the respective kit or kit of parts aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely.
  • 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.
  • 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.
  • 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).
  • HIV Human Immunodeficiency Virus
  • 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
  • the member of the Coronaviridae virus family is a Betacoronavirus or Alphacoronavirus, preferably a SARS-CoV, MERS-CoV, SARS-CoV-2 or SADS- CoV.
  • 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.
  • the pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N 4 -hydroxycytidine, and wherein the viral infection is caused by SARS-CoV-2.
  • a pharmaceutical composition comprising an effective amount of one or more pyrimidine biosynthesis inhibitor and one or more pyrimidine analogue or its prodrug.
  • composition of embodiment 34 comprising about 1 ⁇ mol to 1000 ⁇ mol of the pyrimidine biosynthesis inhibitor and about 1 ⁇ mol to 200 mmol of the pyrimidine analogue or its prodrug.
  • 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 N 4 - hydroxycytidine/EIDD-1931, N 4 -hydroxycytidine-5 ’-isopropyl ester /EIDD-2801/mulnupiravir/MK- 4482, Sofosbuvir, N 4 -aminocytidine, Cidofovir, Stavudine
  • pyrimidine biosynthesis inhibitor is a DHODH inhibitor selected from IMU-838, IMU-935, BAY 2402234 or teriflunomide, wherein the pyrimidine analogue is N 4 -hydroxycytidine.
  • composition comprising one or more pyrimidine biosynthesis inhibitor and one or more pyrimidine analogue or its prodrug.
  • 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 N 4 -hydroxycytidine.
  • kits 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.
  • 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.
  • 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).
  • HIV Human Immunodeficiency Virus
  • 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
  • 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.
  • 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, FK7
  • CTP cytidine triphosphate
  • CTP cytidine triphosphate
  • CPEC cyclopentenyl cytosine
  • 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).
  • 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.
  • EXAMPLE 2 Dose response studies using EIDD-1931 in combination with BAY 2402234
  • EXAMPLE 3 Dose response studies using EIDD-1931 in combination with teriflunomide
  • 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.
  • 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.
  • 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.
  • 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.
  • Vero E6 cells were treated with the DHOH inhibitor IMU-838 in combination with N 4 -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.
  • FIG. 2D 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.
  • 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.
  • 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 N 4 -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.
  • NHC Madin-Darby Canine Kidney
  • pyrimidine analogue NHC and the DHODH inhibitors also synergistically interfere with Influenza A virus replication.

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Abstract

La présente invention concerne de nouvelles stratégies de traitement comprenant des compositions, des méthodes, des utilisations et des kits, dont un kit de pièces, pour le traitement et/ou la prévention d'une infection virale chez un sujet, par exemple, d'une infection à coronavirus, à l'aide d'une polythérapie médicamenteuse. Spécifiquement, la polythérapie médicamenteuse comprend une quantité thérapeutiquement efficace d'un inhibiteur de la biosynthèse de la pyrimidine en combinaison avec un analogue antiviral de la pyrimidine ou son promédicament.
EP22718647.5A 2021-03-26 2022-03-25 Combinaison d'inhibiteur de la biosynthèse de la pyrimidine destinée à être utilisée dans le traitement d'infections virales Pending EP4313150A1 (fr)

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WO2021240429A1 (fr) 2020-05-29 2021-12-02 Janssen Biotech, Inc. Inhibiteurs de la dihydroorotate déshydrogénase de type benzofurane et benzopyrane
WO2021240424A1 (fr) 2020-05-29 2021-12-02 Janssen Biotech, Inc. Inhibiteurs de la dihydroorotate déshydrogénase de type indazole et benzoisoxazole
WO2021240423A1 (fr) 2020-05-29 2021-12-02 Janssen Biotech, Inc. Inhibiteurs de la dihydroorotate déshydrogénase
JPWO2022025174A1 (fr) 2020-07-30 2022-02-03

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