Treating coronavirus infection

Abstract

The present disclosure relates generally to the fields of virology, infectious diseases, and medicine, and relates to compounds, compositions, methods, and kits for the treatment of CoV-mediated diseases, such as those caused by SARS-CoV-2, SAR8, or MERS. More specifically, the present disclosure relates to effective inhibitors of coronaviruses that can treat coronaviruses, including the 2019 novel coronavirus. In one embodiment, the present disclosure provides a novel use of WX-671 as an effective inhibitor of coronaviruses and its use in the preparation of medicaments for treating coronavirus infections in humans. In one embodiment, the present disclosure provides a novel use of ABC294640 as an effective inhibitor of coronaviruses and its use in the preparation of medicaments for treating coronavirus infections in humans.

Description

[Background technology]

[0001] Coronaviruses are lipid-enveloped, positive-stranded RNA viruses (+ssRNA) that replicate within the cytoplasm of cells. Prior to 2002, coronaviruses were not considered to be important human pathogens. Other human coronaviruses, such as HCoV-229E and HCoV-OC43, caused only mild respiratory infections in healthy adults. However, in 2002, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in Guangdong Province, China. While SARS-CoV primarily affected Southeast Asia, particularly throughout China, Hong Kong, Taiwan, Singapore, and Vietnam, the virus was exported outside of these regions.

[0002] In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was detected in patients with severe respiratory disease in Saudi Arabia. Clinical features of MERS-CoV infection in humans range from asymptomatic to very severe pneumonia, potentially resulting in acute respiratory distress syndrome, septic shock, and multiple organ failure leading to death. Since the first reported cases of MERS-CoV infection and its isolation, considerable progress has been made in understanding the epidemiology, ecology, and biology of the virus. Several assays for detecting acute MERS-CoV infection by real-time reverse transcription (RT)-PCR have been developed and are widely used.

[0003] In 2019, a novel coronavirus (nCoV) emerged worldwide and is now known to cause coronavirus disease 2019 (COVID-19). COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, or SARS-CoV-2), a virus closely phylogenetically related to the SARS virus. The World Health Organization (WHO) declared the 2019-2020 coronavirus pandemic a Public Health Emergency of International Concern (PHEIC). For most patients, coronaviruses primarily cause respiratory disease, meaning COVID-19 begins and ends in the patient's lungs. Summary of the Invention

[0004] The present invention relates generally to the fields of virology, infectious diseases, and medicine. In one embodiment, the present invention provides a novel use of ABC294640, either as a free base or as a salt thereof, in the preparation of a medicament for treating coronavirus infections in humans.

[0005] According to embodiments provided herein, a method for the treatment of 2019 coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus is disclosed, which comprises administering to a person in need thereof an effective amount of ABC294640, i.e.,

[0006] [ka] as its free base or as a salt thereof. In one embodiment, ABC294640 is present as the hydrochloride salt. In one embodiment, ABC294640 is combined with a pharmaceutically acceptable carrier material. In one embodiment, the pharmaceutically acceptable carrier material is physiologically buffered saline. In one embodiment, a suspension is formed comprising ABC294640 hydrochloride suspended in buffered saline, and administering comprises delivering the suspension directly to the stomach using a tube. In one embodiment, ABC294640 and optionally the pharmaceutically acceptable carrier material are in a unit dosage form suitable for oral administration. In one embodiment, the dosage form is a solid dosage form. In one embodiment, the solid dosage form is a capsule. In one embodiment, the SARS-CoV-2 virus is wild-type. In one embodiment, the SARS-CoV-2 virus is a naturally occurring coronavirus variant. In one embodiment, the unit dosage form suitable for oral administration is a capsule having 250 mg of ABC294640 hydrochloride, and administering comprises administering two capsules twice daily for at least 10 days for a total daily dose of 1000 mg of ABC294640 hydrochloride. In one embodiment, administration of an effective amount of ABC294640 results in at least a 10% reduction in viral load.

[0007] According to embodiments presented herein, an effective amount of ABC294640, i.e.

[0008] [ka] A method of treatment is disclosed that includes administering ABC294640, as its free base or salt, to a human having coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus. In one embodiment, ABC294640 is present as the hydrochloride salt. In one embodiment, ABC294640 is combined with a pharmaceutically acceptable carrier material. In one embodiment, the pharmaceutically acceptable carrier material is buffered saline. In one embodiment, a suspension is formed that includes ABC294640 hydrochloride suspended in buffered saline, and administering includes delivering the suspension directly to the stomach using a tube. In one embodiment, ABC294640 and optionally the pharmaceutically acceptable carrier material are in a unit dosage form suitable for oral administration. In one embodiment, the dosage form is a solid dosage form. In one embodiment, the solid dosage form is a capsule. In one embodiment, the SARS-CoV-2 virus is wild-type. In one embodiment, the SARS-CoV-2 virus is a naturally occurring coronavirus variant. In one embodiment, the unit dosage form suitable for oral administration is a capsule having 250 mg of ABC294640 hydrochloride, and administering comprises administering two capsules twice daily for at least 10 days for a total daily dose of 1000 mg of ABC294640 hydrochloride.

[0009] According to an embodiment presented herein, there is provided ABC294640, i.e., as its free base or as a salt thereof, for use in the treatment of coronavirus infections.

[0010] [ka] is disclosed.

[0011] According to embodiments provided herein, there is provided a compound, as its free base or as a salt thereof, comprising ABC294640, i.e.,

[0012] [ka] is disclosed.

[0013] According to aspects provided herein, there is disclosed (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide), as its free base or as a salt thereof, for use in the treatment of coronavirus infection.

[0014] According to embodiments provided herein, (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) is disclosed, as its free base or as a salt thereof, for use in the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0015] According to the embodiments presented herein, there is provided a method for the manufacture of a medicament for the treatment of coronavirus infection, namely ABC294640, i.e.

[0016] [ka] The use of either as its free base or as a salt thereof is disclosed.

[0017] According to the embodiments presented herein, ABC294640, i.e., for the manufacture of a medicament for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus, is used.

[0018] [ka] The use of either as its free base or as a salt thereof is disclosed.

[0019] According to embodiments exemplified herein, there is disclosed the use of (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide), as its free base or as a salt thereof, for the manufacture of a medicament for the treatment of coronavirus infection.

[0020] According to embodiments exemplified herein, there is disclosed the use of the compound (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide), as its free base or as a salt thereof, for the manufacture of a medicament for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0021] According to embodiments presented herein, there is provided a method for treating coronavirus infection using ABC294640, i.e.

[0022] [ka] A pharmaceutical composition comprising:

[0023] According to embodiments provided herein, ABC294640, i.e., for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus, is provided.

[0024] [ka] A pharmaceutical composition comprising:

[0025] According to aspects provided herein, a pharmaceutical composition comprising (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide), as its free base or as a salt thereof, for the treatment of coronavirus infection is disclosed.

[0026] According to embodiments provided herein, a pharmaceutical composition comprising (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide), as its free base or as a salt thereof, is disclosed for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0027] According to the embodiments presented herein, ABC294640, i.e.

[0028] [ka] An anti-coronavirus infection agent is disclosed, comprising:

[0029] According to an embodiment shown in the present specification, an anti-coronavirus infection agent comprising (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) as its free base or as a salt thereof is disclosed.

[0030] According to aspects provided herein, a method for treating a human coronavirus infection is disclosed, the method comprising administering a therapeutically effective amount of ABC294640, (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) or a pharmaceutically acceptable salt thereof to a subject in need thereof. In one embodiment, the method further comprises diagnostically confirming that the subject is infected with a human coronavirus prior to administering ABC294640. In one embodiment, ABC294640 is present as the hydrochloride salt. In one embodiment, the coronavirus infection is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0031] According to aspects provided herein, a method of treating COVID-19 (SARS-CoV-2) coronavirus infection is disclosed, the method comprising administering one or more therapeutically effective doses of ABC294640, (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) or a pharmaceutically acceptable salt thereof, to a subject in need thereof for at least 10 days. In one embodiment, the method further comprises diagnostically confirming that the subject is infected with SARS-CoV-2 prior to administering the compound. In one embodiment, ABC294640 is present as the hydrochloride salt. In one embodiment, the total daily dose of ABC294640 is independently selected for each occurrence from about 250 mg to about 1500 mg.

[0032] According to aspects provided herein, a method for treating CoVID-19 (SARS-CoV-2) coronavirus infection is disclosed, the method comprising administering a pharmaceutically effective amount of ABC294640, (3-(4-chloro-phenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide), or a pharmaceutically acceptable salt thereof, to a human subject in need thereof, wherein ABC294640 has the ability to act on sphingosine kinase 2 (SK2), a host cell factor involved in both intracellular viral replication and downstream inflammatory / immune responses.

[0033] According to aspects provided herein, a method of modulating coronavirus replication in a host cell infected with the coronavirus is disclosed, the method comprising administering to the host cell ABC294640, either as its free base or as a salt thereof, in an amount effective to modulate viral replication.

[0034] According to aspects provided herein, the use of ABC294640, as its free base or a salt thereof, in the preparation of a medicament for treating a coronavirus infection is disclosed. In one embodiment, the coronavirus is 2019 novel coronavirus COVID-19. In one embodiment, the coronavirus infection is coronavirus pneumonia. In one embodiment, ABC294640 is present as the hydrochloride salt. In one embodiment, ABC294640 has activity against sphingosine kinase-2, a host cell factor involved in both intracellular viral replication and downstream inflammatory / immune responses.

[0035] According to an embodiment illustrated herein, the invention features a packaged pharmaceutical product that includes a container, a plurality of ABC294640 unit dosage forms suitable for oral administration within the container, and instructions (e.g., a label or insert) associated with the container that indicate administration of ABC294640 to treat coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0036] In one embodiment, the present invention provides a novel use of WX-671 as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as their free base or as their salts, in the preparation of a medicament for treating coronavirus infection in humans.

[0037] According to embodiments provided herein, a method is disclosed for the treatment of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 virus in a human in need thereof, the method comprising administering an effective amount of WX-671, i.e.

[0038] [ka] as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as their free base, or as a salt thereof. In one embodiment, WX-671 is present as the hydrogen sulfate salt. In one embodiment, WX-671 is combined with a pharmaceutically acceptable carrier material. In one embodiment, WX-671 and optionally a pharmaceutically acceptable carrier material are in a unit dosage form suitable for oral administration. In one embodiment, the dosage form is a solid dosage form. In one embodiment, the solid dosage form is a capsule. In one embodiment, the SARS-CoV-2 virus is wild-type. In one embodiment, the SARS-CoV-2 virus is a naturally occurring coronavirus mutant. In one embodiment, 200 mg of WX-671 is administered to a human in need thereof in a single capsule once daily for at least 10 days, for a total dosage of 200 mg per day. In one embodiment, 400 mg of WX-671 is administered to a person in need thereof in two capsules once daily for at least 10 days for a total dose of 400 mg per day. In one embodiment, about 231 mg of WX-671.1 (upamostat) is administered to a person in need thereof in a single capsule once daily for at least 10 days for a total dose equivalent to 200 mg of its free form per day. In one embodiment, about 463 mg of WX-671.1 (upamostat) is administered to a person in need thereof in two capsules once daily for at least 10 days for a total dose equivalent to 400 mg of its free form per day. In one embodiment, administration of an effective amount of WX-671 reduces viral load by at least 10%.

[0039] According to embodiments presented herein, an effective amount of WX-671, i.e.

[0040] [ka] A method of treatment is disclosed, comprising administering WX-671 as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer, or (E / Z)-mixture, and as their free base, or as a salt thereof, to a human having coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus. In one embodiment, WX-671 is present as the hydrogen sulfate salt. In one embodiment, WX-671 is combined with a pharmaceutically acceptable carrier material. In one embodiment, WX-671 and optionally a pharmaceutically acceptable carrier material are in a unit dosage form suitable for oral administration. In one embodiment, the dosage form is a solid dosage form. In one embodiment, the solid dosage form is a capsule. In one embodiment, the SARS-CoV-2 virus is wild-type. In one embodiment, the SARS-CoV-2 virus is a naturally occurring coronavirus mutant. In one embodiment, WX-671.1 (upamostat) is administered as a single capsule containing 200 mg of free base, which single capsule is administered once daily for at least 10 days to a human in need thereof for a total dose of 200 mg per day. In one embodiment, WX-671.1 (upamostat) is administered as two capsules of 200 mg each, which two capsules are administered once daily for at least 10 days to a human in need thereof for a total dose of 400 mg per day.

[0041] According to embodiments exemplified herein, there is provided WX-671, i.e., as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as a free base or as a salt, for use in the treatment of coronavirus infection.

[0042] [ka] is disclosed.

[0043] According to embodiments exemplified herein, there is provided WX-671, i.e., as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as a free base or as a salt, for use in the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0044] [ka] is disclosed.

[0045] According to aspects provided herein, there is disclosed (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide) as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as the free base or a salt thereof, for use in the treatment of coronavirus infection.

[0046] According to aspects provided herein, (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide) is disclosed as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as the free base or a salt thereof, for use in the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0047] According to embodiments exemplified herein, there is provided a method for the manufacture of a medicament for the treatment of coronavirus infection, comprising administering to a subject a compound selected from the group consisting of WX-671, i.e.

[0048] [ka] as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as their free base, or as their salt.

[0049] According to embodiments exemplified herein, there is provided a method for the manufacture of a medicament for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus, comprising administering to said patient WX-671, i.e.,

[0050] [ka] as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as their free base, or as their salt.

[0051] According to embodiments exemplified herein, the use of (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide), as the (L)-enantiomer or the (D)-enantiomer, and as the E-isomer or the (Z)-isomer or the (E / Z)-mixture, and as the free base or a salt thereof, for the manufacture of a medicament for the treatment of coronavirus infection is disclosed.

[0052] According to aspects provided herein, disclosed is the use of (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide) as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as a free base or a salt thereof, for the manufacture of a medicament for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0053] According to embodiments presented herein, there is provided a method for treating coronavirus infection using WX-671, i.e.

[0054] [ka] as the (L)-enantiomer or the (D)-enantiomer, and as the E-isomer or the (Z)-isomer or an (E / Z)-mixture, and as their free base, or as a salt thereof.

[0055] According to embodiments provided herein, there is provided a method for treating coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus, comprising administering to the patient WX-671, i.e.,

[0056] [ka] as the (L)-enantiomer or the (D)-enantiomer, and as the E-isomer or the (Z)-isomer or an (E / Z)-mixture, and as their free base, or as a salt thereof.

[0057] According to aspects presented herein, a pharmaceutical composition comprising (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide) as the (L)-enantiomer or the (D)-enantiomer, and as the E-isomer or the (Z)-isomer or the (E / Z)-mixture, and as a free base or a salt thereof, for the treatment of coronavirus infection is disclosed.

[0058] According to embodiments exemplified herein, a pharmaceutical composition is disclosed for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus, (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide) as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as the free base or a salt thereof.

[0059] According to the embodiments presented herein, WX-671, i.e.

[0060] [ka] as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as their free base, or as a salt thereof.

[0061] According to an embodiment of the present specification, an anti-coronavirus infection agent is disclosed that contains (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide) as the (L)-enantiomer or the (D)-enantiomer, and as the E-isomer or the (Z)-isomer or an (E / Z)-mixture, and as a free base thereof, or a salt thereof.

[0062] According to aspects set forth herein, a method for treating a human coronavirus infection is disclosed. The method includes administering a therapeutically effective amount of a compound selected from N-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxycarbonylpiperazide hydrochloride or its prodrug N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide to a subject in need thereof. The selected compound may exist as the (L)-enantiomer or the (D)-enantiomer, as the E-isomer or the (Z)-isomer, or as an (E / Z)-mixture, as a free base, or as a salt thereof. In one embodiment, the method further includes diagnostically confirming that the subject is infected with a human coronavirus prior to administering the compound. In one embodiment, the coronavirus infection is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In one embodiment, the compound is N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide and is in an orally administrable form. In one embodiment, the compound is N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide hydrochloride and is in an injectable dosage form delivered intravenously or intramuscularly. In one embodiment, the compound is N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide present as a sulfate or hydrogen sulfate salt.

[0063] In one embodiment, the compound is N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide, which exists in the L conformation. In one embodiment, the compound is N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonylpiperazinium hydrogen sulfate. In one embodiment, the compound is N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide, which should be administered at a dosage of 200 mg per day. In one embodiment, the compound is N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide, which should be administered at a dosage of 400 mg per day.

[0064] According to aspects provided herein, a method of treating COVID-19 (SARS-CoV-2) coronavirus infection is disclosed, the method comprising administering to a subject in need thereof one or more therapeutically effective doses of a compound selected from one of N-α-(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxy-carbonylpiperazide hydrochloride or its prodrug, N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide, for at least 14 days, wherein the selected compound may exist as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as a free base or a salt thereof. In one embodiment, the method further includes diagnostically confirming that the subject is infected with SARS-CoV-2 prior to administering the compound. In one embodiment, the total daily dose of the compound N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazinium is independently selected for each occurrence from about 200 mg to about 400 mg. In one embodiment, the compound is N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonylpiperazinium hydrogen sulfate.

[0065] According to an embodiment exemplified herein, a method of treating COVID-19 (SARS-CoV-2) coronavirus infection is disclosed, the method comprising administering to a human subject in need thereof a therapeutically acceptable amount of a compound selected from one of N-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxy-carbonylpiperazide-hydrochloride or its prodrug N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidinophenylalanine-4-ethoxycarbonylpiperazide, wherein the selected compound can exist as the (L)-enantiomer or the (D)-enantiomer, and as the E-isomer or the (Z)-isomer or an (E / Z)-mixture, and as their free base or a salt thereof, and wherein the compound inhibits hemagglutinin (HA)-activating type II transmembrane serine protease (HA). The compound has the ability to bind hemagglutinin-activating type II transmembrane serine protease (TTSP) and reduce coronavirus replication in a human subject following exposure to the coronavirus. In one embodiment, the TTSP is transmembrane protease serine SI member 2 (TMPRSS2). In one embodiment, the TTSP is transmembrane protease serine 11A (TMPRSS11(A)). In one embodiment, the method further includes diagnostically confirming that the subject is infected with SARS-CoV-2 prior to administering the compound. In one embodiment, the compound is N-α-(2,4,6 triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonylpiperazinium hydrogen sulfate.

[0066] According to embodiments exemplified herein, a method for modulating coronavirus replication in a host cell infected with the coronavirus is disclosed, the method comprising administering to the host cell a compound selected from one of N-α(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxy-carbonylpiperazide hydrochloride or its prodrug N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide, wherein the selected compound can be present as the (L)-enantiomer or the (D)-enantiomer, and as the E-isomer or the (Z)-isomer or the (E / Z)-mixture, and as their free base, or as a salt thereof, in an amount effective to modulate viral replication. In one embodiment, the compound is N-α-(2,4,6 triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)-phenylalanine-4-ethoxycarbonylpiperazinium hydrogen sulfate.

[0067] According to aspects provided herein, the use of WX-671 in the preparation of a medicament for treating a coronavirus infection is disclosed. In one embodiment, the coronavirus is the 2019 novel coronavirus COVID-19. In one embodiment, the coronavirus infection is coronavirus pneumonia. In one embodiment, WX-671 has activity against host serine protease inhibitors and blocks spike protein-driven entry into host cells.

[0068] According to an embodiment illustrated herein, the invention features a packaged pharmaceutical product that includes a container, a plurality of WX-671 unit dosage forms suitable for oral administration therein, and instructions (e.g., a label or insert) associated with the container that indicate administration of WX-671 to treat coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus. [Brief explanation of the drawings]

[0069] Embodiments of the present disclosure are further described with reference to the accompanying drawings. The drawings shown are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of embodiments of the present disclosure. [Figure 1] FIG. 1 depicts the human EpiAirway™ cell culture model, referred to herein as human bronchial epithelial cells (HBEC). [Figure 2] FIG. 10 is a graph showing that a dose-dependent reduction in infectious virus production was observed at pharmacologically relevant concentrations in opaganib-treated HBEC cultures infected with SARS-CoV-2 after 3 days of incubation. [Figure 3] FIG. 10 is a graph showing that in opaganib-treated HBEC cultures infected with SARS-CoV-2, limited cytotoxicity was observed after 3 days of incubation across the dose range where potent antiviral effects were observed. [Figure 4A] This graph shows that a dose-dependent decrease in infectious virus production was observed at pharmacologically relevant concentrations in WX-UK1-treated and upamostat-treated HBEC cultures infected with SARS-CoV-2 after 3 days of incubation. Virus was titered via TCID50 assay on apical lavage. Each symbol represents the titer averaged from three replicates tested. [Figure 4B] This graph shows that a dose-dependent reduction in infectious virus production was observed at pharmacologically relevant concentrations in upamostat-treated HBEC cultures infected with SARS-CoV-2 after 3 days of incubation. Virus was titrated via plaque reduction assay on apical lavage. Each symbol represents the titer averaged from three replicates tested. [Figure 5] FIG. 10 shows that limited cytotoxicity was observed in WX-UK1- and upamostat-treated HBEC cultures infected with SARS-CoV-2 after 3 days of incubation across the dose range where potent antiviral effects were observed. [Figure 6] 1, with fractional rate on the y-axis and WX-UK1 concentration on the x-axis. The graph shows how WX-UK1 inhibits the activity of TMPRSS2. [Figure 7] 1, with fractional rate on the y-axis and WX-UK1 concentration on the x-axis. The graph shows how WX-UK1 inhibits the activity of TMPRSS11A. [Figure 8A] Figure 1 shows a graph showing the inhibition of SARS-2-S-driven entry in Calu-3 cells by upamostat and WX-UK1. Calu-3 cells were preincubated with the indicated concentrations of upamostat, WX-UK1, camostat mesylate, or chloroquine and then inoculated with pseudoparticles carrying VSV-SARS-2S proteins. Pseudotype entry was analyzed by determining luciferase activity in cell lysates. Results are shown for a single experiment performed with quadruplicate samples. Error bars indicate standard deviation (SD). [Figure 8B] Figure 1 shows a graph showing the inhibition of SARS-2S-driven entry in Vero-E6 cells by upamostat and WX-UK1. Vero-E6 cells were preincubated with the indicated concentrations of upamostat, WX-UK1, camostat mesylate, or chloroquine and then inoculated with pseudoparticles carrying VSV-SARS-2S proteins. Pseudotype entry was analyzed by determining luciferase activity in cell lysates. Results are shown for a single experiment performed with quadruplicate samples. Error bars indicate standard deviation (SD). [Figure 9]Figure 1 shows a graph showing the inhibition of VSV-g-driven entry in Calu-3 cells by upamostat and WX-UK1. Calu-3 cells were preincubated with the indicated concentrations of upamostat, WX-UK1, camostat mesylate, or chloroquine, and then inoculated with pseudoparticles carrying VSV-g protein. Pseudotype entry was analyzed by determining luciferase activity in cell lysates. Results are shown for a single experiment performed with quadruplicate samples. Error bars indicate standard deviation (SD). [Figure 10] 1 shows Kaplan-Meier curves (mITT sensitivity) of time no longer receiving supplemental oxygen for at least 24 hours after statistical analysis from a randomized, double-blind, placebo-controlled Phase 2a study of opaganib in COVID-19 pneumonia, as described in Example 7. [Figure 11] 1 shows Kaplan-Meier curves (mITT sensitivity) of the time-cumulative incidence of time to 50% decrease from baseline in supplemental oxygen based on oxygen flow rate in L / min after statistical analysis from the randomized, double-blind, placebo-controlled Phase 2a study of opaganib in COVID-19 pneumonia described in Example 7. [Figure 12] FIG. 1 shows a dot plot of percent change from baseline in total supplemental oxygen requirement (area under the curve) using daily oxygen flow (L / min) measurements over 14 days (Day 1-Day 14) after statistical analysis from a randomized, double-blind, placebo-controlled Phase 2a study of opaganib in COVID-19 pneumonia described in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0070] definition As used herein, the term "drug" refers to a pharmaceutical substance that has pharmacological activity or effect on a patient. The terms "drug," "active ingredient," "drug substance," and "compound" are used interchangeably herein.

[0071] As used herein, the term ABC294640 refers to 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide, either as a free base or salt, or as a stereoisomeric or non-stereoisomeric form. For compounds, salts, prodrugs, or solvates that are solid, those skilled in the art will recognize that the compounds, salts, and solvates of the present invention may exist in different crystalline forms, all of which are intended to be within the scope of the present invention. Opaganib, also known as ABC294640 hydrochloride, is one specific salt form of ABC294640.

[0072] As used herein, the term WX-671 refers to (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide) as the (L)-enantiomer or (D)-enantiomer, and as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as the free base or salt thereof. For compounds, salts, prodrugs, or solvates that are solid, those skilled in the art will understand that the compounds, salts, and solvates of the present invention may exist in different crystalline forms, all of which are intended to be within the scope of the present invention. WX-671.1 (upamostat) is one specific crystalline salt form of WX-671.

[0073] As used herein, the term "coronavirus" includes naturally occurring (e.g., wild-type) coronaviruses, naturally occurring coronavirus mutants, laboratory-created coronavirus mutants, including mutants created by selection, mutants created by chemical modification, and genetically engineered mutants (e.g., coronaviruses modified in the laboratory by recombinant DNA methods). In one embodiment, a subject can be tested for viral infection within several days after symptom onset or treatment with the present disclosure by collecting a nasal secretion (nasal or nasopharyngeal (NP) swab), throat (oropharyngeal) swab, blood, or other bodily fluid sample and testing the sample to detect viral antigens or RNA in the blood and other bodily fluids, for example, using an antigen capture enzyme-linked immunosorbent assay (ELISA), using an IgM ELISA (to determine whether the subject has IgM antibodies), using an IgG ELISA (to determine whether the subject has IgG antibodies), using polymerase chain reaction (PCR), or by virus isolation. In one embodiment, the coronavirus is selected from the group consisting of Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and SARS-CoV-2.

[0074] As used herein, "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates the embodiments presented herein or other embodiments that "comprise," "consist of," and "consist essentially of," whether explicitly stated or not.

[0075] The terms "co-administer," "co-administration," or "in combination" are used to describe the administration of a compound of the present invention in combination with at least one other antiviral active agent. The timing of co-administration is best determined by the medical professional treating the patient. It is sometimes desirable for the agents to be administered simultaneously. Alternatively, the drugs selected for combination therapy may be administered to the patient at different times. Of course, when more than one virus or other infection or other condition is present, the present compound may be combined with other agents to treat the other infections or conditions as needed.

[0076] As used herein, the terms "treatment," "treating," and the like are defined as inhibiting viral activity prior to viral infection or after infection, prior to prophylactic administration of a compound in the methods described herein. In one embodiment, the term "treating" refers to administering one or more compounds of the invention to measurably inhibit viral replication in vitro or in vivo, measurably reduce viral load in vitro or in vivo, or reduce at least one symptom associated with having a CoV-mediated disease in a patient. Desirably, the rate of inhibition of replication or reduction in viral load is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, as determined using a suitable assay. Assays for monitoring viral replication include, but are not limited to, cytopathic virus assays, reporter virus and reporter cell assays, viral replicon assays, and gene-targeted virus assays. Viral load testing can be performed using nucleic acid amplification-based tests (NAT or NAAT) and non-nucleic acid-based tests on plasma samples to determine the amount of virus in a given volume, including viral RNA levels and total viral DNA in plasma and tissues. Alternatively, in certain embodiments, treatment is observed by a trained physician as a significant or substantial alleviation of symptoms in patients with CoV-mediated disease. Typically, a reduction in viral replication is achieved by reducing the rate of RNA polymerization, RNA translation, protein processing, or protein modification, or by reducing the activity of molecules involved in any step of viral replication (e.g., proteins encoded by the viral or host genome that are important for viral replication). In one embodiment, the term "treat" refers to the ability of a compound(s) of the present invention to inhibit or suppress the replication of viruses, such as RNA viruses. In one embodiment, the term "treat" refers to the ability of a compound(s) of the present invention to inhibit the cytopathic effects during RNA viral infection.

[0077] In some embodiments, an "effective amount" or "immunostimulatory amount" of a compound of the invention is an amount sufficient to generate a detectable immune response when administered to a subject. In other embodiments, a "protectively effective amount" of an immunogenic composition is an amount sufficient to confer protective immunity to a subject when administered to a subject. In other embodiments, a "therapeutically effective amount" of a compound is an amount sufficient to treat a viral infection, such as by increasing viral clearance levels, when administered to a subject.

[0078] The agents and methods of the present invention can be used to treat subjects in need thereof. In certain embodiments, the subject is a mammal, such as a human or non-human mammal. When administered to an animal, such as a human, the agent is preferably administered as a pharmaceutical composition, e.g., comprising at least one agent of the present invention together with one or a collection of substances with which the at least one agent can be combined. As used herein, the term "pharmaceutically acceptable carrier material" refers to one or a collection of substances with which an agent can be combined, suitable for use in contact with mammalian tissues for therapeutic treatment in a mammal under anticipated exposure conditions. Pharmaceutically acceptable carrier materials are well known in the art and include, for example, inert solid, semi-solid, or liquid fillers, diluents, and encapsulating materials. Pharmaceutically acceptable carrier materials must, of course, be of sufficiently high purity and sufficiently low toxicity to make them suitable for administration to the human or lower animal being treated. The pharmaceutical compositions may be in unit dosage form such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, powders, syrups, suppositories, injections, and the like.

[0079] The term "immune response" refers to the response of cells of the immune system, such as B cells, T cells, macrophages, or polymorphonuclear cells, to a stimulus such as an antigen. An immune response can include any cell of the body that is involved in a host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, an innate immune response or inflammation. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of a disease associated with infection).

[0080] By "more effective" is meant that a treatment exhibits greater efficacy or is less toxic, safer, more convenient, or less expensive than another treatment to which it is being compared. Efficacy can be measured by a skilled practitioner using any standard method appropriate for a given indication.

[0081] As used herein, the term "suitable period" refers to the period of time that begins when a patient begins treatment using the disclosed methods in response to a diagnosis of coronavirus infection and continues throughout treatment until the patient stops treatment due to either a decrease in symptoms associated with coronavirus infection or a laboratory diagnosis indicating that the viral infection is under control. In one embodiment, the suitable period is 1 week. In one embodiment, the suitable period is 1 to 2 weeks. In one embodiment, the suitable period is 2 weeks. In one embodiment, the suitable period is 2 to 3 weeks. In one embodiment, the suitable period is 3 weeks. In one embodiment, the suitable period is 3 to 4 weeks. In one embodiment, the suitable period is 4 weeks. In one embodiment, the suitable period is 4 to 5 weeks. In one embodiment, the suitable period is 5 weeks. In one embodiment, the suitable period is 5 to 6 weeks. In one embodiment, the suitable period is 6 weeks. In one embodiment, the suitable period is 6 to 7 weeks. In one embodiment, the suitable period is 7 weeks. In one embodiment, the suitable period is 7 to 8 weeks. In one embodiment, a suitable period is 8 weeks.

[0082] As used herein, the term "cytopathic effect" refers to changes in cell morphology due to viral infection.

[0083] As used herein, the term "cellular mutation" or "pathogenesis" includes the inhibition of host cell gene expression and includes other cellular changes that contribute to viral pathogenesis in addition to those visible at the microscopic level.

[0084] As used herein, the term "inhibitor" refers to a molecule that affects the activity of an enzyme. The inhibitors of the present invention are reversible, meaning that they form weak interactions with their target enzymes and are easily removed. Reversible inhibitors form transient interactions with the enzyme. The strength of binding between an enzyme and a reversible inhibitor is determined by the dissociation constant (K d ) is defined by K d The smaller the value of K, the stronger the interaction between the enzyme and the inhibitor, and the greater the inhibitory effect. d is K i It is called.

[0085] As used herein, the term "in vitro" refers to procedures performed in an artificial environment, such as, but not limited to, a test tube or cell culture system. One of skill in the art will understand that, for example, an isolated SK enzyme may be contacted with a modulator in an in vitro environment. Alternatively, an isolated cell may be contacted with a modulator in an in vitro environment.

[0086] As used herein, the term "in vivo" refers to procedures performed within a living organism such as, but not limited to, a human, monkey, mouse, rat, rabbit, cow, horse, pig, dog, cat or primate.

[0087] Detailed Description The present invention relates generally to the fields of virology, infectious diseases, and medicine. The present invention features compounds, compositions, methods, and kits for the treatment of CoV-mediated diseases, such as those caused by SARS-CoV-2, SARS, or MERS. More specifically, the present invention relates to effective inhibitors of coronaviruses, including the 2019 novel coronavirus, that can treat coronaviruses. The present invention provides novel uses for compounds as effective inhibitors of coronaviruses, including the 2019 novel coronavirus, and their use in preparing medicaments for treating coronavirus infections in humans.

[0088] ABC294640, [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], is an orally administered sphingosine kinase 2 ("SphK2" or "SK2") inhibitor. ABC294640 has the following structural formula:

[0089] [ka] and can be prepared as a free base, its salt form, and crystalline modifications. U.S. Patent Nos. 7,338,961, 8,063,248, 8,324,237, and 8,557,800, which are incorporated herein by reference, teach these compounds, methods of use, and methods for making the compounds.

[0090] ABC294640, as the hydrochloride salt, has been given the international nonproprietary name (INN) of opaganib and has the following structural formula:

[0091] [ka]

[0092] The molecular formula of opaganib is C 23 H 25ClNO·HCl and has a molecular mass of 417.4 g / mol. Opaganib is a non-hygroscopic white to off-white solid that is substantially insoluble in water and ethyl acetate. In one embodiment, the medicament is prepared by filling a size 1 hard gelatin capsule with opaganib, which further comprises at least one of the following excipients: microcrystalline cellulose, colloidal silicon dioxide, vegetable magnesium stearate, and titanium dioxide. In one embodiment, the opaganib capsule contains 250 mg of ABC294640 as the hydrochloride salt or 228.16 mg of ABC294640 free base. In one embodiment, the opaganib capsule contains 375 mg of ABC294640 as the hydrochloride salt or 342.24 mg of ABC294640 free base.

[0093] In one embodiment, a 250 mg capsule of opaganib contains the drug ABC294640 as the hydrochloride salt with excipients encapsulated in a size 1, gelatin, white opaque body and cap, Konnisnap capsule. In one embodiment, a 375 mg capsule of opaganib contains the drug ABC294640 as the hydrochloride salt with excipients encapsulated in a size 1, gelatin, white opaque body and cap, Konnisnap capsule.

[0094] Opaganib for treating coronavirus infection is generally administered in an amount ranging from about 250 mg to about 1500 mg per day. In one embodiment, 250 mg of opaganib is administered as two capsules twice daily for a total dose of 1000 mg per day. In one embodiment, 250 mg of opaganib is administered as two 500 mg capsules Q12 hours. In one embodiment, patients with a confirmed coronavirus infection are provided with instructions to take a 500 mg dose of opaganib (as two 250 mg capsules) every 12 hours (resulting in 1000 mg of opaganib per day) for up to a total of two weeks, or up to 14 consecutive days.

[0095] After extensive research, the inventors discovered a novel use for opaganib. Opaganib exhibits antiviral, anti-inflammatory, and antithrombotic activity, thus acting on both the cause and effect of COVID-19. Opaganib targets sphingosine kinase-2, a human cellular component involved in viral replication, but not the virus itself. Accumulating evidence of emerging mutations of novel SARS-CoV-2 strains worldwide highlights the importance of this unique mechanism, which potentially minimizes the risk of viral resistance to treatment.

[0096] Provided herein is a packaged pharmaceutical product, also known as a pharmaceutical kit, comprising a container, a plurality of opaganib dosage forms suitable for oral administration therein, and instructions (e.g., a label or insert) associated with the container indicating administration of opaganib to treat coronavirus infection. In one embodiment, the instructions include instructions for performing the above-described method and / or instructions for using the kit. The instructions included in the kit can be attached to the packaging material as a label or can be included as an insert in the packaging. The instructions are typically handwritten or printed material, but are not limited to such. Any medium capable of storing instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges), optical media (e.g., CD ROMs), and the like. As used herein, the term "instructions" can include the address of an internet site providing the instructions.

[0097] WX-671, i.e., (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazide), is an orally active prodrug of the potent serine protease inhibitor WX-UK1 (N-α-(2,4,6-triisopropylphenylsulfonyl)-3-amidino-phenylalanine-4-ethoxycarbonylpiperazide). WX-671 has the following structural formula:

[0098] [ka] and can be prepared as the (L)-enantiomer or (D)-enantiomer, as the E-isomer or (Z)-isomer or (E / Z)-mixture, and as their free base or as a salt thereof.

[0099] WX-671 is a prodrug. As used herein, a prodrug refers to a pharmaceutical composition containing a biologically inactive compound that is metabolized in vivo to produce the active form of the drug. WX-671 is a compound that can be converted in vivo to yield WX-UK1. WX-UK1 can only be administered by intravenous infusion. WX-UK1 is used in many of the experimental in vitro examples described herein. While this disclosure describes the oral WX-671 compound as a pharmaceutical, it should be understood that pharmaceuticals can be made using the intravenously infused compound WX-UK1, which is within the scope and spirit of the present invention. U.S. Patent Nos. 6,861,435, 7,247,724, 7,659,396, and 9,089,532, incorporated herein by reference, disclose WX-UK1 and methods for its preparation.

[0100] WX-671.1, N-α-(2,4,6-triisopropylphenylsulfonyl)-3-hydroxyamidino-(L)phenylalanine-4-ethoxycarbonylpiperazide hydrogen sulfate, also referred to as ethyl 4-{3-[(E)-amino(hydroxyimino)methyl]-N-[(2,4,6-triisopropylphenyl)sulfonyl]-L-phenylalanyl}piperazine-1-carboxylate hydrogen sulfate, is a compound of formula C 32 H 47It has a molecular formula of NOS × HSO and a molecular weight of 727.91 g / mol (free base: 629.83 g / mol) and is described in U.S. Patent Nos. 6,624,169, 7,211,670, 7,247,724, 7,342,018, 7,608,623, 7,659,396, 7,713,980, 7,745,441, 7,807,681, 7,884,206, 7,951,943, 8,492,385, 8,692,761, and RE 46424, which are incorporated herein by reference. The substance WX-671.1 has been given the International Nonproprietary Name (INN) of upamostat.

[0101] The structural formula of WX-671.1 (upamostat) is as follows:

[0102] [ka]

[0103] Upamostat is a non-hygroscopic white to yellowish powder that is freely soluble in dimethyl sulfoxide and soluble in ethanol. This drug substance is very slightly soluble in water or 0.1 M HCl. Solid preparations for oral administration can be prepared as tablets, pills, powders, granules, capsules, etc. These solid preparations are produced by adding at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, to one or more compounds of the present invention. Furthermore, lubricants, such as magnesium stearate and talc, can be used in addition to typical excipients.

[0104] In one embodiment, the pharmaceutical product is prepared by filling upamostat into a hard gelatin capsule further containing at least one of the following excipients: microcrystalline cellulose, hypromellose, anhydrous ethyl alcohol, purified water, and vegetable magnesium stearate. In one embodiment, the upamostat capsule contains 231.26 mg of upamostat hydrogen sulfate (equivalent to 200 mg of the free base). After oral administration, upamostat is converted to the active WX-UK1, which inhibits several serine proteases. Because upamostat can be provided as an oral formulation, it eliminates the drawbacks associated with intravenous administration of other drugs that may be useful in treating coronavirus infections.

[0105] Upamostat for treating coronavirus infections is generally administered in amounts ranging from about 200 mg to about 1000 mg per day. In one embodiment, upamostat is administered as one capsule once daily at a total daily dose of about 231.26 mg (equivalent to 200 mg of the free base). In one embodiment, upamostat is administered as two capsules once daily at a total daily dose of about 462.52 mg (equivalent to 400 mg of the free base). In one embodiment, patients with a confirmed coronavirus infection are provided with instructions to take one upamostat capsule (equivalent to 200 mg of upamostat free base) daily for a total of two consecutive weeks or 14 consecutive days. In one embodiment, patients with a confirmed coronavirus infection are provided with instructions to take two upamostat capsules (equivalent to 400 mg of upamostat free base) daily for a total of two consecutive weeks or 14 consecutive days.

[0106] After much research, the inventors have discovered a new use for upamostat. Without wishing to be bound by theory, it is believed that the serine protease inhibitor WX-UK1, i.e., the active drug of upamostat once it is broken down in the body, has activity against at least one of the serine proteases that appear to be involved in viral spike (S) protein priming. Therefore, the use of protease inhibitors such as WX-UK1 (or its prodrug WX-671) may be effective in reducing CoV activation and spread, thereby providing effective preventative and therapeutic treatment. Thus, WX-UK1 can block SARS-2-S-driven cell entry, thereby inhibiting coronavirus replication. In one embodiment, because infection requires proteolytic activation, which promotes viral interaction with host cell receptors and enhances infectivity and spread, the upamostat of the present invention, when administered in a therapeutically effective amount for a suitable period of time, protects against coronavirus infection.

[0107] Provided herein is a packaged pharmaceutical product, also known as a pharmaceutical kit, including a container, a plurality of upamostat dosage forms suitable for oral administration therein, and instructions (e.g., a label or insert) associated with the container indicating administration of upamostat to treat coronavirus infection. In one embodiment, the instructions include instructions for performing the above-described method and / or instructions for using the kit. The instructions included in the kit can be attached to the packaging material as a label or included as an insert in the packaging. The instructions are typically handwritten or printed material, but are not limited to such. Any medium capable of storing instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges), optical media (e.g., CD-ROMs), and the like. As used herein, the term "instructions" can include the address of an internet site providing the instructions.

[0108] Combination and Alternating Therapies The compounds described herein can be administered in addition to current standard of care for COVID patients, or in combination or alternation with any other compounds or therapies that a healthcare provider deems beneficial to the patient. Combination and / or alternation therapies can be therapeutic, adjunctive, or palliative. When these methods involve administering two or more active agents to a patient, the agents can be administered within 7, 6, 5, 4, 3, 2, or 1 day; within 24, 12, 6, 5, 4, 3, 2, or 1 hour; within 60, 50, 40, 30, 20, 10, 5, or 1 minute; or substantially simultaneously. The methods of the invention can involve administering one or more agents to a patient by oral, systemic, parenteral, topical, intravenous, inhalation, or intramuscular administration.

[0109] It has been observed that COVID patients may progress through various stages of the disease, and standard treatments may differ based on which stage of the disease a patient is currently experiencing or is about to progress to. COVID is notable in that it causes "crosstalk" between the immune and coagulation systems. As the disease progresses, patients may experience an exaggerated immune response, which can lead to many serious potential consequences, including a cytokine storm. Through crosstalk between the immune and coagulation systems, patients may begin to develop blood clots in various areas of the body, including the respiratory system, brain, heart, and other organs. Multiple blood clots requiring anticoagulation therapy have been observed throughout the bodies of COVID patients. It is believed that these clots may cause long-term damage or even permanent damage if not treated to alleviate the disease.

[0110] More specifically, COVID-19 has been described as progressing through three general stages: Stage 1 (early infection), Stage 2 (pulmonary phase), and Stage 3 (hyperinflammatory phase / cytokine storm).

[0111] Stage 1 is characterized by non-specific, often mild symptoms. Viral replication occurs, and immediate treatment with the compounds described herein, possibly in combination or alternation with another antiviral therapy, is appropriate. Interferon beta may also be administered to enhance the natural immune response to the virus. Thus, in one embodiment, an effective amount of the compound of the present invention is used in combination or alternation with interferon beta and / or additional antiviral drugs. Zinc supplements and / or vitamin C may also be administered from time to time at this stage, or as the disease progresses.

[0112] Stage 2 of COVID-19 is the pulmonary phase, during which patients may experience acute hypoxemic respiratory failure. In fact, the primary organ failure of COVID-19 is hypoxemic respiratory failure. It has been shown that moderate immunosuppression via steroids, such as dexamethasone, can be beneficial for patients with acute hypoxemic respiratory failure and / or patients on mechanical ventilation. In one embodiment, the compound of the present invention is used in an effective amount in combination with a corticosteroid, which may be a glucocorticoid. Non-limiting examples include budesonide (Entocort EC), betamethasone (Celstone), prednisone (Predonisone Intensol), prednisolone (Orapred, Prelone), triamcinolone (Aristospan Intra-Articular, Aristaspan Intraleational, Kenalog), methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol), hydrocortisone, or dexamethasone (Dexamethasone Intensol, Dex Pack 10 Day, Dex Pack 13 Day, Dex Pack 6 Day).

[0113] The NS5B inhibitor remdesivir has had mixed results when given to COVID-19 patients. It can only be administered in a hospital setting and by intravenous injection, typically three times daily, making it unsuitable for patients with mild to moderate COVID-19. In one embodiment, a compound of the invention is administered in combination or alternation with remdesivir to enhance the overall antiviral effect.

[0114] Stage 3, the final stage of the disease, is characterized by progressive disseminated intravascular coagulation (DIC), a condition in which small clots develop throughout the bloodstream. This stage can also include multiple organ failure (e.g., vasodilatory shock, myocarditis). It has also been observed that many patients respond to this severe stage of COVID-19 infection with a "cytokine storm." A bidirectional, synergistic relationship appears to exist between DIC and cytokine storm. To combat DIC, patients often receive anticoagulants, which can be, for example, indirect thrombin inhibitors or direct oral anticoagulants (DOACs). Non-limiting examples include low-molecular-weight heparin, warfarin, bivalirudin (Angiomax), rivaroxaban (Xarelto), dabigatran (Pradaxa), apixaban (Eliquis), or edoxaban (Lixiana). In one embodiment, the compounds of the invention are administered in combination or alternation with anticoagulant therapy. In some severe cases of blood clots in COVID patients, TPA (tissue plasminogen activator) can be administered.

[0115] Elevated levels of the cytokine interleukin-6 (IL-6) have been observed to be a harbinger of respiratory failure and death in COVID-19 patients. To combat this surge in immune response, which can constitute a cytokine storm, patients can be administered IL-6-targeting monoclonal antibodies, pharmaceutical inhibitors, or proteolytic agents, such as bispecific compounds that bind IL-6 and proteins that mediate its degradation. Exemplary antibodies include tocilizumab, sarilumab, siltuximab, olokizumab, and clazakizumab. In one embodiment, a compound of the invention is administered in combination or alternation with tocilizumab or sarilumab. Additional non-limiting examples of immunosuppressants used to treat an over-reactive immune system include Janus kinase inhibitors (tofacitinib (Xeljanz)), calcineurin inhibitors (cyclosporine (Neoral, Sandimmune, SangCya)), tacrolimus (Astagraf XL, Envarsus XR, Prograf)), mTOR inhibitors (sirolimus (Rapamune), everolimus (Afinitor, Zortress)), and IMDH inhibitors (azathioprine (Azasan, Imuran), leflunomide (Arava), mycophenolic acid (CellCept, Myfortic)). Additional antibodies and biologics include abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), ixekizumab (Taltz), natalituzumab (Tysabri), rituximab (Rituxan), secukinumab (Cosentyx), tocilizumab (Actemra), ustekinumab (Stelara), vedolizumab (Entyvio), basiliximab (Simulect), and daclizumab (Zinbryta).

[0116] IL-1 blocks the production of IL-6 and other pro-inflammatory cytokines. COVID patients are also sometimes treated with anti-IL-1 therapy, e.g., intravenous administration of anakinra, to reduce the hyperinflammatory response. Anti-IL-1 therapy can generally be a targeted monoclonal antibody, a pharmaceutical inhibitor, or a protein degrader, e.g., a bispecific compound that binds IL-1 and a protein that mediates degradation.

[0117] Patients with COVID-19 often develop viral pneumonia, which can lead to bacterial pneumonia. Patients with severe COVID-19 may also be affected by sepsis or "septic shock." Treatment for bacterial pneumonia or sepsis secondary to COVID-19 includes the administration of antibiotics, such as macrolide antibiotics, including azithromycin, clarithromycin, erythromycin, or roxithromycin. Additional antibiotics include amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, sulfamethoxazole, trimethoprim, amoxicillin, clavulanate, or levofloxacin. Thus, in one embodiment, a compound of the present invention is administered in combination with or alternating with an antibiotic, such as azithromycin. Some of these antibiotics, such as azithromycin, have independent anti-inflammatory properties. Such drugs can be used as anti-inflammatory agents in COVID-19 patients while also exerting therapeutic effects against secondary bacterial infections.

[0118] A unique challenge in treating patients infected with COVID-19 is the relatively long-term need for sedation when patients require mechanical ventilation, which can last for more than 5, 10, or even 14 days. For ongoing pain during this treatment, analgesics can be added sequentially, and for ongoing anxiety, sedatives can be added sequentially. Non-limiting examples of analgesics include acetaminophen, ketamine, and PRN opioids (hydromorphone, fentanyl, and morphine). Non-limiting examples of sedatives include melatonin, atypical antipsychotics with sedative properties (olanzapine, quetiapine), propofol or dexmedetomidine, haloperidol, and phenobarbital. In one embodiment, a compound of the invention is administered in combination with or alternating with an analgesic such as acetaminophen, ketamine, hydromorphone, fentanyl, or morphine. In one embodiment, a compound of the invention is administered in combination or alternation with a sedative agent such as melatonin, olanzapine, quetiapine, propofol, dexmedetomidine, haloperidol, or phenobarbital.

[0119] Investigational drugs for COVID-19 include chloroquine and hydroxychloroquine. In one embodiment, a compound of the invention is administered in combination or alternation with chloroquine or hydroxychloroquine.

[0120] Protease inhibitors such as lopinavir or ritonavir, previously approved for HIV, may also be administered.

[0121] In one embodiment, opaganib is administered in combination with upamostat for use in treating coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus. In one embodiment, opaganib is administered in combination with upamostat for the manufacture of a medicament for the treatment of coronavirus infection. In one embodiment, opaganib is administered in combination with upamostat for the manufacture of a medicament for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0122] Additional drugs that may be used to treat COVID patients include, but are not limited to, favipiravir, fingolimod (Gilenya), methylprednisolone, bevacizumab (Avastin), Actemra (tocilizumab), umifenovir, losartan, and the monoclonal antibody combination REGN3048 and REGN3051, or ribavirin. Any of these drugs or vaccines can be used in combination or alternation with the active compounds provided herein to treat such predisposing viral infections.

[0123] In one embodiment, a compound of the invention is used in an effective amount in combination with an anti-coronavirus vaccine therapy, including, but not limited to, mRNA-1273 (Moderna, Inc.), AZD-1222 (Astra Zeneca and University of Oxford), BNT162 (Pfizer and BioNtech), CoronaVac (Sinovac), NVX-CoV 2372 (NovoVax), SCB-2019 (Sanofi and GSK), ZyCoV-D (Zydus Cadila), and CoVaxin (Bharat Biotech). In another embodiment, a compound of the invention is used in an effective amount in combination with passive antibody therapy or convalescent plasma therapy.

[0124] In one embodiment, the compounds of the invention are used in effective amounts in combination with a 5-HT receptor antagonist, which can alleviate certain symptoms that may be present in patients infected with coronavirus, such as diarrhea.

[0125] SARS-CoV-2 constantly mutates, which increases its virulence and transmission rate. Drug-resistant variants of the virus can emerge after long-term treatment with antiviral agents. Drug resistance can arise through mutations in genes that encode enzymes used in viral replication. The effectiveness of a drug against RNA virus infections in certain cases can be prolonged, enhanced, or restored by administering the compound in combination or alternation with one, and perhaps two or three, other antiviral compounds that induce mutations different from those of the primary drug or act via different pathways.

[0126] The present invention has multiple aspects, which are illustrated by the following non-limiting examples, which are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the invention in any way. [Example]

[0127] Example 1: Evaluation of the antiviral activity of ABC294640 against SARS-CoV-2 in human airway epithelial cells To evaluate whether opaganib can directly inhibit SARS-CoV-2 infection and spread, we designed an in vitro assay in organotypic air-liquid interface (ALI) cultures of human primary bronchial epithelial cells (HBECs, EpiAirway™, MatTek). This human cell culture model system was selected because it contains a pseudostratified epithelial layer that is morphologically and functionally similar to the human airway. This pseudostratified epithelial layer consists of ciliated cells and goblet (mucus-producing) cells exposed to the air from the apical layer. These cells act as the first line of defense against invading viruses and serve as sites of replication. Available evidence also suggests that human bronchial epithelial cells express the host factor (sphingosine kinase-2) targeted by opaganib.

[0128] Test Compound: Opaganib - Test Compound Description: Opaganib [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide, hydrochloride] is an orally available inhibitor of the enzyme sphingosine kinase-2 (SK2). Solvent: DMSO Remdesivir (GS-5734) - positive antiviral control Description: Remdesivir is a nucleotide analogue antiviral prodrug. It exhibits antiviral activity against many variants of EBOV in cell-based assays, with EC50 values ​​ranging from 0.06 to 0.14 μM, and broad-spectrum antiviral activity in vitro against other pathogenic RNA viruses, including SARS-CoV. Solvent: DMSO 100 mg / mL (166.0 mM) Bleomycin (sulfate) - positive cytotoxicity control Description: Bleomycin is a chemotherapeutic agent commonly used in the treatment of Hodgkin's lymphoma and embryonal carcinoma. Bleomycin-induced widespread pulmonary toxicity is a well-known complication of such treatment, the most common of which is bleomycin-induced interstitial pneumonitis (BIP) (see Sleijfer et al., 2001). Bleomycin (BLM) is selected as the best-studied micronucleus (MN) inducer in human lymphocytes, with distinct mechanisms of genotoxicity. Solvent: DMSO 16.67 mg / mL (11.2 mM) method: Cell culture - differentiated human bronchial epithelial cells (HBEC) Normal human bronchial epithelial (HBEC) cells were differentiated by MatTek Corporation (Ashland, MA) and were obtained in kits with either 12-well or 24-well inserts. HBEC cells were placed in 6 mm tissue cultures within transwell inserts. 2 The cells were grown on mesh discs. Three days before shipping, the tissue was transferred to hydrocortisone-free medium. During shipping, the tissue was stabilized on a sheet of agarose, which was removed upon receipt. One insert contained approximately 1.2 x 10 6The cell insert kit (EpiAirway™ AIR-100) was derived from a single donor, #9831, a 23-year-old, healthy, non-smoking, Caucasian male. The cells have a unique property when forming a layer: their apical side is only exposed to air and produces a mucin layer. Upon arrival, according to the manufacturer's instructions, the cell transwell inserts were immediately transferred to individual wells of a 6-well plate, and 1 mL of MatTek's proprietary culture medium (AIR-100-MM) was added to the basolateral side, while the apical side was exposed to a humidified 5% CO2 environment. Before the start of the experiment, the cells were cultured at 37°C for 1 day. After a 16-18 hour equilibration period, the mucin layer secreted from the apical side of the cells was removed by washing with 400 μL of prewarmed TEER buffer. The culture medium was replenished after the washing step. Figure 1 provides a diagram depicting the culture insert and EpiAirway organization.

[0129] Treatment with test compounds: Test compounds were serially diluted from stock solutions (containing DMSO) in assay medium (AIR-ASY-100, MatTek) and placed at room temperature. The test compound dilution method is outlined below (final DMSO <0.5%). HBEC cultures were washed with phosphate-buffered saline (PBS) and incubated at 37°C for 1 hour prior to infection with remdesivir (2 μM), bleomycin sulfate (75.6 μg / mL and 151 μg / mL), and opaganib (six concentrations ranging from 0.05 to 11.25 μg / mL) diluted in assay medium (AIR-100-ASY, MatTek). Assay medium containing DMSO (final DMSO <0.5%, control) and virus-only controls (assay medium only) were added to control wells 1 hour prior to infection. Compounds were added to each insert in triplicate onto the apical layer (0.15 mL) and basal layer (0.85 mL).

[0130] Viral infection and sample processing: After 1 hour of incubation with compound, the apical side of the cultures was washed and then infected with a SARS-CoV-2 clinical isolate (2019-nCoV / USA-WA1 / 2020) at an MOI of 0.1 PFU / cell for 1 hour at 37°C in the presence of compound or assay control medium. After 1 hour of virus incubation, the virus was removed from the apical side, and the cultures were washed once with PBS to remove any unbound virus. The cultures were then incubated with fresh compound for 72 hours at 37°C. At 24 and 48 hours postinfection, the basolateral medium was replaced with 1 mL of fresh medium containing the respective compound.

[0131] At 72 hours post-infection, tissue and medium were harvested for treatment. The top layer was then diluted with 0.4 mL of TEER buffer (Mg 2+ and Ca 2+ The cells were washed with PBS containing 0.1% ethanol and collected, and the virus titer was assessed via a TCID50 (50% tissue culture infectious dose) assay. Eight-fold serial dilutions of the top layer supernatant sample concentrations were added to a 96-well assay plate containing VeroE6 cells (20,000 cells / well). The plate was incubated at 37°C, 5% CO2, and 95% relative humidity. After 3 days (72 ± 4 hours) of incubation, the plate was stained with crystal violet to measure the cytopathic effect (CPE). The virus titer was calculated using the method of Reed and Muench (Reed et al., 1938). This resulted in a TCID 50 The values ​​were determined from triplicate samples.

[0132] A lactate dehydrogenase (LDH) release assay was performed to assess the health of HBEC cells after exposure to opaganib, control compounds, and viral infection. Medium from the bottom outer layer of tissue culture inserts was removed 72 hours postinfection and diluted in LDH storage buffer according to the manufacturer's instructions (Promega). Samples were further diluted in LDH buffer and incubated with an equal volume of LDH detection reagent. Luminescence was recorded after 60 minutes of incubation at room temperature. A cell-free control was included as a negative control to determine culture medium background, and bleomycin was included as a positive cytotoxicity control. Luminescence was reported, and background levels were found to be within the acceptable luminescence range (range 1,000–10,000).

[0133] Furthermore, the top layer of HBEC tissue was collected by adding Trizol LS (Invitrogen) to each culture insert and pipetting up and down several times to lyse the cells, and then stored at −80°C for future RNA and protein expression analysis.

[0134] result: Opaganib is highly active against SARS-CoV-2 in HBEC cultures In this study, normal human bronchial epithelial cells (HBECs) were pretreated in triplicate with opaganib at six different concentrations (ranging from 11.25 to 0.05 μg / mL) in both the apical and basolateral layers of each culture. After pretreatment, HBECs were exposed to SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) for 1 hour, the apical layer was washed to remove unbound virus, and the cultures were then incubated with the compound for 3 days. Three days after infection, the apical layer was washed and assessed for viral load by TCID50 assay. The basolateral medium was collected and assessed for the presence of lactate dehydrogenase (LDH), which is released from damaged cells and serves as an indicator of cell death / viability.

[0135] Opaganib demonstrated potent antiviral activity, inhibiting viral replication in a dose-dependent manner without significantly impairing cell viability. In opaganib-treated HBEC cultures infected with SARS-CoV-2, a dose-dependent decrease in infectious virus production was observed after 3 days of incubation, with complete inhibition observed at opaganib concentrations of 1 μg / mL (a pharmacologically relevant concentration) and above. These results compare favorably with remdesivir, the positive control in the study. Cell viability was assessed in an LDH release assay. To demonstrate the antiviral activity of opaganib against SARS-CoV-2 in a primary human epithelial culture system, we performed an antiviral assay in HBEC cultures grown at an air-liquid interface, which recapitulates the cellular complexity and physiology of the human conducting airways. In opaganib-treated SARS-CoV-2-infected HBEC cultures, a dose-dependent decrease in infectious virus production was observed after 3 days of incubation, with complete inhibition observed at opaganib concentrations of 1 μg / mL (a pharmacologically relevant concentration) and above (Figure 2). These results compare favorably with remdesivir, the positive control in the study.

[0136] Opaganib did not cause cytotoxicity in HBEC cultures across the concentration range where potent antiviral effects were observed (Figure 3). Collectively, these data demonstrate that opaganib has potent antiviral activity against SARS-CoV-2 in primary human lung cultures without compromising cell membrane integrity, consistent cell viability, and drug safety, further demonstrating the promising potential of opaganib for treating patients with COVID-19.

[0137] Neither 50% inhibition nor 50% cytotoxicity was achieved over the concentration range tested. At the lowest concentration tested, 0.05 μg / mL, greater than 90% inhibition of infectious virus production was achieved. At the highest concentration tested, 11.25 μg / mL, cells remained viable throughout the experiment. Using the high and low concentration ranges from this experiment, we calculated the selectivity index (SI), a ratio that measures the window between cytotoxicity and antiviral activity by dividing the antiviral activity value (AVA) by the toxicity (TOX) value (AVA / TOX). The SI value is 225. If a wider range of concentrations were tested, the SI value would be expected to be larger.

[0138] Example 2: Evaluation of the antiviral activity of upamostat and WX-UK1 against SARS-CoV-2 in human airway epithelial cells To evaluate whether SARS-CoV-2 infection and spread could be directly inhibited by upamostat and WX-UK1, we designed an in vitro assay in organotypic air-liquid interface (ALI) cultures of primary human bronchial epithelial cells (HBECs, EpiAirway™, MatTek). This human cell culture model system was selected because it contains a pseudostratified epithelial layer that is morphologically and functionally similar to the human airway. This pseudostratified epithelial layer consists of ciliated cells and goblet (mucus-producing) cells exposed to the air from the apical layer. These cells act as the first line of defense against invading viruses and serve as sites of replication. Available evidence also suggests that human bronchial epithelial cells express host factors targeted by upamostat (e.g., TMPRSS2).

[0139] Test Compound: Upamostat - Test Compound Description: Upamostat ethyl 4-{3-[(E)-amino(hydroxyimino)methyl]-N-[(2,4,6-triisopropylphenyl)sulfonyl]-L-phenylalanyl}-piperazine-1-carboxylate hydrogen sulfate. Solvent: DMSO WX-UK1-Test Compound Description: WX-UK1 Ethyl 4-[(25)-3-(3-carbamimidoylphenyl)-2-[(2,4,6-triisopropylphenyl)sulfonylamino]propanoyl]piperazine-1-carboxylate. Solvent: DMSO Camostat mesilate - test compound Description: Camostat mesylate (CM) 4-[[4-[(aminoiminomethyl)amino]benzoyl]oxy]benzeneacetic acid 2-(dimethylamino)-2-oxoethyl ester methanesulfonate, FOY 305, FOY-S 980, foipan mesylate. Camostat is a synthetic, orally bioavailable serine protease.

[0140] Solvent: DMSO Bleomycin (sulfate) - positive cytotoxicity control Description: Bleomycin is a chemotherapy agent commonly used in the treatment of Hodgkin's lymphoma and embryonal carcinoma. Bleomycin-induced widespread pulmonary toxicity is a well-known complication of such treatment, the most common of which is bleomycin-induced interstitial pneumonia (BIP) (see Sleijfer et al., 2001). Bleomycin (BLM) is selected as the best-studied micronucleus (MN) inducer in human lymphocytes, with distinct mechanisms of genotoxicity. Solvent: DMSO 16.67 mg / mL (11.2 mM)

[0141] method: Cell culture - differentiated human bronchial epithelial cells (HBEC) Normal human bronchial epithelial (HBEC) cells were differentiated by MatTek Corporation (Ashland, MA) and were obtained in kits with either 12-well or 24-well inserts. HBEC cells were placed in 6 mm tissue cultures within transwell inserts. 2The cells were grown on mesh discs. Three days before shipping, the tissue was transferred to hydrocortisone-free medium. During shipping, the tissue was stabilized on a sheet of agarose, which was removed upon receipt. One insert contained approximately 1.2 x 10 6 The cell insert kit (EpiAirway™ AIR-100) was derived from a single donor, #9831, a 23-year-old, healthy, non-smoking, Caucasian male. The cells have a unique property when forming a layer: their apical side is only exposed to air and produces a mucin layer. Upon arrival, according to the manufacturer's instructions, the cell transwell inserts were immediately transferred to individual wells of a 6-well plate, and 1 mL of MatTek's proprietary culture medium (AIR-100-MM) was added to the basolateral side, while the apical side was exposed to a humidified 5% CO2 environment. Before the start of the experiment, the cells were cultured at 37°C for 1 day. After a 16-18 hour equilibration period, the mucin layer secreted from the apical side of the cells was removed by washing with 400 μL of prewarmed TEER buffer. The culture medium was replenished after the washing step. Figure 1 provides a diagram depicting the culture insert and EpiAirway organization.

[0142] Treatment with test compounds: Test compounds were serially diluted from stock solutions (containing DMSO) in assay medium (AIR-ASY-100, MatTek) and placed at room temperature. The test compound dilution method is outlined below (final DMSO should be less than 0.5%). HBEC cultures were washed with phosphate-buffered saline (PBS) and incubated with bleomycin sulfate (75.6 and 151 μg / mL), upamostat (six concentrations ranging from 0.12 to 30.00 μg / mL), WX-UK1 (3.33, 10, and 30.00 μg / mL), or camostat (0.5, 5, and 25 μg / mL) diluted in assay medium (AIR-100-ASY, MatTek) for 1 h at 37°C prior to infection. For control wells, assay medium containing DMSO (final DMSO less than 0.5%, control) and virus-only control (assay medium only) were added 1 hour before infection. Compounds were added to each insert in triplicate onto the apical layer (0.15 mL) and basal layer (0.85 mL).

[0143] Viral infection and sample processing: After 1 hour of incubation with compound, the apical side of the cultures was washed and then infected with a SARS-CoV-2 clinical isolate (2019-nCoV / USA-WA1 / 2020) at an MOI of 0.1 PFU / cell for 1 hour at 37°C in the presence of compound or assay control medium. After 1 hour of virus incubation, the virus was removed from the apical side, and the cultures were washed once with PBS to remove any unbound virus. The cultures were then incubated with fresh compound for 72 hours at 37°C. At 24 and 48 hours postinfection, the basolateral medium was replaced with 1 mL of fresh medium containing the respective compound.

[0144] At 72 hours post-infection, tissue and medium were harvested for treatment. The top layer was then diluted with 0.4 mL of TEER buffer (Mg 2+ and Ca 2+The cells were washed with PBS containing 0.1% ethanol and collected, and the virus titer was assessed via TCID50 (50% tissue culture infectious dose) assay. Eight-fold serial dilutions of the top layer supernatant sample concentrations were added to 96-well assay plates containing VeroE6 cells (20,000 cells / well). The plates were incubated at 37°C, 5% CO2, and 95% relative humidity. After incubation for 3 days (72 ± 4 hours), the plates were stained with crystal violet to measure cytopathic effect (CPE). The virus titer was calculated using the method of Reed and Muench (Reed et al., 1938). TCID 50 Values ​​were determined from triplicate samples. Plaque reduction assays were performed to confirm the results of the TCID50 assay. Briefly, 10-fold serial dilutions of the top layer supernatant sample concentrations were added to 24-well assay plates (100,000 cells / well) containing VeroE6 cells for the plaque reduction assay. The plates were incubated at 37°C, 5% CO2, and 95% relative humidity. After 3 days (72 ± 4 hours) of incubation, the plates were fixed with 5% neutral buffered formalin and stained with crystal violet to visualize plaques. Titers were calculated in PFU / mL using the following formula: Titer (PFU / mL) = Number of plaques × 10 希釈回数 x 10 (to get numbers in mL since 100 μL of diluted sample was added). Assays were performed in duplicate, with a second assay performed with virus + DMSO and 0.2 μg / mL upamostat to evaluate additional sample dilutions.

[0145] A lactate dehydrogenase (LDH) release assay was performed to assess the health of HBEC cells after exposure to opaganib, control compounds, and viral infection. Medium from the bottom outer layer of tissue culture inserts was removed 72 hours postinfection and diluted in LDH storage buffer according to the manufacturer's instructions (Promega). Samples were further diluted in LDH buffer and incubated with an equal volume of LDH detection reagent. Luminescence was recorded after 60 minutes of incubation at room temperature. A cell-free control was included as a negative control to determine culture medium background, and bleomycin was included as a positive cytotoxicity control. Luminescence was reported, and background levels were found to be within the acceptable luminescence range (range 1,000–10,000).

[0146] Furthermore, the top layer of HBEC tissue was collected by adding Trizol LS (Invitrogen) to each culture insert and pipetting up and down several times to lyse the cells, and then stored at −80°C for future RNA and protein expression analysis.

[0147] result: Upamostat and WX-UK1 are highly potent antiviral inhibitors of SARS-CoV-2 in human bronchial epithelial tissue cultures. In this study, normal human bronchial epithelial cells (HBECs) were pretreated in triplicate with six different concentrations of upamostat (ranging from 0.12 to 30.0 μg / mL) and three different concentrations of WX-UK1 (ranging from 3.33 to 30.0 μg / mL) in both the apical and basolateral regions of each culture. After pretreatment, HBECs were exposed to SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) and incubated with the compounds for three days. Three days after infection, the apical layer was washed and assessed for viral load by TCID50 assay. The basolateral medium was collected and assessed for the presence of lactate dehydrogenase (LDH), which is released from damaged cells and serves as an indicator of cell death / viability. For comparison, three concentrations of camostat (ranging from 0.5 to 25.0 μg / mL), an established TMPRSS2 inhibitor, were included.

[0148] Both upamostat and WX-UK1 exhibited potent antiviral activity, inhibiting replication in a dose-dependent manner (except at the highest dose of each compound) without significantly impairing cell viability. Reductions in viral load by three and four orders of magnitude, measured by TCID50, were observed at the lowest concentrations of upamostat (0.12 μg / mL) and WX-UK1 (3.33 μg / mL), respectively. Both upamostat and WX-UK1 induced similar reductions in viral titers at 3 days postinfection. Cell viability, assessed in an LDH release assay, was reported to be completely unimpaired except at the highest concentrations evaluated for upamostat and WX-UK1.

[0149] To demonstrate the antiviral activity of upamostat and WX-UK1 against SARS-CoV-2 in a human primary epithelial culture system, we performed antiviral assays in HBEC cultures grown at an air-liquid interface, which recapitulate the cellular complexity and physiology of the human conducting airways. After 3 days of incubation, a dose-dependent reduction in infectious virus production (confirmed by TCID50 and plaque reduction assays) was observed at pharmacologically relevant concentrations in upamostat- and WX-UK1-treated HBEC cultures infected with SARS-CoV-2 (Figures 4A and 4B). These results compare favorably with those of camostat, a known TMPRSS2 inhibitor.

[0150] An EC50 estimate was calculated from the plaque reduction assay results. The highest concentration tested inhibited virus production by more than 50%. GraphPad was used to estimate the EC50 from the available data. The estimated EC50 was 0.02 μg / mL. The following formula was used to calculate the % inhibition after converting TCID50 values ​​to estimated PFU values, as described below: % Inhibition = ((value - mean virus control value) / (mean cell control value - mean virus control value) * 100) The % inhibition values ​​were then analyzed via GraphPad according to the instructions below. The -X value is the upamostat concentration. The -Y value is the response. -Select "Dose vs. Response Curve." Select Analysis, Nonlinear Regression, and the Dose vs. Response (Stimulus) set of equations, and select Variable Slope for Dose vs. Response. All other default settings were accepted. The resulting EC50 was calculated to be 0.02 μg / mL.

[0151] Viral replication was inhibited by upamostat, WX-UK1, and camostat (except at the highest concentration tested) without significantly impairing cell viability, as measured by LDH release. To measure LDH release from nonviable cells, medium from the bottom outer layer of tissue culture inserts was removed 72 hours postinfection and diluted with LDH storage buffer according to the manufacturer's instructions (Promega). Samples were further diluted in LDH buffer and incubated with an equal volume of LDH detection reagent. Luminescence was recorded after 60 minutes of incubation at room temperature. A cell-free control was included as a negative control to determine culture medium background, and bleomycin (151 μg / mL) was included as a positive cytotoxicity control. Data are plotted using luminescence values ​​minus the cell-free control values ​​(mean luminescence reading of 6936). Using a physiologically relevant human respiratory tissue model, this data demonstrates upamostat's potential to strongly inhibit SARS-CoV-2 viral replication with limited cytotoxicity in HBEC cultures across the dose range where potent antiviral effects are observed (Figure 5), further demonstrating its promising potential for treating patients with COVID-19. A 50% cytotoxic concentration (CC50) for cell cultures was generated with available data to determine the compound concentration required to reduce absorbance by 50% in treated cells compared to control cells. Using luminescence data generated via the MTT assay, the calculated CC50 value for upamostat was 46.37 μM (or 29.2 μg / mL). At this CC50 concentration, an EC50 concentration less than 4.6 μM (or 2.9 μg / mL) would result in an SI value (CC50 / EC50) greater than 10.

[0152] Example 3: Evaluation of the in vivo efficacy of ABC294640 against ARDS-induced thrombosis This study evaluated the efficacy of ABC294640 in reducing the incidence of thromboembolic adverse events in situ in acute respiratory distress syndrome (ARDS) conditions using a rat venous congestion model. This assay was designed to measure thrombotic risk after LPS-induced lung injury. LPS-induced lung injury is one of the most commonly used rodent models of ARDS and has been described to mimic the neutrophilic inflammatory response observed in ARDS patients. The mechanism of LPS-induced ARDS is based on endothelial cell damage and a systemic inflammatory response.

[0153] The venous stasis (Wessler) test in animals has been widely used for over 40 years as a laboratory measure of in vivo coagulability and has proven invaluable in assessing the thrombogenicity of various blood products.

[0154] At 3 hours, 24, 48, and 72 hours post-injection, the test compound was administered by oral gavage at a dose of 250 mg / kg. An appropriate amount of LPS from E. coli (O55:B5) was diluted with saline to a final concentration of 400 μg / mL. This solution was administered by intratracheal instillation (0.5 mL / kg). The vehicle consisted of PBS pH 7.4 ± 0.1. ABC294640 was weighed and transferred into the vehicle (PBS, 0.375%, pH 7.4) to obtain a final solution of 25 mg / mL. The ABC294640 solution was stirred at room temperature for 10 minutes before administration. This solution was administered by oral gavage (250 mg / kg, 10 mL / kg).

[0155] Male Sprague-Dawley rats weighing 275-400 g were used in this study. Animals were randomly assigned to treatment groups by the study director. Food and water were available ad libitum. Observations of behavior and general health were conducted until sacrifice. Body weights were recorded before injection and at 24, 48, and 72 hours after injection.

[0156] Arterial oxygen saturation (SpO2) and heart rate were recorded before and 24, 48, and 72 hours after infusion using a mouse pulse oximeter color probe (MouseOx Plus system, Starr Life Sciences) attached to conscious mice. Rats were also introduced into a plethysmograph chamber environment on the same schedule as for SpO2. Functional respiratory parameters were assessed using a whole-body plethysmograph (VivoFlow, SCIREQ). Analyzed functional respiratory parameters included respiratory rate, PenH (pulmonary congestion index), and inspiratory / expiratory time measurements. Blood samples were also collected before the terminal procedure for complete blood counts and cytokine level assessment.

[0157] In this study, ARDS was induced by intratracheal instillation of LPS. Throughout the ARDS induction and development process, animals were administered vehicle or ABC294640 by oral gavage (at 3, 24, 48, and 72 hours after instillation). Following 72 hours of conscious measurement, rats were anesthetized, and venous ischemia was performed on the inferior vena cava (4 hours after the 72-hour oral gavage). Ischemia was maintained for 30 minutes. The segment was then excised, and its contents were scored. Subsequently, the animals were euthanized by exsanguination.

[0158] After exsanguination, a tracheotomy was performed, the chest cavity was opened, and the lungs were exposed. The trachea was then connected to the cannula of the perfusion system. A solution of cold 1x PBS and 1x protease inhibitor was infused through the trachea while the left lung was clamped. Bronchoalveolar lavage fluid (BALF) was then performed on the right lobe of the lung and collected for further analysis. Total cell counts, along with differential cell counts, and total protein content in the BALF samples were assessed. An aliquot of BALF was retained for quantification of chemokine / cytokine levels in the BALF.

[0159] The left lobe of the lung was excised. The freshly harvested left lobe was weighed wet to determine the left lung weight and left lung index (left lung weight / body weight × 100). The lower part of the left lobe was used to determine the left lung wet / dry ratio, an index of pulmonary edema. The remaining portion of the left lung was homogenized and aliquoted for quantification of protein content.

[0160] Induction of lung injury by LPS: 1. Prior to LPS or saline infusion, functional respiratory parameters in all rats were assessed by whole-body plethysmography, and SpO2 was assessed in conscious rats fitted with a color-probe pulse oximeter. Rats were first acclimated to the plethysmograph chamber before physiological assessment. Respiratory rate, PenH, and inspiratory / expiratory time measurements were analyzed. 2. Rats were anesthetized with 2.5% isoflurane USP in oxygen (Abbot Laboratories, Montreal, Canada). Rats were then intubated and LPS or saline was delivered via intratracheal instillation. 3. The rats were allowed to recover from anesthesia and returned to their cages. 4. Three hours after injection, the first dose of ABC294640 was administered by oral gavage (see Table 1 below).

[0161] [Table 1] 5. Rats were assessed regularly to ensure animal welfare (general behavior and daily body weights). 6. Animals were also dosed by oral gavage at 24, 48, and 72 hours post-injection. 7. SpO2 and whole body plethysmography were also assessed at 24, 48, and 72 hours post-infusion. 8. Prior to the venous arrest procedure, blood samples were taken from the jugular vein for measurement of complete blood counts and cytokine levels. 9. Four hours after the last dose, rats were anesthetized with 2.5% isoflurane USP in oxygen (Abbot Laboratories, Montreal, Canada). The procedure was performed on a homeothermic blanket to control body temperature. 10. The rat's inferior vena cava was exposed and two loose sutures were placed 1 cm apart. Any collateral vessels in the isolated segment were ligated. 11. Hemostasis was maintained in situ for 30 minutes. 12. The venous stasis segment was removed, opened longitudinally, emptied onto filter paper, and photographed. Any existing thrombus was removed and dried on filter paper. The thrombus was measured, weighed, and scored on a scale of 0 to 4 (see Table 2).

[0162] [Table 2] Following venous hemostasis, the animals were euthanized by exsanguination, and bronchoalveolar lavage fluid (BALF) was collected from the right lung. To do so, the muscles above the trachea were incised before a tracheotomy was performed. The thoracic cavity was opened to expose the lungs, and the trachea was connected to the cannula of the perfusion system. The left lung was clamped while 15 mL (3 × 5 mL) of cold 1x PBS and 1x protease inhibitor solution was instilled through the trachea, and bronchoalveolar lavage fluid (BALF) was collected from the right lobe of the lung. BALF was collected for further analysis. The total cell count in the BALF sample was assessed, along with differential cell counts. An aliquot of BALF was retained for quantification of chemokine / cytokine levels in the BALF. 14. The left lung was then harvested and weighed for left lung weight and left lung index calculations. Lung tissue edema was assessed using wet / dry ratio calculations. The lower part of the left lung was weighed separately (wet weight) and used to determine the lung wet / dry ratio. After drying at 60°C for at least 24 hours, it was reweighed (dry weight).

[0163] Each parameter (listed below) was compiled for each group and presented in a bar graph with appropriate statistical analysis. 1. Weight change 2- Saturation (SpO2) and heart rate (bpm) 3-Respiratory parameters: Inhalation / exhalation time Tidal volume and expiratory volume breathing rate PenH 4. BALF total cell count and cell differential 5. BALF cytokine / chemokine levels 6-Left lung weight and index 7- Lung wet / dry ratio 8- Total lung protein content in lung homogenate

[0164] LPS induces a significant increase in lung weight, which is associated with inflammation and lethality. This increase is associated with severe edema, as indicated by a significant increase in the wet / dry ratio. Lung weight increase was greater in the LPS-vehicle group compared with the LPS-vehicle group at 4 hours post-gavage at 72 hours. ABC294640 administered at 250 mg / kg demonstrated a reduction in thrombosis, as evidenced by a reduction in clot length, weight, and total thrombus score.

[0165] Example 4: Evaluation of TMPRSS2 and TMPRSS11(A) as targets for WX-UK1 inhibition Several enzymes belonging to different protease families can be hijacked by CoV S protein for priming. The pH-dependent cysteine ​​proteases cathepsin L, TMPRSS2, and TMPRSS11A, as well as the serine protease furin, can prime the S protein during viral entry into target cells. To determine whether any of the TTSPs are relevant targets for inhibition by upamostat, we performed analyses including structural modeling / prediction, structural analysis, and a review of relevant literature.

[0166] Mammalian expression systems for TMPRSS2 and TMPRS11A were purchased from MyBioSource (MBS1193731 and MBS1345824, respectively). Proteins were reconstituted to 1 mg / mL according to the manufacturer's instructions and run on gels with the reconstituted proteins. A fresh stock solution of WX-UK1 (100 mM WX-UK1 in 100% DMSO) was made. The concentrated stock was diluted to 1 mM in HBS buffer before further dilution in the assay.

[0167] Enzyme inhibitors can interact with enzymes and / or enzyme-substrate complexes in several different ways, slowing the rate of enzyme-catalyzed reactions. For each mode of inhibition, the inhibitor's dissociation constant, Ki, can be calculated, which reflects the strength of the interaction between the enzyme and the inhibitor. The Ki of an inhibitor is similar to the Km of the substrate. A small Ki value reflects tight binding of the inhibitor to the enzyme, while a larger Ki value reflects weaker binding. The exact formula used to calculate Ki depends on the mode of inhibition, which is the V for the enzyme in the presence of the inhibitor. max The "apparent" values ​​and Km of V in the absence of any inhibitors max and Km values ​​(Equation 2 below).

[0168] The chromogenic substrate chosen for these studies was the S-2288 substrate. i The K value was determined by measuring the effect of WX-UK1 on human serine protease cleavage of a chromogenic substrate. i For the determination of K values, a range of concentrations of WX-UK1 were preincubated with the target human serine protease before initiating the reaction by adding a chromogenic substrate. Reaction rates were determined from the slope using linear regression and normalized to that of the uninhibited reaction. Normalized activity was plotted against WX-UK1 concentration, and then the K was calculated. i Values ​​were obtained by nonlinear regression using Equation 1.

[0169]

number

[0170] K M Parameters were obtained using the standard Michaelis-Menten kinetic equation. Serine proteases were added to a series of suitable concentrations of substrate high enough to obtain experimental Vmax values. The ensuing reaction rates were plotted against the substrate concentration, and KM values ​​were derived using the Michaelis-Menten equation (2).

[0171]

number

[0172] All experiments were performed in HBS (30 mM Hepes, pH = 7.4; 150 mM NaCl; 0.5% BSA) at 37 °C in at least triplicate. Reactions were monitored at 405 nm at 2 readings / min for at least 45 min. Because WX-UK1 was stored in 100% DMSO, an uninhibited DMSO-control was included in all experiments to rule out any unwanted DMSO effects on protease activity.

[0173] Inhibition of human TMPRSS2 by WX-UK1 Figure 6 shows the fitted curve of Equation 1, with fractional rate on the y-axis and WX-UK1 concentration on the x-axis. The graph shows how WX-UK1 inhibits the activity of TMPRSS2. This results in a K i was determined to be 2.9±0.04(3) μM.

[0174] Inhibition of human TMPRSS11a by WX-UK1 Figure 7 shows the fitted curve of Equation 1, with fractional rate on the y-axis and WX-UK1 concentration on the x-axis. The graph shows how WX-UK1 inhibits the activity of TMPRSS11A. This results in a K i was determined to be 0.39±0.01(3) μM.

[0175] Table 3 lists the inhibition constants, Ki values, of WX-UK1 against a panel of proteases.

[0176] [Table 3]

[0177] Example 5: Evaluation of the effects of upamostat and WX-UK1 on SARS-CoV-2 spike protein-mediated entry Studies were conducted to evaluate the inhibitory effects of both upamostat and WX-UK1 on the cell entry of replication-defective, single-cycle vesicular stomatitis virus (VSV) particles pseudotyped with the SARS-CoV-2 spike protein (VSVpp + SARS-2-S Δ18) or the glycoprotein of vesicular stomatitis virus (VSV) as a control. Δ18 refers to the deletion of 18 C-terminal amino acids in the S protein, which increases pseudotyping efficiency without affecting ACE2 or TMPRSS2 usage. Pseudotype entry and its inhibition were evaluated in Calu-3 and Vero-E6 cells. Calu-3 cells, a lung-derived human carcinoma cell line, allow S-driven entry of SARS-CoV-2 in a TMPRSS2-dependent manner. Agents that inhibit TMPRSS2, including camostat (Hoffmann et al., 2020), a known TMPRSS2 inhibitor shown to inhibit SARS-CoV-2 infection of cultured lung cells, are predicted to inhibit S-driven entry in this model. Vero cells, a green monkey kidney cell line, allow SARS-CoV-2 spike-driven entry in a TMPRSS2-independent but cathepsin L-dependent manner. Agents that increase the acidic intracellular endosomal pH, including chloroquine, are predicted to inhibit entry in this model. Entry driven by the G protein of vesicular stomatitis virus (VSV) served as a specificity control (VSV-G-driven entry is dependent on low pH and is therefore sensitive to chloroquine but not camostat).

[0178] method: For pseudotyping, vesicular stomatitis virus pseudotypes (VSVpp) were generated according to published protocols (Berger Rentsch and Zimmer, 2011). Briefly, 293T cells transfected to express the viral surface glycoprotein under study contained the VSV-G open reading frame, VSV * Instead of DG-fLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhausen, Switzerland), a replication-deficient VSV vector containing an expression cassette for enhanced green fluorescent protein (eGFP) and firefly luciferase was inoculated. After a 1-h incubation period at 37°C, the inoculum was removed, the cells were washed with PBS, and then medium supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant, CRL-2700, from ATCC) was added to neutralize residual input virus (no antibody was added to cells expressing VSV-G). Pseudotyped particles were harvested 16 h postinoculation, cleared from cell debris by centrifugation, and used in experiments.

[0179] For transduction, target cells were grown in 96-well plates until they reached 50%–75% confluency before inoculation with the respective pseudotypes. In experiments involving protease inhibitors, target cells were treated with the respective chemicals 2 h prior to transduction. Transduction efficiency was quantified 16 h posttransduction by measuring firefly luciferase activity in cell lysates using a commercially available substrate (Beetle-Juice, PA) and a Hidex Sense plate luminometer (Hidex). The transduction assay measures entry of a single-cycle vesicular stomatitis virus (VSV) carrying the SARS-CoV-2 spike.

[0180] result: The ability of upamostat and WX-UK1 to inhibit entry of pseudotypes bearing SARS-CoV-S and VSV-G was evaluated in Calu-3 (human lung cancer cells) and Vero-E6 cells. Calu-3 cells, a lung-derived human cancer cell line, allow SARS-CoV-2 spike-driven entry in a TMPRSS2-dependent manner and thus camostat-sensitive. Vero cells, a kidney cell line derived from African green monkeys, allow SARS-CoV-2 spike-driven entry in a cathepsin L-dependent and chloroquine-sensitive manner. Entry driven by the G protein of vesicular stomatitis virus (VSV) served as a specificity control (VSV-G-driven entry is dependent on low pH and therefore chloroquine-sensitive, but not camostat-sensitive).

[0181] Upamostat and WX-UK1 inhibit SARS-CoV-2 S protein-mediated entry in human lung cancer cells (Calu-3) and green monkey kidney cells (VeroE6) with moderate efficiency. When tested against VSVpp + SARS-2-SΔ18 in Calu-3 cells, both WX-UK1 and upamostat showed moderate inhibitory activity, although this was lower than that of camostat, another serine protease inhibitor (Figure 8A). Moderate inhibitory activity was still observed for upamostat and WX-UK1 when tested in Vero-E6 cells, which lack surface TMPRSS2. Camostat was inactive in this context, whereas the highest concentration of chloroquine potently inhibited S protein-driven entry (Figure 8B). WX-UK1 and upamostat moderately inhibited VSV-G entry in Calu-3 cells, suggesting a broader range of activity for upamostat (Figure 9). All three compounds, except chloroquine, were inactive when tested against VSV-G in Vero-76 cells. Overall, these results indicate that WX-UK1 and upamostat inhibit SARS-CoV-2 spike-driven entry into Calu-3 and Vero cells with moderate efficiency. Due to the nature of the model, specific estimation of actual in vitro or in vivo inhibitory concentrations is not possible.

[0182] Example 6: Treatment of COVID-19 with pneumonia with opaganib Patients diagnosed with COVID-19 infection, who developed pneumonia and did not require mechanical ventilation or who were mechanically ventilated for 24 hours or less were evaluated in this inpatient study.

[0183] Main purpose: 1) To evaluate the safety and tolerability of opaganib administered at 500 mg Q12 hours in patients hospitalized with COVID-19 infection. To assess viral shedding in response to opaganib treatment.

[0184] Secondary / exploratory may include one or more of the following: 1) To assess vital signs in patients hospitalized with COVID-19 infection and receiving opaganib treatment. 2) To evaluate clinical improvement in patients hospitalized with COVID-19 infection who are treated with opaganib. 3) To assess the need for mechanical ventilation in patients who were not mechanically ventilated at baseline if treated with opaganib. 4) To assess improvement of hypoxia via SpO2 / FiO2 or PaO2 / FiO2 ratios and SpO2 on room air. Return to room air or to a specified SpO2 oxygen saturation on room air.

[0185] Assessment of changes in viral load on treatment, and changes in D-dimer, cardiac troponin, LDH, and ferritin levels.

[0186] research design The study included one active treatment arm. All eligible patients hospitalized with COVID-19 pneumonia who did not require mechanical ventilation or had been on mechanical ventilation for less than 24 hours received open-label opaganib at 500 mg twice daily over 12 hours. Patients entered a screening period of up to 1 week. Eligible patients entered a treatment period of up to 2 weeks. All participants were followed for 2 weeks after receiving their last dose of study drug at the end of the 2-week treatment period, after two consecutive daily viral swab tests for COVID-19 virus were negative before day 14, or after early discontinuation of drug administration before day 14. The maximum duration of study participation was 35 days (7 weeks). Study participants received opaganib daily, two 250 mg capsules (500 mg) q12 h, for a maximum total of 14 days (2 weeks) or until two consecutive daily nasopharyngeal viral swab tests were negative for COVID-19, whichever occurred first. Opaganib was administered with food (after a light to moderate meal) and followed by 240 mL (8 fluid ounces) of water. If patients were only able to receive opaganib via nasogastric tube, the capsule contents were suspended in 20 cc of normal saline solution and pushed through the tube, followed by a thorough flush with sterile water. If patients were tube-fed, opaganib was administered shortly (approximately 15–30 minutes) after the tube meal.

[0187] Key participation criteria: 1. Adult male or female, aged 18 to 75 years old. 2. Proven COVID-19 infection and pneumonia, but not requiring mechanical ventilation or requiring mechanical ventilation for 24 hours or less at the time of informed consent. 3. The patient, guardian, or legally acceptable representative has signed an IRB-approved informed consent form.

[0188] Main exclusion criteria: 1. New York Heart Association Class III or IV heart disease, myocardial infarction within the past 6 months, unstable arrhythmia, or evidence of ischemia on ECG 2. Any comorbid condition that, in the investigator's judgment, may add to the risk of treatment. 3. Pregnant (serologically positive) or breastfeeding women 4. Unwillingness or inability to comply with the procedures required by this protocol. 5. AST (SGOT) or ALT (SGPT) is greater than 2.5 x the upper limit of normal (ULN) 6. Bilirubin level greater than 1.5 × ULN (unless the elevated bilirubin is due to Gilbert's syndrome) 7. Serum creatinine >2.0 × ULN 8. Absolute neutrophil count is 1000 / mm 3 Less than 9. Platelet count is 75,000 / mm 3 Less than 10. Hemoglobin level is less than 8.0 g / dL 11.Currently taking warfarin, apixaban, argatroban, or rivaroxaban 12.Currently engaging in drug or alcohol abuse

[0189] Research evaluation: The following will be monitored daily (see Table 4): ·Concomitant medication review Adverse events Physical inspection Vital signs (temperature, blood pressure, heart rate, respiratory rate, and oxygen saturation by pulse oximeter) Clinical symptoms (cough, difficulty breathing, nausea, vomiting, diarrhea) Nasopharyngeal virus swab Serum chemistry CBC with differential ·Chest X-ray Urine tests

[0190] [Table 4] 1 On day 14 or earlier if two consecutive daily viral swab tests are negative for SARS-CoV-2. 2 Temperature, blood pressure, heart rate, respiratory rate, and oxygen saturation by pulse oximetry will be assessed. 3 Assess for cough, dyspnea, nausea, vomiting, and diarrhea. 4 Women of childbearing potential. A negative serum pregnancy test must be obtained within 3 days prior to randomization.

[0191] Dosage forms and modes of administration: Opaganib was supplied as 250 mg capsules containing 250 mg of opaganib along with excipients in white, opaque, hard gelatin capsules.

[0192] Opaganib was administered orally (or via nasogastric tube, if appropriate) as two capsules (500 mg) every 12 hours for up to 2 weeks, with each dose administered with a meal or 15–30 minutes after a meal via tube, if appropriate.

[0193] Study endpoints: Primary Safety Endpoint: 1) Adverse events, laboratory tests, physical examination, and vital signs 2) The proportion of patients who achieved two consecutive negative nasopharyngeal viral swabs by day 14 of opaganib treatment

[0194] Secondary / Exploratory Endpoints: 1) Percentage of patients showing improvement in vital signs (based on improvement in one or more of the following: temperature, heart rate, respiratory rate, or oxygen saturation) 2) Percentage of patients showing clinical improvement (based on improvement in one or more of the following symptoms: cough, dyspnea, nausea, vomiting, diarrhea) Percentage of patients who were not mechanically ventilated at baseline who do not require mechanical ventilation by the end of 2 weeks of study drug-free follow-up

[0195] result: Results were obtained from seven patients approved for special use. These patients had moderate to severe COVID-19-associated pneumonia with hypoxia who were provided with supplemental oxygen. Patients received opaganib plus standard of care, including hydroxychloroquine (HCQ) as background therapy in six of the seven patients. As can be seen in Table 5 below, with the exception of patient #7, who was only treated for 1 day due to diarrhea that may or may not have been related to opaganib (he was also given HCQ and azithromycin), 6 moderate to severe patients improved significantly, 5 patients returned to breathing room air, and 3 patients were discharged. All six patients showed a reduction in C-reactive protein (CRP), and all six patients showed measurable clinical improvement, including reduced use of supplemental oxygen and higher lymphocyte counts. All patients were started on 250 mg opaganib administered 12 hours each day for the first 3 days, then increased to 500 mg opaganib administered 12 hours each day. Although only six patients were included, these preliminary findings demonstrate clinical improvement in the first COVID-19 patients treated with opaganib and provide preliminary support for the tolerability of opaganib use in patients with COVID-19.

[0196] [Table 5] * Improvement within one day of starting treatment, and return to room air without blood testing. RA-room air, D / C-discharge, SOB-shortness of breath

[0197] Five patients were included in the analysis, and a control group with patients of the same gender and severity (baseline characteristics) was used for comparison. Univariate comparisons between groups were performed using chi-square tests for categorical variables and t-tests or Mann-Whitney U tests, as appropriate, for continuous variables. Time variables were compared using Cox proportional hazards regression adjusted for age and background diseases. Changes in CRP and lymphocyte counts were compared using a general linear model with repeated measures with Bonferroni correction for multiple comparisons.

[0198] Patients treated with opaganib had a significantly faster increase in lymphocyte counts. All other clinical outcomes, while not statistically significant, tended to favor the treatment group: the mean time to discontinuation of high-flow nasal cannula (HFNC) was 10 days in treated patients versus 15 days in controls (HR = 0.3, 95% CI: 0.07-1.7, p = 0.2); time to return to ambient air was 13 days versus 14.5 days (HR = 0.4, 95% CI: 0.15-1.5); and no treated patients required mechanical ventilation, compared with 33% of controls. In this small group of severely ill COVID-19 patients, opaganib was safe and well tolerated, with improvements in both clinical and laboratory parameters in all treated patients. The efficacy of opaganib for COVID-19 infection should be further tested in randomized, placebo-controlled trials.

[0199] Example 7: Randomized, Double-Blind, Placebo-Controlled, Phase 2a Study of Opaganib in COVID-19 Pneumonia Main purpose: To assess total oxygen requirement (area under the curve) using daily supplemental oxygen flow rates (L / min) over a 14-day period (days 1-14). Secondary Objectives: 1) To assess the time taken to achieve a 50% decrease from baseline in supplemental oxygen based on oxygen flow rate in L / min. 2) To assess the proportion of patients who no longer require supplemental oxygen for at least 24 hours by day 14. 3) To assess the proportion of afebrile patients on day 14. 4) To evaluate the time to negative swab test results for SARS-CoV-2 by PCR. 5) To assess the proportion of patients whose swab tests were negative for SARS-CoV-2 by PCR by day 14. 6) Evaluate the need for intubation and mechanical ventilation up to day 14. 7) Assess the time to need for mechanical ventilation. 8) To assess the proportion of patients with at least one measurement of fever at baseline (defined as a temperature greater than 38.0°C [100.4°F]) who are afebrile (defined as a temperature less than 37.2°C [99°F]) at day 14. 9) To assess 30-day mortality after reaching baseline.

[0200] Exploratory purpose: To assess changes in systemic markers of inflammation (D-dimer, cardiac troponin, C-reactive protein [CRP], lactate dehydrogenase [LDH], and ferritin).

[0201] Safety Objectives: To evaluate the safety and tolerability of opaganib administered orally at a dose of 500 mg Q12 hours for up to 14 days in patients with COVID-19 pneumonia.

[0202] Study population: The study population consisted of patients diagnosed with COVID-19 infection who developed pneumonia, defined as radiopacities on chest x-ray, and required supplemental oxygen. Patients must be hospitalized and at baseline status at least during screening (Day 1).

[0203] Study design and description: This was a phase 2a, proof-of-concept, multicenter, randomized, double-blind, parallel-arm, placebo-controlled study. After informed consent was obtained, patients entered a 3-day or less screening phase to determine eligibility. Forty-two eligible patients were randomized to receive opaganib added to standard of care or placebo added to standard of care in a 1:1 randomization ratio. Treatment assignment remained blinded to patients, investigators, and hospital staff, as well as the sponsor. Because there is no consensus for a definitive treatment specifically targeting the SARS-CoV-2 virus that causes COVID-19 (Wilson, 2020), standard of care referred to therapies implemented during the COVID-19 pandemic, whether locally, at large centers, or by individual physicians.

[0204] Study participants received either two 250 mg capsules of opaganib (500 mg) or a matching placebo every 12 hours in addition to standard care (pharmacological and / or supportive care). The study drug was to be administered daily for 14 days (Days 1-14) unless the patient was discharged from the hospital without requiring supplemental oxygen (in which case the study drug was administered only on Day 10).

[0205] All participants were followed for 4 weeks after the last dose of study drug, which could occur at the end of the 2-week double-blind treatment phase or after early discontinuation, based on patient or physician decision. The maximum duration of study participation was up to 45 days (including up to 3 days of screening, the 2-week double-blind treatment phase, and 4 weeks of study drug-free follow-up).

[0206] Stratification: Patients were stratified based on a minimization algorithm that considered the following three parameters: age at screening, ≥70 years (yes or no); HbA1c at screening, ≥6.5 (yes or no); oxygen requirement at baseline, requiring noninvasive positive pressure ventilation (e.g., via BIPAP, CPAP) (yes or no).

[0207] Eligibility Criteria: Participation: 1. Adult male or female, aged 18 to 80 years old 2. COVID-19 infection confirmed by RT-PCR assay of a throat sample (nasopharyngeal or oropharyngeal) and pneumonia defined as radiopaque chest X-ray. 3. Patient requires supplemental oxygen at baseline 4. The patient, guardian, or legal representative has signed an IRB-approved informed consent form.

[0208] Exclude: 1. Any comorbid condition that, in the investigator's judgment, may add to the risk of treatment. 2. Requires intubation and mechanical ventilation 3. Oxygen saturation is 95% or higher on room air 4. Any pre-existing respiratory condition requiring intermittent or continuous ambulatory oxygen prior to admission 5. Patient is unlikely to survive beyond 72 hours in the clinical judgment of the investigator 6. Pregnant (serum test positive within 3 days prior to randomization) or breastfeeding women 7. Unwillingness or inability to comply with the procedures required by this protocol. 8. A corrected QT (QTc) interval on the electrocardiogram (ECG), calculated using the Friedericia formula (QTcF), of greater than 470 ms for women and greater than 450 ms for men 9.AST (SGOT) or ALT (SGPT) is greater than 2.5 x the upper limit of normal (ULN) 10. Bilirubin level greater than 1.5 × ULN (unless elevated bilirubin is due to Gilbert's syndrome) 11. Serum creatinine greater than 2.0 × ULN 12. Absolute neutrophil count is 1000 / mm 3 Less than 13. Platelet count is 75,000 / mm 3 Less than 14. Hemoglobin level less than 8.0 g / dL 15. Currently taking a drug that is a sensitive CYP3A4, CYP2C9, or CYP2C19 substrate and has a narrow therapeutic index 16. Currently taking drugs that are strong inducers or inhibitors of CYP2D6 and CYP3A4 17.Currently taking warfarin, apixaban, argatroban, or rivaroxaban 18.Currently engaging in drug or alcohol abuse 19. Currently participating in clinical studies evaluating pharmacological treatments, including antiviral studies

[0209] Number of targets: A total of 49 patients were screened in the study, of which 42 were randomized (23 assigned to opaganib and 19 to placebo), with 7 screening failures. Two patients were randomized in each group but were not treated. Nineteen opaganib patients and 16 placebo patients completed treatment (day 14). Three opaganib patients and two placebo patients discontinued treatment early. Two patients in the opaganib group experienced adverse events that led to termination of study drug administration, and one placebo patient discontinued due to an adverse event.

[0210] Screening / Baseline Assessment: Signed informed consent Eligibility determination Complete medical history (including onset of COVID-19 symptoms) -Evaluation of combination drugs System baseline review Physical examination Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation by pulse oximeter) If the patient is ambulatory, weight Oxygen requirement (L / min) 12-lead electrocardiogram ·Chest X-ray Nasopharyngeal or oropharyngeal swab for PCR testing of SARS-CoV-2 Serum chemistry CRP, D-dimer, LDH, ferritin, cardiac troponin HbA1c CBC with differential Urine tests A serum pregnancy test within 3 days prior to treatment (for women of childbearing potential)

[0211] Research evaluation: The following were monitored and recorded daily as part of the standard of care: Concomitant medications Adverse events Intermediate physical examination Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation by pulse oximeter) Oxygen requirement (L / min)

[0212] The following are preferably monitored less frequently as part of standard care: For patients receiving concomitant hydroxychloroquine, a 12-lead ECG (if permitted by hospital treatment guidelines during COVID-19) is required approximately 3 hours after the first study drug dose on Day 1, any time on Days 2 and 4, and at the end of treatment (either Day 10, Day 14, or early discontinuation of study drug). If the patient is on a monitor (including remote or Holter monitor), investigators are encouraged to collect QT interval duration data. Nasopharyngeal or oropharyngeal viral swab for SARS-CoV-2 PCR testing every 1-3 days Weekly serum chemistry Serum CRP, D-dimer, LDH, ferritin, and cardiac troponin were measured weekly. · CBC with differential once a week Chest X-ray by physician decision

[0213] Study endpoints: major Total oxygen requirement (area under the curve) using daily supplemental oxygen flow rates (L / min) over 14 days (days 1–14).

[0214] secondary 1) Time taken to achieve a 50% decrease from baseline in supplemental oxygen based on oxygen flow rate in L / min. 2) By day 14, the percentage of patients no longer receiving supplemental oxygen for at least 24 hours. 3) Time until two consecutive swab tests, at least 24 hours apart, are negative for SARS-CoV-2 by PCR. 4) The percentage of patients with at least two consecutive negative swab test results who subsequently remain negative for SARS-CoV-2 by PCR on day 14. 5) Percentage of patients requiring intubation and mechanical ventilation by the end of 2 weeks of study drug-free follow-up 6) Time to intubation or mechanical ventilation 7) Percentage of patients with at least one measurement of fever at baseline (defined as a temperature greater than 38.0°C [100.4°F]) who are afebrile (defined as a temperature less than 37.2°C [99°F]) on day 14. 8) Mortality rate: death from any cause at 30 days.

[0215] exploratory 1) Mean change in systemic markers of inflammation (D-dimer, cardiac tronin, C-reactive protein [CRP], procalcitonin [PCT], lactate dehydrogenase [LDH], and ferritin) from baseline on day 14

[0216] safety 1) Incidence of all treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs) 2) Assessment of vital signs 3) Evaluation of laboratory parameters (chemistry and hematology parameters) 4) Electrocardiogram (ECG) evaluation

[0217] Statistical methods: The primary efficacy objective of the study was to evaluate the effect of opaganib on total supplemental oxygen requirement (area under the curve) using daily oxygen flow (L / min) measurements over 14 days (Days 1–14). The primary efficacy endpoint was calculated for each patient using the trapezoidal rule to calculate the area under the curve for supplemental oxygen requirement through Day 14 after subtracting the baseline oxygen requirement for each day. Days on which supplemental oxygen was not required were recorded as 0. If several values ​​for oxygen requirement (L / min) were recorded for a given day, the highest of these values ​​was taken for the primary analysis. In the primary analysis, for patients who died before Day 14 or required intubation and mechanical ventilation, missing daily values ​​were assigned a maximum supplemental oxygen requirement flow rate of 8 L / min. For patients discharged from the hospital before Day 14, if values ​​were not collected by the site after discharge, the supplemental oxygen requirement (L / min) on the day of discharge was assigned for each day thereafter until Day 14.

[0218] The primary analysis was based on the modified intent-to-treat (mITT) population, consisting of all patients randomized and treated with at least one dose of study drug (the population included 40 subjects, 22 in the opaganib group and 18 in the placebo group). Descriptive statistics for baseline-adjusted AUC are presented by group, along with the mean difference between groups, along with 95% confidence intervals. Supplemental oxygen requirements were collected up to day 14, even if patients discontinued treatment before day 14 but continued in the study until day 14. Furthermore, loss to follow-up, such as missing vital signs up to day 14, was assumed to be unlikely. Therefore, in the primary analysis, if all supplemental oxygen values ​​were missing after treatment discontinuation, the last value was assumed to be carried forward until day 14, or until death if death occurred before that date. Sensitivity analyses to the above missing data handling approaches were performed using the AUC summary statistics approach, in which group AUC was calculated from the estimated parameters of the repeated measures model.

[0219] Within the mITT cohort, two subjects withdrew their consent due to grade 1 gastrointestinal adverse events. Additionally, one subject did not require any supplemental oxygen at baseline before starting treatment, but was excluded from some analyses as required by the statistical analysis plan (SAP). Therefore, the post-hoc activity analysis population ("mITT sensitivity") excluded these three patients from the analysis, resulting in 37 subjects, 19 of whom received opaganib and 18 received placebo. Results from both the mITT population (including these three subjects) and the mITT sensitivity population showed similar activity trends.

[0220] result: Topline results from the study found opaganib to be safe, with no significant safety differences between the opaganib and placebo arms. Overall, fewer patients in the opaganib arm experienced serious adverse events (SAEs) than in the placebo arm. In this small sample size, there were few intubation or fatal events, and these were balanced between the two arms.

[0221] The opaganib-treated arm demonstrated a consistent trend of greater improvement in reduction of oxygen requirements by end of treatment on day 14 across key primary and secondary efficacy outcomes correlated with clinical improvement as defined by the World Health Organization (WHO) ordinal scale.

[0222] There was a greater improvement versus the control group in the proportion of patients who reached room air and no longer required oxygen support by day 14 (52.6% vs. 22.2%). Figure 10 shows the Kaplan-Meier curve (mITT sensitivity) for the time it takes to no longer receive supplemental oxygen for at least 24 hours.

[0223] There was a significant improvement over the control arm in the proportion of patients achieving a 50% reduction in supplemental oxygen by day 14 (89.5% vs. 66.7%). Figure 11 shows the Kaplan-Meier curve (mITT sensitivity) of the cumulative incidence of time to a 50% reduction in supplemental oxygen from baseline based on oxygen flow rate in L / min.

[0224] Relative to the control group, a higher proportion of patients were discharged by day 14 (73.7% vs. 55.6%).

[0225] There was a greater decrease from baseline in mean total oxygen requirement (AUC) over 14 days (68.0% vs. 46.7%). Figure 12 shows a dot plot of the percent change from baseline in total supplemental oxygen requirement (area under the curve) using daily oxygen flow (L / min) measurements over 14 days (Days 1-14).

[0226] Example 8: Randomized, Double-Blind, Placebo-Controlled International Phase 2 / 3 Study - Opaganib in COVID-19 Pneumonia Main purpose: The proportion of patients requiring intubation and mechanical ventilation by day 14 will be assessed.

[0227] Secondary Objectives: 1) To assess change based on the WHO ordinal scale of clinical improvement. 2) Evaluate the time to intubation or mechanical ventilation. 3) If high oxygen flow is not an available option, assess the time taken to transition from high oxygen flow via, for example, nasal cannula or CPAP to low oxygen flow via nasal cannula. 4) To assess the proportion of patients who no longer require supplemental oxygen for at least 24 hours by day 14. 5) To assess total oxygen requirement (area under the curve) using daily supplemental oxygen flow rates (L / min) over a 14-day period (days 1-14). 6) To assess the time to two consecutive negative swab tests for SARS-CoV-2 by PCR. 7) To assess the proportion of patients with two consecutive swab tests negative for SARS-CoV-2 by PCR on day 14. 8) To assess the proportion of patients with at least one measurement of fever at baseline (defined as a temperature greater than 38.0°C [100.4°F]) who are afebrile (defined as a temperature less than 37.2°C [99°F]) at day 14. 9) To assess 30-day mortality after reaching baseline.

[0228] Exploratory purpose: To assess changes in systemic markers of inflammation (D-dimer, cardiac troponin, C-reactive protein [CRP], lactate dehydrogenase [LDH], and ferritin) over the 14-day treatment period.

[0229] Safety Objectives: To evaluate the safety and tolerability of opaganib administered orally at a dose of 500 mg Q12 hours for up to 14 days in patients with severe COVID-19 pneumonia.

[0230] Study population: The study population will consist of patients diagnosed with COVID-19 infection, defined as severe based on eligibility criteria to align with current region-specific diagnostic guidance. Specifically, if high-flow oxygen is not an available option, patients must have, at a minimum, pneumonia incident to SARS-CoV-2, radiographic evidence of pneumonia on a chest x-ray or CT scan, and require supplemental oxygen with high-flow oxygen via nasal cannula or CPAP. Patients must be hospitalized at least during screening and at baseline (Day 1).

[0231] Study design and description: This is a phase 2 / 3, multicenter, randomized, double-blind, parallel-arm, placebo-controlled study with an adaptive design utilizing futility assessment. The study will be conducted at up to approximately 40 clinical sites in Italy, other EU countries, Russia, Brazil, Mexico, and the United States.

[0232] After informed consent is obtained, patients will enter a screening phase of no more than 3 days to determine eligibility. Approximately 270 eligible patients will be randomized to receive opaganib added to standard of care or placebo added to standard of care in a 1:1 randomization ratio. Treatment assignment will remain blinded to patients, investigators, hospital staff, and the sponsor. As approvals and / or guidance for treating COVID-19 evolve, for this protocol, standard of care will be defined by treatment recommendation schemes according to disease severity based on local diagnostic and guideline documents, such as the Temporary Methods Recommendation: Prevention, Diagnosis, and Treatment of Novel Coronavirus Infection (COVID-19) from the EU Commission, European Medicines Agency (EMA), Heads of Medicines Agency (HMA), and FDA, and as updated with the latest version of the recommendations.

[0233] Study participants will receive either 2 x 250 mg capsules (500 mg) of opaganib or matching placebo every 12 hours at any given site, in addition to standard of care (pharmacological and / or supportive care as defined above). Study drug will be administered daily for 14 days (Days 1-14). All participants will be followed for 28 days after the last dose of study drug, which may occur on Day 14, or after early discontinuation based on patient or physician decision.

[0234] Randomization strategy: The standard of care administered to patients may also vary between sites, as treatments in the recommended treatment scheme according to disease severity may differ based on local diagnostic and guideline documents, such as the Temporary Method Recommendations: Prevention, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19) from the EU Commission, the European Medicines Agency (EMA), the Heads of Medicines Agency (HMA), and the FDA. Randomization will be determined at the individual site level to ensure a balance of standard treatment regimens in both treatment arms.

[0235] Preliminary fitness analysis of futility: An open-label futility interim analysis will be performed when approximately 100 subjects (approximately 50 subjects from each group) will be evaluated for the primary endpoint to determine the probability of rejecting the null hypothesis of no effect and to determine whether it is futility to continue the study. Criteria will be determined and documented prospectively in the final version of the Statistical Analysis Plan (SAP) prior to the interim analysis.

[0236] Data Safety Monitoring Board: To ensure the safety of trial participants, a data safety monitoring board (DSMB) will meet to monitor the safety of the study. DSMB meetings to review safety data are scheduled after 25%, 50%, and 75%, or approximately 70, 135, and 200 randomized patients have reached Day 7 and Day 14, respectively. The DSMB will also be responsible for communicating the results of the futility analysis (futility / non-futility) to the sponsor, which will be performed by an independent, unblinded statistician.

[0237] Stratification: Patients will be stratified based on meeting three or more high-risk clinical parameters for COVID-19 outcomes at baseline (yes or no). The parameters are: 1) age at screening, 60 years or older (yes or no); 2) male gender (yes or no); 3) HbA1c at screening 6.5 or higher (yes or no); 4) hypoxemia without a proportional increase in the work of breathing (increased respiratory rate, increased nasal flaring, and / or increased use of respiratory muscles, including the diaphragm); 5) known underlying chronic lung disease (yes or no); 6) known heart disease or hypertension (yes or no); and 7) BMI of 28.0 kg / m². 2 (Yes or No) 8) Do you have any known kidney disease? (Yes or No)

[0238] Treatment and Administration: Opaganib 500 mg or matching placebo Q12 hours. Opaganib or placebo, made into a suspension form, can be administered to the patient's stomach via a nasogastric tube.

[0239] Research period: The maximum duration of study participation was up to 45 days (up to 3 days of screening, up to 14 days of double-blind treatment, and 28 days of study drug-free follow-up).

[0240] Eligibility Criteria: Participation: 1. Adult male or female, aged 18 to 80 years old. 2. Proven COVID-19 infection by RT-PCR assay of a throat sample (nasopharyngeal or oropharyngeal) and pneumonia defined as radiopaqueness on chest X-ray or CT scan. 3. Patient requires high-flow supplemental oxygen or CPAP at baseline if high-flow oxygen is not an available option. 4. Patients will use adequate contraception throughout the study and until 3 months after the last dose of study drug. 5. The patient or legal representative has signed a written informed consent form approved by the IRB / ethics committee.

[0241] Exclude: 1. Any comorbid condition that, in the investigator's judgment, may add to the risk of treatment. 2. Requires intubation and mechanical ventilation 3. Oxygen saturation is greater than 95% on room air. 4. Any pre-existing respiratory condition requiring intermittent or continuous ambulatory oxygen prior to admission 5. Patient is unlikely to survive beyond 72 hours in the clinical judgment of the investigator. 6. Pregnant (positive serum or urine test within 3 days prior to randomization) or breastfeeding women. 7. Unwillingness or inability to comply with the procedures required by this protocol. 8. A corrected QT (QTc) interval on the electrocardiogram (ECG), calculated using the Friedericia formula (QTcF), of greater than 470 ms for women and greater than 450 ms for men. 9. AST (SGOT) or ALT (SGPT) is greater than 2.5 x the upper limit of normal (ULN). 10. Total bilirubin level greater than 1.5 × ULN (unless the elevated bilirubin is due to Gilbert's syndrome). 11. Serum creatinine greater than 2.0 × ULN. 12. Absolute neutrophil count is 1000 / mm 3 To be less than. 13. Platelet count is 75,000 / mm 3 To be less than. 14. Hemoglobin < 8.0 g / dL 15. Currently taking a drug that is a sensitive CYP3A4, CYP2C9, or CYP2C19 substrate and has a narrow therapeutic index. 16. Currently taking medications that are strong inducers or inhibitors of CYP2D6 and CYP3A4. 17. Currently taking warfarin, apixaban, argatroban, or rivaroxaban due to drug-drug interactions based on CYP450 metabolism. 18. Currently abusing drugs or alcohol. 19. Currently participating in clinical studies evaluating pharmacological treatments, including antiviral studies.

[0242] Screening / Baseline Assessment: Signed informed consent from the patient or their legal representative Eligibility determination Complete medical history (including onset of COVID-19 symptoms) -Evaluation of combination drugs System baseline review Physical examination Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation by pulse oximeter) If the patient is ambulatory, weight Oxygen requirement (L / min) FiO2 (estimated value) 12-lead electrocardiogram Chest X-ray or CT scan Nasopharyngeal or oropharyngeal swab for PCR testing of SARS-CoV-2 Serum chemistry CRP, D-dimer, LDH, ferritin, cardiac troponin HbA1c CBC with differential Urine tests Serum or urine pregnancy test within 3 days prior to treatment (for women of childbearing potential)

[0243] Research evaluation: The following will be monitored and recorded daily as part of the standard of care: Concomitant medications Adverse events Intermediate physical examination Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation by pulse oximeter) Oxygen flow rate setting (L / min) FiO2 (estimated or known if patient is ventilated)

[0244] The following are preferably monitored less frequently as part of standard care: For patients on chloroquine / hydroxychloroquine / mefloquine combination, a 12-lead ECG (if permitted by hospital treatment guidelines during COVID-19) is required approximately 3 hours after the first study drug dose on Day 1, any time on Days 2 and 4, and at the end of treatment (either Day 14 or early discontinuation of study drug). If the patient is on a monitor (including remote or Holter monitor), investigators are encouraged to collect QT interval duration data. Nasopharyngeal or oropharyngeal viral swab test for SARS-CoV-2 PCR testing every 3 days Weekly serum chemistry Serum CRP, D-dimer, LDH, ferritin, and cardiac troponin were measured weekly. · CBC with differential once a week Chest X-ray or CT scan, as determined by a doctor

[0245] Study endpoints: major Percentage of patients requiring intubation and mechanical ventilation by day 14

[0246] secondary 1) Percentage of patients with a 2 or more category improvement on the WHO Basic Scale of Clinical Improvement. 2) Time to intubation or mechanical ventilation. 3) If high oxygen flow is not an available option, e.g., the time taken to go from high oxygen flow via nasal cannula or CPAP to low oxygen flow via nasal cannula. 4) By day 14, the percentage of patients no longer receiving supplemental oxygen for at least 24 hours. 5) Total oxygen requirement (area under the curve) using daily supplemental oxygen flow rates (L / min) over 14 days (days 1-14). 6) Time until two consecutive swab tests, at least 24 hours apart, are negative for SARS-CoV-2 by PCR. 7) Percentage of patients with at least two consecutive negative swab test results for SARS-CoV-2 by PCR on day 14 8) Percentage of patients with at least one measurement of fever at baseline (defined as a temperature greater than 38.0°C [100.4°F]) who are afebrile (defined as a temperature less than 37.2°C [99°F]) on day 14. 9) Mortality from any cause 30 days after baseline

[0247] exploratory Mean change in systemic markers of inflammation (D-dimer, cardiac tronin, C-reactive protein [CRP], procalcitonin [PCT], lactate dehydrogenase [LDH], and ferritin) from baseline on day 14

[0248] safety 1) Incidence of all treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs) 2) Assessment of vital signs 3) Evaluation of laboratory parameters (chemistry and hematology parameters) 4) Electrocardiogram (ECG) evaluation

[0249] Drugs banned during the study: The following medications are prohibited during the study, including the 28-day follow-up period: Drugs that are sensitive CYP3A4, CYP2C9, or CYP2C19 substrates and have a narrow therapeutic index are prohibited. Strong inducers or inhibitors of CYP2D6 and 3A4 are prohibited.

[0250] Warfarin, apixaban, argatroban, and rivaroxaban are prohibited due to drug interactions based on CYP450 metabolism.

[0251] Stopping rules: If, at any time during the study, a participant experiences any of the following adverse events (using grading criteria defined in the revised NCI Common Terminology for Adverse Events [CTCAE v. 5.0]), study drug will be stopped: Any neuropsychiatric adverse event of grade 3 severity Hallucinations of any severity (any grade) Grade 3 nausea Grade 3 severity vomiting Grade 2 severe creatinine increase

[0252] Statistical methods: The primary analysis was based on a composite failure (yes / no) variable indicating whether the subject required intubation and mechanical ventilation or died by study day 14.

[0253] In the rare case where a patient's outcome is unknown (the patient is lost to follow-up), this will also be counted as a treatment failure for the purposes of the primary analysis. If a patient starts a new investigational therapy for COVID-19 within 14 days, this will also be considered as a treatment failure in the primary analysis.

[0254] The number and percentage of subjects with adverse events are tabulated by treatment group. 95% confidence intervals are constructed for each rate. The Cochran Mantel-Haenzel (CMH) trial compares the rate of adverse events between two groups using the study stratification factors used for randomization, and the corresponding risk difference estimates are presented with 95% confidence intervals. Exact confidence intervals are used where appropriate.

[0255] The significance level for this test is two-sided 5%. In the case of small numbers of events (fewer than 5 events in any study arm), Fisher's exact test will be used.

[0256] The number and percentage of each failure type (intubation and mechanical ventilation) is described for each group.

[0257] The primary analysis was based on the modified intent to treat (mITT) population, consisting of all patients randomized and treated with at least one dose of study drug.

[0258] DSMB futility review In January 2021, the independent Data and Safety Monitoring Board (DSMB) unanimously recommended continuing the study after a pre-specified futility review of open-label efficacy data from the first 135 patients and safety data from the first 175 patients treated in the study.

[0259] Example 9: Randomized, Double-Blind, Placebo-Controlled, Phase 2 / 3 Study of Opaganib, a Sphingosine Kinase-2 (SK2) Inhibitor, or Placebo for the Treatment of COVID-19 Disease This study will evaluate the activity of opaganib versus placebo for the treatment of patients with COVID-19 who, in the investigator's judgment, do not require hospitalization.

[0260] Main purpose: Opaganib vs. placebo in time to sustained recovery from disease. Patients are considered recovered if they meet the following criteria: 1) Afebrile (core temperature <38.0°C) for at least 48 hours without the use of antipyretics. 2) All symptoms have resolved or returned to pre-illness levels (e.g., if the patient had baseline respiratory problems before the onset of COVID-19), except for the following: a. Malaise, anosmia, anageusia, or dysgeusia that can persist at a level similar to that during the acute illness, i.e., at the same level according to symptom questionnaires; b. Chest pain, cough, or dyspnea, if present, must be at least one grade lower than at the start of treatment and no worse than Grade 1 (mild).

[0261] Sustained recovery is recovery according to the above definition and maintained for at least 28 days or until the end of the study (whichever comes first).

[0262] Secondary Objectives: Comparisons will be made between the active treatment group and placebo for: 1) The proportion of patients who were PCR negative on days 8, 15, 29, and 57 after starting treatment (landmark analysis), 2) time to resolution of individual disease-related symptoms present at baseline; 3) the occurrence of new disease-related symptoms under study; 4) incidence of pneumonia during the study among patients without baseline pneumonia (clinically diagnosed); 5) Changes in laboratory markers of disease severity, i.e., oxygen saturation, CRP, lymphocyte count, cardiac troponin, and D-dimer levels, from baseline to time points measured on the study; 6) adverse events, 7) Hospitalization for overall and COVID-19-related indicators within 8 weeks after the first dose of study drug; 8) Mortality 30 days after the first dose of study drug.

[0263] exploratory 1) Percentage of patients reporting a household contact who developed symptomatic, PCR-confirmed COVID-19 by day 57; 2) Serum IgM and IgG antibody levels against SARS-CoV-2 57 days after initiation of treatment.

[0264] Safety: Throughout the course of the trial, patients will be followed for adverse events, including both clinical and laboratory events.

[0265] In particular, toxicities that result in dosage reduction or discontinuation of therapy are followed and tabulated.

[0266] Group: Participation criteria: 1. Symptomatic patients with confirmed COVID-19 according to RT-PCR assay of respiratory tract samples. 2. Patients must either be symptomatic or have tested positive by RT-PCR within 3 days of randomization. 3. Men and women aged 18 and over. 4. At baseline, the laboratory parameters listed below are worse than NCI CTCAE v5.0 grade 2, with exceptions noted below. -Bilirubin less than or equal to 1.5 times the upper limit of normal (ULN, Grade 1 only). -AST (SGOT) and ALT (SGPT) must be 5.0 x ULN or less. - Serum creatinine ≤1.5 × ULN (Grade 1) - Albumin ≥ 2.0 g / dL. 5.- Acceptable hematological status: Absolute neutrophil count 1000 cells / mm 3 That's all. - Platelet count 50,000 / mm 3 That's all. -Hemoglobin level ≥ 8.0 g / dL. 6. Clinically acceptable glycemic control in the opinion of the investigator. 7. INR and partial thromboplastin time (PTT) each less than or equal to 1.5 x ULN (i.e., Grade 1), unless the patient is taking dabigatran or heparin. 8. Oxygen saturation of 92% or higher measured by pulse oximeter on room air 9. Negative pregnancy test (women of childbearing potential). 10. Women of childbearing potential and men with female partners of childbearing potential must agree to use an acceptable method of contraception throughout the study and for at least 2 months after the last dose of study drug. 11. Ability to complete daily journal entries independently. 12. Patients must provide informed consent.

[0267] Exclusion criteria: 1. The patient requires acute hospitalization according to clinician assessment. 2. Pregnant or breastfeeding women. 3. Unwillingness or inability to comply with the procedures required by this protocol. 4. The patient requires supplemental oxygen 5. Patient is currently receiving, has received within the past 7 days, or is expected to receive at any time during the study, remdesivir, chloroquine, hydroxychloroquine, azithromycin, or other specific antiviral therapy for COVID-19, or systemic corticosteroids equivalent to 20 mg prednisone daily / 3 mg dexamethasone daily or greater. 6. Patient is currently receiving, or has received within 30 days prior to screening, any other investigational drug for any investigational indication, including approved agents given for that indication (e.g., anti-cytokine therapy). 7. Patients are currently taking or are expected to start taking Warafin, apixaban (Elquis), or rivaroxaban (Xarelto). Patients may also be taking or may start taking dabigatran (Pradaxa), standard or low molecular weight heparin at the time of the study.

[0268] design This is a randomized, double-blind, placebo-controlled, parallel-group study of opaganib compared with placebo in patients with symptomatic COVID-19 who do not require hospitalization. The study will include an interim analysis for early termination due to futility or an increase in sample size, as indicated by early results.

[0269] Methodology: Part B: Study participants will receive either two 250 mg capsules (500 mg) of opaganib or a matching placebo every 12 hours. Patients will be stratified by the number of the following conditions (none, one, or two or more): age 65 years or older; presence of the following medical conditions of concern (hypertension, chronic lung disease, obesity [BMI 30 or higher], diabetes, heart failure, coronary artery disease, thrombotic events (current or history), renal disease). Patients will also be stratified by the region in which they are treated (US vs. non-US). They will then be randomized 1:1 to receive active drug or placebo. Patients will complete daily questionnaires about symptoms, including adverse events, vital signs including temperature and pulse oximetry, and a log of medications taken daily for the first four weeks of the study and three times weekly thereafter. Viral swabs and blood for safety laboratory and pharmacodynamic markers will be obtained during home visits by healthcare professionals. After completing treatment, patients will be followed up until 57 days after randomization.

[0270] Drugs banned during the study: The following medications are prohibited during the study, including the 28-day follow-up period: Drugs that are sensitive CYP3A4, CYP2C9, or CYP2C19 substrates and have a narrow therapeutic index are prohibited. Strong inducers or inhibitors of CYP2D6 and 3A4 are prohibited.

[0271] Warfarin, apixaban, argatroban, and rivaroxaban are prohibited due to drug interactions based on CYP450 metabolism.

[0272] Example 10: Randomized, Double-Blind, Placebo-Controlled Phase 2 / 3 Study of Upamostat, a Serine Protease Inhibitor, or Placebo for the Treatment of COVID-19 Disease This study will evaluate the activity of upamostat versus placebo for the treatment of patients with COVID-19 who, in the investigator's judgment, do not require hospitalization.

[0273] Main purpose: Part A of the study: Determination of safety and tolerability of two dose levels and decision regarding upamostat dose for Part B. Changes in disease marker severity will be assessed, but this will not be the primary factor in determining which dose will be pursued. Time to recovery will also be calculated, however, due to the small sample size and expected variability of results, clinically meaningful differences may not be seen.

[0274] Part B of the Study: Comparison of Upamostat and Placebo in Time to Sustained Recovery from Illness Patients are considered recovered if they meet the following criteria: 1) Afebrile (core temperature <38.0°C) for at least 48 hours without the use of antipyretics. 2) All symptoms have resolved or returned to pre-illness levels (e.g., if the patient had baseline respiratory problems before the onset of COVID-19), except for the following: c. Malaise, anosmia, anageusia, or dysgeusia that can persist at a level similar to that during the acute illness, i.e., at the same level according to symptom questionnaires; d. Chest pain, cough, or dyspnea, if present, must be at least one grade lower than at the start of treatment and no worse than Grade 1 (mild).

[0275] Sustained recovery is recovery according to the above definition and maintained for at least 28 days or until the end of the study (whichever comes first).

[0276] Secondary Objectives: Comparisons will be made between the active treatment group and placebo for: 1) The proportion of patients who were PCR negative on days 8, 15, 29, and 57 after starting treatment (landmark analysis), 2) time to resolution of individual disease-related symptoms present at baseline; 3) the occurrence of new disease-related symptoms under study; 4) incidence of pneumonia during the study among patients without baseline pneumonia (clinically diagnosed); 5) Changes in laboratory markers of disease severity, i.e., oxygen saturation, CRP, lymphocyte count, cardiac troponin, and D-dimer levels, from baseline to time points measured on the study; 6) adverse events, 7) Hospitalization for overall and COVID-19-related indicators within 8 weeks after the first dose of study drug; 8) Mortality 30 days after the first dose of study drug.

[0277] exploratory 1) Percentage of patients reporting a household contact who developed symptomatic, PCR-confirmed COVID-19 by day 57; 2) Serum IgM and IgG antibody levels against SARS-CoV-2 57 days after initiation of treatment.

[0278] Safety: Throughout the course of the trial, patients will be followed for adverse events, including both clinical and laboratory events.

[0279] In particular, toxicities that result in dosage reduction or discontinuation of therapy are followed and tabulated.

[0280] Group: Participation criteria: 1. Symptomatic patients with confirmed COVID-19 according to RT-PCR assay of respiratory tract samples. 2. Patients must either be symptomatic or have tested positive by RT-PCR within 3 days of randomization. 3. Men and women aged 18 and over. 4. At baseline, the laboratory parameters listed below are worse than NCI CTCAE v5.0 grade 2, with exceptions noted below. -Bilirubin less than or equal to 1.5 times the upper limit of normal (ULN, Grade 1 only). -AST (SGOT) and ALT (SGPT) must be 5.0 x ULN or less. - Serum creatinine ≤1.5 × ULN (Grade 1) - Albumin ≥ 2.0 g / dL. 5. Acceptable hematological status: Absolute neutrophil count 1000 cells / mm 3 That's all. - Platelet count 50,000 / mm 3 That's all. -Hemoglobin level ≥ 8.0 g / dL. 6. Clinically acceptable glycemic control in the opinion of the investigator. 7. INR and partial thromboplastin time (PTT), each less than or equal to 1.5 x ULN (i.e., Grade 1), unless the patient is taking dabigatran or heparin. 8. Oxygen saturation of 92% or higher measured by pulse oximeter on room air 9. Negative pregnancy test (women of childbearing potential). 10. Women of childbearing potential and men with female partners of childbearing potential must agree to use an acceptable method of contraception throughout the study and for at least 2 months after the last dose of study drug. 11. Ability to complete daily journal entries independently. 12. Patients must provide informed consent.

[0281] Exclusion criteria: 1. The patient requires acute hospitalization according to clinician assessment. 2. Pregnant or breastfeeding women. 3. Unwillingness or inability to comply with the procedures required by this protocol. 4. The patient requires supplemental oxygen 5. Patient is currently receiving, has received within the past 7 days, or is expected to receive at any time during the study, remdesivir, chloroquine, hydroxychloroquine, azithromycin, or other specific antiviral therapy for COVID-19, or systemic corticosteroids equivalent to 20 mg prednisone daily / 3 mg dexamethasone daily or greater. 6. Patient is currently receiving, or has received within 30 days prior to screening, any other investigational drug for any investigational indication, including approved agents given for that indication (e.g., anti-cytokine therapy). 7. Patients are currently taking or are expected to start taking Warafin, apixaban (Elquis), or rivaroxaban (Xarelto). Patients may also be taking or may start taking dabigatran (Pradaxa), standard or low molecular weight heparin at the time of the study.

[0282] design This is a randomized, double-blind, placebo-controlled, parallel-group study of upamostat compared with placebo in patients with symptomatic COVID-19 who do not require hospitalization. The study uses a Phase 2 / 3 operationally seamless design methodology for dose selection (Part A) and an inference-free confirmatory Phase 3 trial (Part B). The Phase 3 portion includes an interim analysis or sample size increase for early termination due to futility, as indicated by initial results.

[0283] Methodology: Part A: After admission to the study, patients will be stratified by age, i.e., under 65 years of age or 65 years of age or older. They will then be randomized 1:1:1 to one of the following treatment groups: 1. Upamostat 200 mg, 2 capsules qd (n=20), 2. One capsule of upamostat 200 mg and one matching placebo capsule qd (n=20) 3. Placebo 2 capsules qd (n=20).

[0284] To maintain blinding, patients are given two bottles of medication and are instructed to take one pill from each bottle each day, both pills taken at the same time.

[0285] The medication should be taken with water, with or without food.

[0286] Patients are to take the drug for 14 days or until one of the following occurs: Adverse events, whether related to the study drug or unrelated, that, in the investigator's judgment, require discontinuation of treatment; The patient or investigator decides that it is in the patient's best interest to stop treatment.

[0287] An interim analysis will be performed by the Data Safety Monitoring Board (DSMB) after a total of 60 patients have completed Part A. If the DSMB determines that the safety of both regimens is similar, the Part B accrual will continue at the 400 mg qd dose. If safety is more favorable with the 200 mg qd regimen, the escalation in Part B will continue at the 200 mg qd dose.

[0288] Part B: Based on the safety results from Part A, a treatment regimen of either 200 mg or 400 mg (i.e., one or two 200 mg capsules) will be selected. Patients enrolled in Part B will be stratified by the number of the following conditions (none, one, or two or more): age 65 years or older; presence of the following medical conditions of concern (hypertension, chronic lung disease, obesity [BMI 30 or higher], diabetes, heart failure, coronary artery disease, thrombotic events (current or history), and renal disease). Patients will also be stratified by the region in which they will be treated (US vs. non-US). They will then be randomized 3:2 to active drug or placebo on the schedule selected based on Part A. A total of approximately 250 additional patients will be enrolled in Part B of the study, with 150 receiving the active drug and 100 receiving placebo. Therefore, by combining both parts of the study, a total of 170 patients will receive the active drug at the dose selected in Part A and 120 receiving placebo. However, the analysis will be performed independently for Part A and Part B.

[0289] Patients will complete daily questionnaires about symptoms, including adverse events, vital signs including temperature and pulse oximetry, and a log of medications taken daily for the first 4 weeks of the study and three times weekly thereafter. Viral swabs and blood for safety laboratory and pharmacodynamic markers will be obtained during home visits by medical personnel. After completion of treatment, patients will be followed up until 57 days after randomization.

[0290] statistics: Part A of the study will test two dose levels of active drug and placebo. The regimen for Part B of the study will be selected based on the incidence and severity of toxicity in each active group and the overall safety assessment by the DSMB. If there are no significant differences in toxicity between the two active groups, the default choice for continuation into Part B will be the 400 mg daily regimen.

[0291] Efficacy data from Parts A and B will be analyzed separately.

[0292] The overall sample size may be expanded based on interim study results.

[0293] Sample size was determined based on the primary endpoint, time to sustained recovery from COVID-19 illness, as defined by the primary objective. To detect a hazard ratio of 1.5 comparing the active and placebo groups in a 3:2 allocation ratio, a total of 201 recovery events was calculated to provide 80% power using a log-rank test at a two-sided significance level of 0.05. Assuming an 80% sustained recovery rate by the end of follow-up (assuming equal follow-up for all enrolled patients), the minimum number of patients enrolled in Part B would be a total of 250: 150 in the active arm of the regimen incorporated into Part B of the study and 100 in the placebo arm.

[0294] Industrial Applicability The present invention provides an anti-coronavirus agent, which comprises, as an active ingredient:

[0295] [ka] The present invention also provides an anti-SARS agent comprising the above-described anti-coronavirus agent, and a method for treating SARS using the anti-coronavirus agent. The present invention enables the treatment of diseases caused by coronaviruses, particularly SARS-related coronaviruses.

[0296] The present invention provides an anti-coronavirus agent, which comprises, as an active ingredient:

[0297] [ka] The present invention includes compounds represented by the following formula: as their (L)-enantiomer or (D)-enantiomer, as their E-isomer or (Z)-isomer, or as an (E / Z)-mixture, as their free base, or as a salt thereof. The present invention also provides anti-SARS agents containing the above-mentioned anti-coronavirus agents, and methods for treating SARS using the anti-coronavirus agents. The present invention enables the treatment of diseases caused by coronaviruses, particularly SARS-related coronaviruses.

[0298] All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are apparent to those skilled in molecular biology, medicine, immunology, pharmacology, virology, or related fields are intended to be within the scope of the present invention. The present application also includes the following aspects. [Aspect 1] 1. A method for the treatment of coronavirus disease, comprising administering an effective amount of ABC294640, i.e.

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Claims

1. ABC294640, i.e., 【Chemistry 1】 as its free base or as a salt thereof, and further comprising a pharmaceutically acceptable carrier material, for the treatment of coronavirus disease, the medicament is in a solid dosage form suitable for oral administration; The coronavirus disease is coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus; The medicine.

2. The method of claim 1, wherein the SARS-CoV-2 virus is wild-type.

3. The method of claim 1, wherein the SARS-CoV-2 virus is a naturally occurring coronavirus variant.

4. The medicament according to any one of claims 1 to 3, wherein the medicament comprises the hydrochloride salt of ABC294640.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the solid dosage form is a capsule.

6. 6. The pharmaceutical composition of claim 5, wherein the capsule contains 250 mg of ABC294640 hydrochloride, and two of the capsules are administered twice daily for at least 10 days for a total daily dose of ABC294640 hydrochloride of 1000 mg.

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