Treatment of Coronavirus Infection

Abstract

The present disclosure generally relates to the fields of virology, infectious diseases, and medicine, and describes compounds, compositions, methods, and kits for treating 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, which can treat coronaviruses, including the 2019 novel coronavirus. In one embodiment, the present disclosure provides a new use of WX-671 as an effective inhibitor of coronaviruses and its application in the preparation of a medicament for treating human coronavirus infection. In one embodiment, the present disclosure provides a new use of ABC294640 as an effective inhibitor of coronaviruses and its application in the preparation of a medicament for treating human coronavirus infection.

Description

[0001] Divisional Information

[0002] This application is a divisional application of the patent application with the application number 202180022144.5 and the invention title "Treatment of Coronavirus Infections" filed on March 8, 2021. Technical Field

[0003] This application relates to the field of medicine, and more particularly to the treatment of coronavirus infections. Background Art

[0004] Coronaviruses are lipid-enveloped positive-strand RNA viruses (+ssRNA) that replicate in the cytoplasm. Prior to 2002, coronaviruses were not considered important human pathogens. Other human coronaviruses such as HCoV-229E and HCoV-OC43 only cause mild respiratory infections in healthy adults. However, in 2002, the severe acute respiratory syndrome coronavirus (SARS-CoV) emerged.

[0005] In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was detected in a patient with severe respiratory disease in Saudi Arabia. The clinical features of human MERS-CoV infection range from asymptomatic to very severe pneumonia and can progress to acute respiratory distress syndrome, septic shock, and multi-organ failure leading to death. Since the first case of MERS-CoV infection was reported and the virus was isolated, significant 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.

[0006] In 2019, a novel coronavirus (nCoV) emerged in the world, and it is currently known to cause coronavirus disease 2019 (COVID-19). COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS coronavirus-2 or SARS-CoV-2), which is phylogenetically closely related to the SARS virus. The World Health Organization (WHO) declared the 2019 - 2020 coronavirus outbreak a public health emergency of international concern (PHEIC). For most patients, COVID-19 begins and ends in the lungs because coronaviruses mainly cause respiratory diseases. Summary of the Invention

[0007] The present invention generally relates to the fields of virology, infectious diseases, and medicine. In one embodiment, the present invention provides a new use of ABC294640 as a free base or as a salt thereof in the preparation of a medicament for the treatment of human coronavirus infections.

[0008] According to aspects shown herein, a method for treating coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is disclosed, the method comprising administering to a person in need an effective amount of ABC294640,

[0009] either as a 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 physiological buffered saline. In one embodiment, a suspension is formed comprising ABC294640 hydrochloride suspended in physiological buffered saline, and the administration comprises using a tube to directly deliver the suspension to the stomach. In one embodiment, ABC294640 and an optional 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 wherein the administration comprises administering two capsules twice a day for at least 10 days, with a total daily dose of 1000 mg of ABC294640 hydrochloride. In one embodiment, administering an effective amount of ABC294640 results in at least a 10% reduction in viral load.

[0010] According to aspects shown herein, a method of treatment is disclosed, the method comprising administering an effective amount of ABC294640,

[0011] Administered to a human suffering from coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus, either as a 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 physiological buffered saline. In one embodiment, a suspension is formed comprising ABC294640 hydrochloride salt suspended in physiological buffered saline, and administration comprises delivering the suspension directly to the stomach using a tube. In one embodiment, ABC294640 and an optional 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 salt, and wherein administration comprises administering two capsules twice a day for at least 10 days, with a total daily dose of 1000 mg of ABC294640 hydrochloride salt.

[0012] According to aspects shown herein, ABC294640 is disclosed,

[0013] either as a free base or as a salt thereof, for use in the treatment of coronavirus infections.

[0014] According to aspects shown herein, ABC294640 is disclosed,

[0015] either as a 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.

[0016] According to aspects shown herein, (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide), either as a free base or as a salt thereof, is disclosed for use in the treatment of coronavirus infections.

[0017] According to aspects shown herein, (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide), either as a free base or as a salt thereof, is disclosed for use in the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0018] According to aspects shown herein, ABC294640 is disclosed,

[0019] Use as a free base or as a salt thereof for the manufacture of a medicament for the treatment of coronavirus infection.

[0020] According to aspects shown herein, ABC294640 is disclosed,

[0021] Use as a 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.

[0022] According to aspects shown herein, use of (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) as a free base or as a salt thereof for the manufacture of a medicament for the treatment of coronavirus infection is disclosed.

[0023] According to aspects shown herein, use of the compound (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) as a 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 is disclosed.

[0024] According to aspects shown herein, a pharmaceutical composition for the treatment of coronavirus infection is disclosed, the pharmaceutical composition comprising ABC294640,

[0025] as a free base or as a salt thereof.

[0026] According to aspects shown herein, a pharmaceutical composition for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is disclosed, the pharmaceutical composition comprising ABC294640,

[0027] as a free base or as a salt thereof.

[0028] According to aspects shown herein, a pharmaceutical composition for the treatment of coronavirus infection is disclosed, the pharmaceutical composition comprising (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) as a free base or as a salt thereof.

[0029] According to aspects shown herein, a pharmaceutical composition for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is disclosed, the pharmaceutical composition comprising (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) as a free base or as a salt thereof.

[0030] According to aspects shown herein, an anti-coronavirus infection agent is disclosed, which comprises ABC294640,

[0031] either as a free base or as a salt thereof.

[0032] According to aspects shown herein, an anti-coronavirus infection agent is disclosed, which comprises (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)-amide) either as a free base or as a salt thereof.

[0033] According to aspects shown herein, a method for treating human coronavirus infection is disclosed, which comprises administering a therapeutically effective amount of ABC294640 (3-(4-chlorophenyl)-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).

[0034] According to aspects shown herein, a method for treating COVID-19 (SARS-CoV-2) coronavirus infection is disclosed, which comprises administering one or more therapeutically effective doses of ABC294640 (3-(4-chlorophenyl)-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 from about 250 mg to about 1500 mg each time it is administered.

[0035] According to aspects shown herein, a method for treating COVID-19 (SARS-CoV-2) coronavirus infection is disclosed, which comprises administering a pharmaceutically effective amount of ABC294640 (3-(4-chlorophenyl)-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 the host cell factor sphingosine kinase-2 (SK2), which is involved in intracellular viral replication and downstream inflammatory / immune responses.

[0036] According to aspects shown herein, a method of modulating the replication of a coronavirus in a host cell infected with the coronavirus is disclosed, the method comprising administering to the host cell an amount of ABC294640, as a free base or as a salt thereof, effective to modulate viral replication.

[0037] According to aspects shown herein, the use of ABC294640, as a free base or as a salt thereof, in the preparation of a medicament for the treatment of 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, ABC294640 is present as the hydrochloride salt. In one embodiment, ABC294640 has activity against the host cell factor sphingosine kinase-2, which is involved in intracellular viral replication and downstream inflammatory / immune responses.

[0038] According to aspects shown herein, the present invention features a packaged pharmaceutical product. The packaged pharmaceutical product includes a container, a plurality of unit dosage forms of ABC294640 suitable for oral administration in the container, and a legend (e.g., a label or an insert) associated with the container and indicating the administration of ABC294640 for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0039] In one embodiment, the present invention provides a new use of WX-671 as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as a free base or as a salt thereof in the preparation of a medicament for the treatment of human coronavirus infection.

[0040] According to aspects shown herein, a method of treating coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus in a person in need thereof is disclosed, the method comprising administering an effective amount of WX-671,

[0041] As the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as its salt. In one embodiment, WX-671 is present as the bisulfate. In one embodiment, WX-671 is combined with a pharmaceutically acceptable carrier material. In one embodiment, WX-671 and an optional 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, 200 mg of WX-671 is administered to a person in need in a single capsule once daily for at least 10 days, with a total daily dose of 200 mg. In one embodiment, 400 mg of WX-671 is administered to a person in need in two capsules once daily for at least 10 days, with a total daily dose of 400 mg. In one embodiment, approximately 231 mg of WX-671.1 (upamostat) is administered to a person in need in a single capsule once daily for at least 10 days, with a total daily dose equivalent to 200 mg in free form. In one embodiment, approximately 463 mg of WX-671.1 (upamostat) is administered to a person in need as two capsules once daily for at least 10 days, with a total daily dose equivalent to 400 mg in free form. In one embodiment, administering an effective amount of WX-671 results in at least a 10% reduction in viral load.

[0042] According to aspects shown herein, a method of treatment is disclosed that comprises administering an effective amount of WX-671,

[0043] Administered to a human suffering from coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as its salt. In one embodiment, WX-671 is present as the bisulfate. In one embodiment, WX-671 is combined with a pharmaceutically acceptable carrier material. In one embodiment, WX-671 and an optional 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, WX-671.1 (umostatin) is administered as a single capsule containing 200 mg of the free base, and wherein the single capsule is administered once daily to a person in need for at least 10 days, with a total daily dose of 200 mg. In one embodiment, WX-671.1 (umostatin) is administered as two capsules, each capsule containing 200 mg, and wherein the two capsules are administered once daily to a person in need for at least 10 days, with a total daily dose of 400 mg.

[0044] According to aspects shown herein, WX-671 is disclosed,

[0045] as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as its salt, for treating coronavirus infections.

[0046] According to aspects shown herein, WX-671 is disclosed,

[0047] as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as its salt, for treating coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0048] According to aspects shown herein, (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidinophenylalanine-4-ethoxycarbonylpiperazine) is disclosed as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as its salt, for treating coronavirus infections.

[0049] According to aspects shown herein, (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidinophenylalanine-4-ethoxycarbonylpiperazine) is disclosed as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof, which is used for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0050] According to aspects shown herein, WX-671 is disclosed.

[0051] Use as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof for the manufacture of a medicament for the treatment of coronavirus infections.

[0052] According to aspects shown herein, WX-671 is disclosed.

[0053] Use as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the 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.

[0054] According to aspects shown herein, (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidinophenylalanine-4-ethoxycarbonylpiperazine) is disclosed as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof for the manufacture of a medicament for the treatment of coronavirus infections.

[0055] According to aspects shown herein, (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidinophenylalanine-4-ethoxycarbonylpiperazine) is disclosed as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the 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.

[0056] According to aspects shown herein, a pharmaceutical composition for the treatment of coronavirus infections is disclosed, the pharmaceutical composition comprising WX-671.

[0057] As the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof.

[0058] According to aspects shown herein, a pharmaceutical composition for treating coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is disclosed, the pharmaceutical composition comprising WX-671,

[0059] as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof.

[0060] According to aspects shown herein, a pharmaceutical composition for treating coronavirus infection is disclosed, the pharmaceutical composition comprising (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine) as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof.

[0061] According to aspects shown herein, a pharmaceutical composition for treating coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is disclosed, the pharmaceutical composition comprising (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine) as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof.

[0062] According to aspects shown herein, an anti-coronavirus infection agent is disclosed, the anti-coronavirus infection agent comprising WX-671,

[0063] as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof.

[0064] According to aspects shown herein, an anti-coronavirus infection agent is disclosed, the anti-coronavirus infection agent comprising (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine) as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof.

[0065] According to aspects shown herein, a method for treating human coronavirus infection is disclosed, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from N-α(2,4,6-triisopropylbenzenesulfonyl)-3-guanidino-phenylalanine-4-ethoxy-carbonylpiperazine hydrochloride or a prodrug thereof, N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine, wherein the selected compound can exist as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof. In one embodiment, the method further comprises 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-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine and is in an orally administrable form. In one embodiment, the compound is N-α(2,4,6-triisopropylbenzenesulfonyl)-3-guanidino-phenylalanine-4-ethoxy-carbonylpiperazine hydrochloride and is in an injectable form for intravenous or intramuscular delivery. In one embodiment, the compound is N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine present as the sulfate or bisulfate salt.

[0066] In one embodiment, the compound is N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine in the L-stereoisomeric conformation. In one embodiment, the compound is N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-(L)-phenylalanine-4-ethoxycarbonylpiperazinium bisulfate. In one embodiment, the compound is N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine and is to be administered at a dose of 200 mg per day. In one embodiment, the compound is N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine and is to be administered at a dose of 400 mg per day.

[0067] According to aspects shown herein, a method of treating a 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 N-α(2,4,6-triisopropylbenzenesulfonyl)-3-guanidino-phenylalanine-4-ethoxy-carbonylpiperazine-hydrochloride or its prodrug N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine for at least 14 days, wherein the selected compound can exist as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as its salt. 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, the total daily dose of the compound N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine is independently selected from about 200 mg to about 400 mg at each presentation. In one embodiment, the compound is N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-(L)-phenylalanine-4-ethoxycarbonylpiperazinium hydrogen sulfate.

[0068] According to aspects shown herein, a method for treating a 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 N-α(2,4,6-triisopropylbenzenesulfonyl)-3-guanidino-phenylalanine-4-ethoxy-carbonylpiperazine-hydrochloride or its prodrug N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-phenylalanine-4-ethoxycarbonylpiperazine, wherein the selected compound can exist as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as its salt, and the compound has the ability to bind to a hemagglutinin (HA)-activated type II transmembrane serine protease (TTSP), thereby reducing coronavirus replication in the human subject after exposure to the coronavirus. In one embodiment, the TTSP is transmembrane protease serine 2 (TMPRSS2). In one embodiment, the TTSP is transmembrane protease serine 11A (TMPRSS11(A)). 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, the compound is N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyguanidino-(L)-phenylalanine-4-ethoxycarbonylpiperazinium hydrogen sulfate.

[0069] According to aspects shown herein, a method of modulating the replication of a coronavirus in a host cell infected with the coronavirus is disclosed, the method comprising administering to the host cell an amount of a compound effective to modulate viral replication selected from N-α(2,4,6-triisopropylbenzenesulfonyl)-3-amidinophenylalanine-4-ethoxycarbonylpiperazine hydrochloride or a prodrug thereof, N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidinophenylalanine-4-ethoxycarbonylpiperazine, wherein the selected compound can exist as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof. In one embodiment, the compound is N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidinophenylalanine-(L)-4-ethoxycarbonylpiperazinium hydrogen sulfate.

[0070] According to aspects shown herein, the use of WX-671 in the preparation of a medicament for the treatment of 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 host cell entry.

[0071] According to aspects shown herein, the present invention features a packaged pharmaceutical product. The packaged pharmaceutical product comprises a container, a plurality of unit dosage forms of WX-671 suitable for oral administration in the container, and a legend (e.g., a label or insert) associated with the container and indicating the administration of WX-671 for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The presently disclosed embodiments will be further explained with reference to the accompanying drawings. The drawings shown are not necessarily to scale, but will generally focus on illustrating the principles of the presently disclosed embodiments.

[0073] Figure 1 Depicts the human EpiAirway TM cell culture model, herein referred to as human bronchial epithelial cells (HBEC).

[0074] Figure 2 is a graph showing a dose-dependent decrease in the infectious virus yield observed at pharmacologically relevant concentrations after 3 days of incubation in an opaganib-treated SARS-CoV-2-infected HBEC culture.

[0075] Figure 3This is a graph showing limited cytotoxicity within the dose range where potent antiviral effects were observed after 3 days of incubation in SARS-CoV-2-infected HBEC cultures treated with opaganib.

[0076] Figure 4A This is a graph showing a dose-dependent decrease in infectious virus production observed after 3 days of incubation in SARS-CoV-2-infected HBEC cultures treated with WX-UK1 and ulinastatin at pharmacologically relevant concentrations. Viruses were titrated in apical wash fluids via TCID50 assay. Each symbol represents the titer, averaged from 3 replicates tested.

[0077] Figure 4B This is a graph showing a dose-dependent decrease in infectious virus production observed after 3 days of incubation in SARS-CoV-2-infected HBEC cultures treated with ulinastatin at pharmacologically relevant concentrations. Viruses were titrated in apical wash fluids via plaque reduction assay. Each symbol represents the titer, averaged from 3 replicates tested.

[0078] Figure 5 This is a graph showing limited cytotoxicity within the dose range where potent antiviral effects were observed after 3 days of incubation in SARS-CoV-2-infected HBEC cultures treated with WX-UK1 and ulinastatin.

[0079] Figure 6 This is Curve-Fit Equation 1, with fractional velocity on the y-axis and WX-UK1 concentration on the x-axis. This graph shows how WX-UK1 inhibits the activity of TMPRSS2.

[0080] Figure 7 This is Curve-Fit Equation 1, with fractional velocity on the y-axis and WX-UK1 concentration on the x-axis. This graph shows how WX-UK1 inhibits the activity of TMPRSS11A.

[0081] Figure 8A and Figure 8B This is a graph showing the inhibition of SARS-2-S-driven entry by ulinastatin and WX-UK1 in Calu-3 cells and Vero-E6 cells. Figure 8A Calu-3 cells or Figure 8B Vero-E6 cells were pre-incubated with the indicated concentrations of ulinastatin, WX-UK1, camostat mesylate, or chloroquine, and then inoculated with pseudoparticles carrying the VSV-SARS-2S protein. Pseudotyped entry was analyzed by measuring luciferase activity in cell lysates. Results of a single experiment performed in quadruplicate samples are shown. Error bars represent the standard deviation (SD).

[0082] Figure 9Figure showing the inhibition of VSV-g-driven entry in Calu-3 cells by ulinastatin and WX-UK1. Calu-3 cells were pre-incubated with the indicated concentrations of ulinastatin, WX-UK1, camostat mesylate or chloroquine, and then inoculated with pseudoparticles carrying the VSV-g protein. Pseudotyped entry was analyzed by measuring luciferase activity in cell lysates. Results of a single experiment performed in quadruplicate samples are shown. Error bars represent standard deviation (SD).

[0083] Figure 10 Kaplan-Meier curves (mITT sensitivity) showing the time at which supplemental oxygen was no longer required for at least 24 hours after the statistical analysis of the randomized, double-blind, placebo-controlled phase 2a study of olaparib in COVID-19 pneumonia described in Example 7.

[0084] Figure 11 Kaplan-Meier curves (mITT sensitivity) showing the cumulative incidence over time of a 50% reduction in supplemental oxygen from baseline based on oxygen flow rate in L / min after the statistical analysis of the randomized, double-blind, placebo-controlled phase 2a study of olaparib in COVID-19 pneumonia described in Example 7.

[0085] Figure 12 Dot plot showing the percentage change from baseline of the total supplemental oxygen requirement (area under the curve) measured using daily oxygen flow rate (L / min) over 14 days (days 1 to 14) after the statistical analysis of the randomized, double-blind, placebo-controlled phase 2a study of olaparib in COVID-19 pneumonia described in Example 7.

[0086] Definition

[0087] As used herein, the term "agent" refers to a pharmaceutical substance having pharmacological activity (i.e., the effect of the agent on an individual). The terms "agent", "active ingredient", "pharmaceutical substance" and "compound" are used interchangeably herein.

[0088] As used herein, the term ABC294640 refers to [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide] in free base or salt form or in stereoisomeric or non-stereoisomeric form. In cases where the compound, salt, prodrug or solvate is a solid, those skilled in the art will understand that the compounds, salts and solvates of the present invention can exist in different crystalline forms, all of which are intended to be within the scope of the present invention. Olaparib, also known as ABC294640 hydrochloride, is a specific salt form of ABC294640.

[0089] As used herein, the term WX-671 refers to (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidino-phenylalanine-4-ethoxycarbonylpiperazine) as the (L)- or (D)-enantiomer and E- or Z-isomer or (E / Z)-mixture, and as the free base or as its salt. In the case where the compound, salt, prodrug or solvate is a 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 (ulimorelin) is a specific crystalline salt form of WX-671.

[0090] As used herein, the term "coronavirus" includes naturally occurring (e.g., wild-type) coronaviruses; naturally occurring coronavirus variants; and coronavirus variants generated in the laboratory, including variants generated by selection, variants generated by chemical modification, and genetically modified variants (e.g., coronaviruses modified in the laboratory by recombinant DNA methods). In one embodiment, within a few days after the onset of symptoms or after treatment according to the present disclosure, a subject can be tested for viral infection by collecting nasal secretions (nasal or nasopharyngeal (NP) swabs), throat (oropharyngeal) swabs, blood or other body fluid samples and using, for example, antigen capture enzyme-linked immunosorbent assay (ELISA), using IgM ELISA (to determine whether the subject has IgM antibodies), using IgG ELISA (to determine whether the subject has IgG antibodies), using polymerase chain reaction (PCR), or testing the samples for viral antigens or RNA 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.

[0091] As used herein, the terms "comprising", "including", "having", "may / can", "containing" and their variants are intended to be open transitional phrases, terms or words that do not exclude the possibility of additional acts or structures. The singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments of "comprising the embodiments or elements presented herein", "consisting of the embodiments or elements presented herein", and "consisting essentially of the embodiments or elements presented herein", whether or not explicitly recited.

[0092] The terms "co-administered", "co - administered", or "combined" are used to describe the administration of the compounds of the invention in combination with at least one other antiviral agent. The timing of co - administration is preferably determined by a medical professional treating the patient. Sometimes it is desirable to administer these agents simultaneously. Alternatively, the drugs selected for combination therapy may be administered to the patient at different times. Of course, when there is more than one virus or other infection or other condition, the compounds of the invention may be combined with other agents as needed to treat that other infection or condition.

[0093] As related to the present invention, terms such as "treatment" are defined as inhibiting viral activity before prophylactic administration of a compound in the methods described herein, before a viral infection, or after an infection has occurred. In one embodiment, the term "treatment" means administering one or more compounds of the invention to measurably inhibit viral replication in vitro or in vivo, measurably reduce the viral load in cells in vitro or in vivo, or reduce at least one symptom associated with a patient having a CoV - mediated disease. Ideally, the inhibition of replication or reduction of 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%, 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 of plasma samples can be performed using nucleic acid amplification - based tests (NAT or NAAT) and non - nucleic - acid - based tests 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, a trained physician observes a marked or significant alleviation of symptoms in a patient having a CoV - mediated disease. Generally, the reduction of viral replication is achieved by decreasing the rate of RNA polymerization, RNA translation, protein processing or modification, or by decreasing the activity of molecules involved in any step of viral replication (e.g., proteins important for viral replication or molecules encoded by the genome of the virus or the host). In one embodiment, the term "treatment" refers to the ability of one or more compounds of the invention to inhibit or arrest the replication of a virus such as an RNA virus. In one embodiment, the term "treatment" refers to the ability of one or more compounds of the invention to inhibit the cytopathic effect during an RNA virus infection.

[0094] In some embodiments, an “effective amount” or “immunostimulatory amount” of a compound of the invention is an amount sufficient to produce a detectable immune response when administered to a subject. In other embodiments, a “protective effective amount” of an immunogenic composition is an amount sufficient to confer protective immunity to a subject when administered to the subject. In other embodiments, a “therapeutically effective amount” of a compound is an amount sufficient to treat a viral infection such as increasing the rate of virus clearance when administered to a subject.

[0095] The agents and methods of the invention can be used to treat a subject 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, which comprises, for example, at least one agent of the invention and a substance or collection of substances capable of combining with the at least one agent. As used herein, the term “pharmaceutically acceptable carrier material” refers to a substance or collection of substances capable of combining with an agent, which is suitable for contact with mammalian tissue under the intended exposure conditions for the purpose of therapeutic treatment of mammals. Pharmaceutically acceptable carrier materials are well known in the art and include, for example, inert solids, semi-solids or liquid fillers, diluents, encapsulating materials. Of course, the pharmaceutically acceptable carrier material must have a sufficiently high purity and a sufficiently low toxicity such that they are suitable for administration to the human or lower animal being treated. The pharmaceutical composition can be in unit dosage forms, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, powders, syrups, suppositories, injections, etc.

[0096] 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 body cells involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. An immune response includes, but is 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 an infection-related disease).

[0097] “More effective” means that a treatment exhibits greater efficacy, or less toxicity, greater safety, greater convenience, or lower cost, compared to another treatment being compared. Efficacy can be determined by those skilled in the art using any standard method suitable for a given indication.

[0098] As used herein, the term "suitable time period" refers to the time period starting from when a patient begins to use the method of the present disclosure for the diagnosis and treatment of coronavirus infection, throughout the treatment until the patient stops the treatment due to alleviation of symptoms associated with coronavirus infection or due to a laboratory diagnosis indicating that the viral infection is under control. In one embodiment, the suitable time period is one (1) week. In one embodiment, the suitable time period is between one (1) week and two (2) weeks. In one embodiment, the suitable time period is two (2) weeks. In one embodiment, the suitable time period is between two (2) weeks and three (3) weeks. In one embodiment, the suitable time period is three (3) weeks. In one embodiment, the suitable time period is between three (3) weeks and four (4) weeks. In one embodiment, the suitable time period is four (4) weeks. In one embodiment, the suitable time period is between four (4) weeks and five (5) weeks. In one embodiment, the suitable time period is five (5) weeks. In one embodiment, the suitable time period is between five (5) weeks and six (6) weeks. In one embodiment, the suitable time period is six (6) weeks. In one embodiment, the suitable time period is between six (6) weeks and seven (7) weeks. In one embodiment, the suitable time period is seven (7) weeks. In one embodiment, the suitable time period is between seven (7) weeks and eight (8) weeks. In one embodiment, the suitable time period is eight (8) weeks.

[0099] As used herein, the term "cytopathic effect" refers to the morphological changes of cells caused by viral infection.

[0100] As used herein, the term "cytopathogenesis" or "pathogenesis" includes the inhibition of host cell gene expression and, in addition to those changes visible at the microscopic level, also includes other cellular changes contributing to viral pathogenesis.

[0101] 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, which means that they form weak interactions with their target enzymes and are easily removed. Reversible inhibitors form transient interactions with enzymes. The binding strength between an enzyme and a reversible inhibitor is defined by the dissociation constant (K d ). The smaller the K d value, the stronger the interaction between the enzyme and the inhibitor, and the greater the inhibitory effect. When talking about enzyme inhibition, K d is referred to as K i .

[0102] As used herein, the term "in vitro" refers to procedures performed in an artificial environment, such as, but not limited to, in a test tube or cell culture system. One of ordinary skill in the art will understand that, for example, an isolated SK enzyme can be contacted with a modulator in an in vitro environment. Alternatively, an isolated cell can be contacted with a modulator in an in vitro environment.

[0103] 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, canine, feline, or primate. Detailed Description

[0104] The present invention generally relates to the fields of virology, infectious diseases, and medicine. The present invention features compounds, compositions, methods, and kits for treating CoV-mediated diseases (e.g., diseases caused by SARS-CoV-2, SARS, or MERS). More specifically, the present invention relates to effective inhibitors of coronaviruses, which can treat coronaviruses, including the novel coronavirus 2019. The present invention provides new uses of the compounds as effective inhibitors of coronaviruses, including the novel coronavirus 2019, and their use in the preparation of drugs for treating human coronavirus infections.

[0105] ABC294640, [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide], is an orally administered inhibitor of sphingosine kinase 2 ("SphK2" or "SK2"). ABC294640 is represented by the following structural formula:

[0106] And can be prepared in the form of the free base, its salts, and crystalline variants. U.S. Patents 7,338,961, 8,063,248, 8,324,237, and 8,557,800 teach these compounds, their uses, and their preparation methods, which are incorporated herein by reference.

[0107] ABC294640 as the hydrochloride salt has been given the International Nonproprietary Name (INN) of opaganib and is represented by the following structural formula:

[0108]

[0109] The molecular formula of opaganib is C 23 H 25ClN2O·HCl, with a molecular weight of 417.4 g / mol. Opaganib is a non-hygroscopic white to off-white solid that is practically insoluble in water and ethyl acetate. In one embodiment, a medicament is prepared by filling opaganib in hard gelatin size 1 capsules, which also contain at least one of the following excipients: microcrystalline cellulose; colloidal silicon dioxide; magnesium stearate of vegetable origin; titanium dioxide. In one embodiment, an opaganib capsule contains 250 mg of ABC294640 hydrochloride or 228.16 mg of ABC294640 free base. In one embodiment, an opaganib capsule contains 375 mg of ABC294640 hydrochloride or 342.24 mg of ABC294640 free base.

[0110] In one embodiment, an opaganib 250 mg capsule contains the medicament ABC294640 as the hydrochloride salt and excipients encapsulated in a coni-snap gelatin capsule with a size 1 white opaque body and cap. In one embodiment, an opaganib 375 mg capsule contains the medicament ABC294640 as the hydrochloride salt and excipients encapsulated in a coni-snap gelatin capsule with a size 1 white opaque body and cap.

[0111] Opaganib for the treatment of coronavirus infection is generally administered in an amount in the range of about 250 mg to about 1500 mg per day. In one embodiment, 250 mg of opaganib is administered as two capsules, twice a day, for a total daily dose of 1000 mg. In one embodiment, 250 mg of opaganib is administered as two capsules, 500 mg, Q12 hours. In one embodiment, a patient with a confirmed coronavirus infection is provided with instructions to take a single 500 mg dose (as two 250 mg capsules) of opaganib every 12 hours (thus 1000 mg of opaganib per day) for up to 2 consecutive weeks or up to 14 consecutive days.

[0112] After extensive research, the present inventors have discovered a new use for opaganib. Opaganib has demonstrated antiviral, anti-inflammatory, and antithrombotic activities - acting on both the cause and effect of COVID-19. Opaganib targets sphingosine kinase-2, a human cellular component involved in viral replication, rather than the virus itself. A large body of evidence of new SARS-CoV-2 mutations emerging globally emphasizes the importance of this unique mechanism, which potentially minimizes the risk of the virus developing resistance to treatment.

[0113] The present disclosure provides a packaged pharmaceutical product, also referred to as a pharmaceutical kit, which includes a container, a plurality of olaparib dosage forms suitable for oral administration in the container, and a legend (such as a label or insert) associated with the container and indicating the administration of olaparib for the treatment of coronavirus infection. In one embodiment, the legend includes instructions for practicing the above methods and / or how to use the kit. The instructions included in the kit can be affixed to the packaging material as a label or can be included as a packaging insert. Although the instructions are typically written or printed materials, they are not limited thereto. The present disclosure contemplates any medium capable of storing the instructions and communicating them to the end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, cassette tapes), optical media (e.g., CD ROM), etc. As used herein, the term "instructions" can include the address of an Internet website providing the instructions.

[0114] WX-671 (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidinophenylalanine-4-ethoxycarbonylpiperazine) is an orally active prodrug of the potent serine protease inhibitor WX-UK1 (N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-amidinophenylalanine-4-ethoxycarbonylpiperazine). WX-671 is represented by the following structural formula:

[0115] And can be prepared as the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as the free base or as a salt thereof.

[0116] WX-671 is a prodrug. As used herein, a prodrug refers to a pharmaceutical composition that includes 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 provide WX-UK1. WX-UK1 can only be administered by intravenous infusion. WX-UK1 is used in many of the in vitro experimental examples described herein. Although the present disclosure describes the oral WX-671 compound as a drug, it should be understood that the drug can be prepared using the intravenous infusion compound WX-UK1, which is within the scope and spirit of the present invention. U.S. Patents 6,861,435, 7,247,724, 7,659,396, and 9,089,532 disclose WX-UK1 and its preparation methods, which are incorporated herein by reference.

[0117] WX-671.1, N-α-(2,4,6-triisopropylbenzenesulfonyl)-3-hydroxyamidinyl-(L)-phenylalanine-4-ethoxycarbonylpiperazine hydrogen sulfate, also known as 4-{3-[(E)-amino(hydroxyimino)methyl]-N-[(2,4,6-triisopropylphenyl)sulfonyl]-L-phenylalanyl}piperazine-1-carboxylic acid ethyl ester hydrogen sulfate, has the molecular formula C32H47N5O6S×H2SO4 and a molecular weight of 727.91 g / mol (free base: 629.83 g / mol). U.S. Patents 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 RE46424 disclose these compounds, their uses, and methods of preparation, and these patents are incorporated herein by reference. The substance WX-671.1 has been given the International Nonproprietary Name (INN) of ulimorelin.

[0118] The structural formula of WX-671.1 (ulimorelin) is as follows:

[0119]

[0120] Ulimorelin is a non-hygroscopic white to pale yellow powder that is freely soluble in dimethyl sulfoxide and soluble in ethanol. The drug substance is very slightly soluble in water or 0.1 M HCl. Solid formulations for oral administration can be prepared as tablets, pills, powders, granules, capsules, etc. These solid formulations are manufactured by adding at least one excipient such as starch, calcium carbonate, sucrose, lactose, or gelatin to one or more compounds of the present invention. In addition, lubricants such as magnesium stearate and talc can be used in addition to the typical excipients.

[0121] In one embodiment, a drug is prepared by filling ulimorelin in a hard gelatin capsule that also contains at least one of the following excipients: microcrystalline cellulose; hypromellose; anhydrous ethanol; purified water; and magnesium stearate. In one embodiment, the ulimorelin capsule contains 231.26 mg of ulimorelin hydrogen sulfate (equivalent to 200 mg of free base). After oral administration, ulimorelin is converted to the active WX-UK1, which inhibits several serine proteases. Since ulimorelin can be provided as an oral formulation, it can avoid the disadvantages associated with the intravenous administration of other drugs that may be used to treat coronavirus infections.

[0122] Ummostat for treating coronavirus infection is generally administered in an amount in the range of about 200 mg to about 1000 mg per day. In one embodiment, ummostat is administered as one capsule once a day, with a total daily dose of about 231.26 mg (equivalent to 200 mg of free base). In one embodiment, ummostat is administered as two capsules once a day, with a total daily dose of about 462.52 mg (equivalent to 400 mg of free base). In one embodiment, a patient with a confirmed coronavirus infection is provided with instructions to take one ummostat capsule (equivalent to 200 mg of ummostat free base) per day for 2 consecutive weeks or 14 consecutive days. In one embodiment, a patient with a confirmed coronavirus infection is provided with instructions to take two ummostat capsules (equivalent to 400 mg of ummostat free base) per day for 2 consecutive weeks or 14 consecutive days.

[0123] After extensive research, the present inventors have discovered a new use of ummostat. Without being bound by theory, it is believed that the serine protease inhibitor WX-UK1 (the active drug of ummostat once it decomposes in the body) has activity against at least one serine protease among the serine proteases that seem to be responsible for the serine protease triggered by the viral spike (S) protein. Therefore, the use of protease inhibitors such as WX-UK1 (or its prodrug WX-671) can effectively reduce CoV activation and transmission, thereby producing effective prophylactic and therapeutic treatments. Therefore, WX-UK1 is capable of blocking SARS-2-S-driven cell entry, and thus, as a result, coronavirus replication will be inhibited. In one embodiment, since infection requires proteolytic activation of proteins that promote the interaction between the virus and the host cell receptor, thereby enhancing infectivity and transmission, the ummostat of the present invention will protect against coronavirus infection when administered in a therapeutically effective amount and for a suitable period of time.

[0124] The present disclosure provides a packaged pharmaceutical product, also referred to as a drug kit, which includes a container, a plurality of ummostat dosage forms suitable for oral administration in the container, and a legend (such as a label or insert) associated with the container and indicating the administration of ummostat for treating coronavirus infection. In one embodiment, the legend includes instructions for implementing the above methods and / or how to use the kit. The instructions included in the kit can be attached to the packaging material as a label or can be included as a package insert. Although the instructions are typically written or printed materials, they are not limited thereto. The present disclosure contemplates any medium capable of storing the instructions and communicating them to the end user. Such media include, but are not limited to, electronic storage media (e.g., disk, tape, cassette), optical media (e.g., CD ROM), etc. As used herein, the term "instructions" can include the address of an Internet website providing the instructions.

[0125] Combination Alternating Therapy

[0126] The compounds described herein can be administered on top of the current standard of care for COVID patients, or in combination with or alternating with any other compound or therapy that a healthcare provider deems beneficial to the patient. The combination and / or alternative therapies can be therapeutic, adjuvant, or palliative. When the method involves administering more than one active agent to a patient, the agents can be administered within 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day; within 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour; within 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, or 5 minutes 1 minute; or substantially simultaneously. The methods of the invention can include administering one or more agents to a patient by oral, systemic, parenteral, topical, intravenous, inhalation, or intramuscular administration.

[0127] It has been observed that COVID patients can experience different disease stages, and the standard of care can vary based on what disease stage the patient presents or progresses to. COVID is notable for the development of "crosstalk" between the immune system and the coagulation system. As the disease progresses, the patient's immune system may overreact, which can lead to many serious effects, including cytokine storms. Via the crosstalk between the immune system and the coagulation system, the patient can start to form blood clots in various areas of the body, including the respiratory system, brain, heart, and other organs. Multiple blood clots have been observed throughout the body in COVID patients, thus requiring anticoagulant therapy. It is believed that without treatment and disease mitigation, these blood clots can lead to long-term or even permanent damage.

[0128] More specifically, COVID-19 has been described as going through three general stages of the disease: Stage 1 (early infection), Stage 2 (lung stage), and Stage 3 (hyperinflammatory stage / cytokine storm).

[0129] Stage 1 is characterized by non-specific and usually mild symptoms. Viral replication is occurring and is suitable for immediate treatment with the compounds described herein, and may be combined with or alternated with another antiviral therapy. Interferon-β can also be administered to enhance the innate immune response to the virus. Thus, in one embodiment, the compounds of the invention are used in combination with or alternated with interferon-β and / or additional antiviral drugs in an effective amount. Zinc supplements and / or vitamin C are sometimes also administered during this stage or as the disease progresses.

[0130] The second stage of COVID-19 is the pulmonary stage, during which patients may experience acute hypoxemic respiratory failure. In fact, the main organ failure in COVID-19 is hypoxemic respiratory failure. It has been shown that moderate immunosuppression via steroids such as dexamethasone can be beneficial to patients with acute hypoxemic respiratory failure and / or patients on mechanical ventilation. In one embodiment, the compounds of the present invention are used in combination with corticosteroids, which can be glucocorticoids, in an effective amount. Non-limiting examples are budesonide (Entocort EC), betamethasone (Celestone), prednisone (Prednisone Intensol), prednisolone (Orapred, Prelone), triamcinolone (Aristospan Intra-Articular, Aristospan Intralesional, Kenalog), methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol), hydrocortisone or dexamethasone (Dexamethasone Intensol, DexPak 10Day, DexPak 13Day, DexPak6Day).

[0131] When administered to COVID-19 patients, the results of the NS5B inhibitor remdesivir have been mixed. It can only be administered in a hospital setting and only by intravenous injection, usually three times a day, which makes it unsuitable for mild to moderate COVID-19 patients. In one embodiment, the compounds of the present invention are administered in combination with or alternately with remdesivir to enhance the overall antiviral effect.

[0132] Phase 3, the final phase of the disease, is characterized by progressive disseminated intravascular coagulation (DIC), a condition in which small blood clots form throughout the bloodstream. This phase may also include multi-organ failure (e.g., vasodilatory shock, myocarditis). A "cytokine storm" has also been observed in many patients in this severe phase of COVID-19 infection. There appears to be a two-way, synergistic relationship between DIC and the cytokine storm. To counteract DIC, anticoagulants are typically administered to the patient, which can be, for example, indirect thrombin inhibitors or direct oral anticoagulants ("DOACs"). Non-limiting examples are low molecular weight heparin, warfarin, bivalirudin (Angiomax), rivaroxaban (Xarelto), dabigatran (Pradaxa), apixaban (Eliquis), or edoxaban (Lixiana). In one embodiment, the compounds of the present invention are administered in combination with or alternating with anticoagulant therapy. In some severe coagulation cases in COVID patients, TPA (tissue plasminogen activator) may be administered.

[0133] It has been observed that high levels of the cytokine interleukin-6 (IL-6) are precursors of respiratory failure and death in COVID-19 patients. To treat this surge in the immune response that may constitute a cytokine storm, monoclonal antibodies, drug inhibitors, or proteolysis agents targeting IL-6 can be administered to the patient, such as bispecific compounds that bind IL-6 and also bind a protein that mediates degradation. Examples of antibodies include tocilizumab, sarilumab, siltuximab, olokizumab, and clazakizumab. In one embodiment, the compounds of the invention are administered in combination with or alternately with tocilizumab or sarilumab. Additional non-limiting examples of immunosuppressive drugs for treating an overreactive 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), mycophenolate mofetil (CellCept, Myfortic)). Additional antibodies and biologics include abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), ixekizumab (Taltz), natalizumab (Tysabri), rituximab (Rituxan), secukinumab (Cosentyx), tocilizumab (Actemra), ustekinumab (Stelara), vedolizumab (Entyvio), basiliximab (Simulect), and daclizumab (Zinbryta).

[0134] IL-1 blocks the production of IL-6 and other pro-inflammatory cytokines. COVID patients are sometimes also treated with anti-IL-1 therapies to reduce the over-inflammatory response, such as intravenous administration of anakinra. Anti-IL-1 therapies can generally be, for example, targeted monoclonal antibodies, drug inhibitors, or proteolysis agents, such as bispecific compounds that bind IL-1 and also bind a protein that mediates degradation.

[0135] COVID patients typically develop viral pneumonia, which can lead to bacterial pneumonia. Patients with severe COVID-19 may also be affected by sepsis or "septic shock". Treatment of secondary bacterial pneumonia or sepsis in COVID includes 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, the compounds of the present invention are administered in combination with or alternately with an antibiotic such as azithromycin. Some of these antibiotics, such as azithromycin, have independent anti-inflammatory properties. Such drugs can be used both as anti-inflammatory agents in COVID patients and also have a therapeutic effect on secondary bacterial infections.

[0136] A unique challenge in treating patients infected with COVID-19 is the need for relatively long-term sedation if the patient requires mechanical ventilation, which may last up to or greater than 5 days, 10 days, or even 14 days. For pain that persists during this treatment, analgesics can be added sequentially, and for persistent 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-dominant properties (olanzapine, quetiapine), propofol, or dexmedetomidine, haloperidol, and phenobarbital. In one embodiment, the compounds of the present invention are administered in combination with or alternately with a pain reliever such as acetaminophen, ketamine, hydromorphone, fentanyl, or morphine. In one embodiment, the compounds of the present invention are administered in combination with or alternately with a sedative such as melatonin, olanzapine, quetiapine, propofol, dexmedetomidine, haloperidol, or phenobarbital.

[0137] Investigational drugs for COVID-19 include chloroquine and hydroxychloroquine. In one embodiment, the compounds of the present invention are administered in combination with or alternately with chloroquine or hydroxychloroquine.

[0138] Protease inhibitors previously approved for HIV can also be administered, such as lopinavir or ritonavir.

[0139] In one embodiment, opaganib is administered in combination with ulinastatin for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus. In one embodiment, opaganib is administered in combination with ulinastatin to manufacture a medicament for the treatment of coronavirus infection. In one embodiment, opaganib is administered in combination with ulinastatin to manufacture a medicament for the treatment of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus.

[0140] Additional drugs that can 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 of REGN3048 and REGN3051, or ribavirin. Any of these drugs or vaccines can be used in combination with or alternately with the active compounds provided herein to treat virus infections sensitive thereto.

[0141] In one embodiment, the compounds of the present invention are used in combination with an anti-coronavirus vaccine therapy in an effective amount, which includes but is not limited to mRNA-1273 (Moderna, Inc.), AZD-1222 (AstraZeneca and University of Oxford), BNT162 (Pfizer and BioNTech), CoronaVac (Sinovac), NVX-CoV2372 (Novavax), SCB-2019 (Sanofi and GSK), ZyCoV-D (Zydus Cadila), and CoVaxin (BharatBiotech). In another embodiment, the compounds of the present invention are used in combination with a passive antibody therapy or a convalescent plasma therapy in an effective amount.

[0142] In one embodiment, the compounds of the present invention are used in combination with a 5-HT receptor antagonist in an effective amount, which can relieve certain symptoms that may be present in patients infected with the coronavirus, such as diarrhea.

[0143] SARS-CoV-2 mutates continuously, which increases its virulence and transmission rate. Drug-resistant variants of the virus may emerge after long-term treatment with antiviral agents. Drug resistance may be caused by gene mutations encoding enzymes used for viral replication. In some cases, the efficacy of a drug against RNA virus infections can be extended, enhanced, or restored by combining or alternating the administration of the compound with another and possibly even two or three other antiviral compounds that induce different mutations or act via a pathway different from that of the primary drug.

[0144] The present invention has multiple aspects illustrated by the following non-limiting examples. The purpose of providing the following examples is to illustrate the various embodiments of the present invention and is not meant to limit the present invention in any way.

[0145] Example

[0146] Example 1 : Evaluation of the antiviral activity of ABC294640 against SARS-CoV-2 in human airway epithelial cells

[0147] We designed an in vitro evaluation in organotypic air-liquid interface (ALI) cultures of primary human bronchial epithelial cells (HBEC; EpiAirway TM , MatTek) to evaluate whether the infection and transmission of SARS-CoV-2 could be directly inhibited by opaganib. This human cell culture model system was chosen because it contains a pseudostratified epithelial layer that is morphologically and functionally similar to the pseudostratified epithelial layer of the human airway and consists of ciliated and goblet (mucus-producing) cells that are exposed to air at the apical layer. These cells serve as the first line of defense against invading viruses and as replication sites. Available evidence also indicates that human bronchial epithelial cells express the host factor (sphingosine kinase-2) targeted by opaganib.

[0148] Test Compound:

[0149] Olaparib - Test Compound

[0150] 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

[0151] Remdesivir (GS - 5734) - Positive Antiviral Control

[0152] Description: Remdesivir is a nucleotide analogue antiviral prodrug. It exhibits antiviral activity against multiple variants of EBOV, with EC50 values in the range of 0.06 - 0.14 μM in cell-based assays, and exhibits broad-spectrum antiviral activity against other pathogenic RNA viruses, including SARS-CoV, in vitro. Solvent: DMSO 100 mg / mL (166.0 mM)

[0153] (Bleomycin (Sulfate)) - Positive Cytotoxic Control

[0154] Description: Bleomycin is a chemotherapeutic agent commonly used in the treatment of Hodgkin lymphoma and embryonal carcinoma. The broad-spectrum pulmonary toxicity caused by bleomycin has been well-described as a complication of such therapies, with the most common variant being bleomycin-induced pneumonitis (BIP) (Sleijfer et al., 2001). Bleomycin (BLM) was chosen as the most well-studied micronucleus (MN) inducer with different genotoxic mechanisms in human lymphocytes. Solvent: DMSO 16.67 mg / mL (11.2 mM)

[0155] Method :

[0156] Cell Culture - Differentiated Human Bronchial Epithelial Cells (HBEC)

[0157] Normal human bronchial epithelial (HBEC) cells are differentiated by MatTek Corporation (Ashland, MA) and provided in kits with either 12-well or 24-well inserts. HBEC cells grow on a 6 mm² mesh disk in the transwell insert. Three days before shipment, the tissue is transferred to medium without hydrocortisone. During transportation, the tissue is stabilized on an agarose sheet, which is removed upon receipt. One insert is estimated to consist of approximately 1.2×10 6 cells. The cell insert kit (EpiAirway TM AIR-100) is derived from a single donor, #9831, a 23-year-old, healthy, non-smoking Caucasian male. These cells have unique properties in terms of the epithelium, with only the apical side exposed to air and producing a mucin layer. Upon arrival, according to the manufacturer's instructions, the cell transwell insert is immediately transferred to individual wells of a 6-well plate, and 1 mL of MatTek's proprietary medium (AIR-100-MM) is added to the basolateral side, while the apical side is exposed to a humidified 5% CO2 environment. The cells are cultured at 37 °C for one day before the experiment begins. After a 16 - 18 hour equilibration period, the mucin layer secreted from the apical side of the cells is removed by washing with 400 μL of pre-warmed TEER buffer. The medium is replenished after the washing step. Figure 1 A description of the culture insert and EpiAirway tissue is provided.

[0158] Treatment with Test Compound :

[0159] Serial dilute the test compound from a stock solution (containing DMSO) in the assay medium (AIR-ASY-100, MatTek) and place it at room temperature. The overview of the test compound dilution is as follows (final DMSO < 0.5%). Wash the HBEC culture with phosphate-buffered saline (PBS) and incubate it at 37°C for 1 hour with remdesivir (2 μM), bleomycin sulfate (75.6 μg / ml and 151 μg / ml), and olaparib (6 concentrations ranging from 0.05 μg / ml to 11.25 μg / ml) diluted in the assay medium (AIR-100-ASY, MatTek) before infection. For the control wells, add the assay medium containing DMSO (final DMSO < 0.5%; control) and the virus-only control (assay medium only) 1 hour before infection. Add the compound in triplicate to the apical layer (0.15 mL) and the basal layer (0.85 mL) of each insert.

[0160] Viral Infection and Sample Processing :

[0161] After incubating with the compound for 1 hour, wash the apical side of the culture, and then infect it with the SARS-CoV-2 clinical isolate (2019-nCoV / USA-WA1 / 2020) at an MOI of 0.1 PFU / cell in the presence of the compound or the assay control medium at 37°C for 1 hour. After 1 hour of virus incubation, remove the virus from the apical side and wash the culture once with PBS to remove any unbound virus. Then incubate the culture with fresh compound at 37°C for 72 hours. At 24 hours and 48 hours post-infection, replace the basolateral medium with 1 mL of fresh medium containing the corresponding compound.

[0162] At 72 hours post-infection, collect the tissue and medium for processing. Wash the apical layer with 0.4 mL of TEER buffer (PBS containing Mg 2+ and Ca 2+ ) and collect it for evaluation of virus titer via TCID50 (50% tissue culture infective dose) assay. Add serial eight-fold dilutions of the apical layer supernatant sample concentrations to a 96-well assay plate (20,000 / well) containing Vero E6 cells. Incubate the plate at 37°C, 5% CO2, and 95% relative humidity. After incubating for 3 days (72 ± 4 hours), stain the plate with crystal violet to measure the cytopathic effect (CPE). Calculate the virus titer using the method of Reed and Muench (Reed et al., 1938). The TCID 50 value is determined from triplicate samples.

[0163] To evaluate the health status of HBEC cells after exposure to olaparib, control compounds, and viral infection, a lactate dehydrogenase (LDH) release assay was performed. The medium was removed from the basolateral layer of the tissue culture insert 72 hours post-infection and diluted in LDH storage buffer according to the manufacturer's instructions (Promega). The samples were further diluted with LDH buffer and incubated with an equal volume of LDH detection reagent. Luminescence was recorded after a 60-minute incubation at room temperature. A cell-free control was included as a negative control to determine the medium background, and bleomycin was included as a positive cytotoxicity control. Luminescence was reported, with the background level found to be within an acceptable luminescence range (range 1,000 - 10,000).

[0164] Additionally, the apical layer of the HBEC tissue was collected by adding Trizol LS (Invitrogen) to each culture insert and pipetting up and down several times to lyse and collect the cells, which were stored at -80 °C for future RNA and protein expression analysis.

[0165] Result :

[0166] Olaparib has High Activity against SARS - CoV - 2 in HBEC Cultures

[0167] In this study, normal human bronchial epithelial cells (HBEC) were pretreated with 6 different concentrations of olaparib (11.25 μg / ml - 0.05 μg / ml) on the apical and basolateral sides of each culture, in triplicate. Once pretreated, the HBEC were exposed to SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) for 1 hour, the apical layer was washed to remove unbound virus, and then the cultures were incubated with the compounds for 3 days. Three days post-infection, the apical layer was washed and the viral load was evaluated by TCID50 assay. The basolateral medium was collected and the presence of lactate dehydrogenase (LDH), released from damaged cells and used as an indicator of cell death / vitality, was evaluated.

[0168] Opanib demonstrated potent antiviral activity, where viral replication was inhibited in a dose-dependent manner without significantly compromising cell viability. In Opanib-treated SARS-CoV-2-infected HBEC cultures, after 3 days of incubation, a dose-dependent decrease in infectious virus production was observed, with complete inhibition starting from Opanib 1 μg / ml (pharmacologically relevant concentration). These results were favorable compared to the positive control remdesivir in the study. Cell viability was evaluated as in the LDH release assay. To demonstrate the antiviral activity of Opanib against SARS-CoV-2 in a human primary epithelial culture system, we conducted antiviral assays in HBEC cultures that were grown at the air-liquid interface and recapitulated the cellular complexity and physiology of the human conducting airway. In Opanib-treated SARS-CoV-2-infected HBEC cultures, after 3 days of incubation, a dose-dependent decrease in infectious virus production was observed, with complete inhibition starting from Opanib 1 μg / ml (pharmacologically relevant concentration) Figure 2 ). These results were favorable compared to the positive control remdesivir in the study.

[0169] Opanib did not cause cytotoxicity in HBEC cultures within the concentration range where potent antiviral effects were observed( Figure 3 ). In summary, these data demonstrate that Opanib has potent antiviral activity against SARS-CoV-2 in primary human lung cultures without compromising cell membrane integrity, which is a measure of cell viability and drug safety, thus further demonstrating the promising potential of Opanib for treating COVID-19 patients.

[0170] Neither 50% inhibition nor 50% cytotoxicity was achieved within the tested concentration range. At the lowest tested concentration of 0.05 μg / ml, greater than 90% inhibition of infectious virus production was achieved. At the highest tested concentration of 11.25 μg / ml, the cells remained viable throughout the experiment. When calculating the selectivity index (SI) using the high and low concentration ranges from this experiment, the SI value was 225, which is a ratio measuring the window between cytotoxicity and antiviral activity by dividing the antiviral activity value (AVA) by the toxicity (TOX) value (AVA / TOX). Larger SI values are expected if a wider range of concentrations is tested.

[0171] Example 2 : Evaluation of the Antiviral Activity of Ulinastatin and WX-UK1 against SARS-CoV-2 in Human Airway Epithelial Cells

[0172] We designed experiments in human primary bronchial epithelial cells (HBEC; EpiAirway TM, in vitro evaluation in organotypic air-liquid interface (ALI) cultures from MatTek) to evaluate whether the infection and spread of SARS-CoV-2 can be directly inhibited by ulinastatin and WX-UK1. This human cell culture model system was chosen because it contains a pseudostratified epithelial layer that is morphologically and functionally similar to the pseudostratified epithelial layer of the human airway and consists of ciliated and goblet (mucus-producing) cells exposed to air at the apical layer. These cells serve as the first line of defense against invading viruses and as replication sites. Available evidence also indicates that human bronchial epithelial cells express host factors (e.g., TMPRSS2) targeted by ulinastatin.

[0173] Test Compound :

[0174] Ulinastatin - Test Compound

[0175] Description: Ulinastatin - Ethyl 4-{3-[(E)-amino(hydroxyimino)methyl]-N-[(2,4,6-triisopropylphenyl)sulfonyl]-L-phenylalanyl}-piperazine-1-carboxylate hydrogen sulfate.

[0176] Solvent: DMSO

[0177] WX - UK1 - Test Compound

[0178] Description: WX-UK1 - Ethyl 4-[(2S)-3-(3-formamidophenyl)-2-[(2,4,6-triisopropylphenyl)sulfonylamino]propanoyl]piperazine-1-carboxylate.

[0179] Solvent: DMSO

[0180] Camostat Mesylate - Test Compound

[0181] Description: Camostat mesylate (CM) 2-(Dimethylamino)-2-oxoethyl 4-[[4-[(aminoiminomethyl)amino]benzoyl]oxy]phenylacetate mesylate; FOY 305; FOY-S980; Foipan mesylate. Camostat is a synthetic orally bioavailable serine protease.

[0182] Solvent: DMSO

[0183] (Bleomycin (Sulfate)) - Positive Cytotoxic Control

[0184] Description: Bleomycin is a chemotherapeutic agent commonly used in the treatment of Hodgkin lymphoma and embryonal carcinoma. The broad-spectrum pulmonary toxicity caused by bleomycin has been well-described as a complication of such therapies, with the most common variant being bleomycin-induced pneumonitis (BIP) (Sleijfer et al., 2001). Bleomycin (BLM) was selected as the most well-studied micronucleus (MN) inducer in human lymphocytes with different genotoxic mechanisms.

[0185] Solvent: DMSO 16.67 mg / mL (11.2 mM)

[0186] Method :

[0187] Cell Culture - Differentiated Human Bronchial Epithelial Cells (HBEC)

[0188] Normal human bronchial epithelial (HBEC) cells were differentiated by MatTek Corporation (Ashland, MA) and provided in kits with either 12-well or 24-well inserts. HBEC cells were grown on 6 mm^2 mesh disks in transwell inserts. Three days before shipment, the tissue was transferred to medium without hydrocortisone. During transportation, the tissue was stabilized on an agarose wafer, which was removed upon receipt. One insert was estimated to consist of approximately 1.2×10 6 cells. The cell insert kit (EpiAirway TM AIR-100) was derived from a single donor, #9831, a 23-year-old, healthy, non-smoking Caucasian male. These cells have unique properties in terms of the stratification, with only the apical side exposed to air and producing a mucin layer. Upon arrival, the cell transwell inserts were immediately transferred to individual wells of a 6-well plate according to the manufacturer's instructions, and 1 mL of MatTek's proprietary medium (AIR-100-MM) was added to the basolateral side while the apical side was exposed to a humidified 5% CO2 environment. The cells were cultured at 37 °C for one day before the experiment began. 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 pre-warmed TEER buffer. The medium was replenished after the washing step. Figure 1 A description of the culture inserts and EpiAirway tissue is provided.

[0189] Treatment with Test Compound :

[0190] Serial dilute the test compound from a stock solution (containing DMSO) in the assay medium (AIR-ASY-100, MatTek) and place it at room temperature. The test compound dilution is outlined below (final DMSO < 0.5%). Wash the HBEC cultures with phosphate-buffered saline (PBS) and incubate them at 37 °C for 1 hour with bleomycin sulfate (75.6 μg / ml and 151 μg / ml), ulinastatin (6 concentrations ranging from 0.12 μg / ml to 30.00 μg / ml), WX-UK1 (3.33 μg / ml, 10 μg / ml, and 30.00 μg / ml), or camostat (0.5 μg / ml, 5 μg / ml, and 25 μg / ml) diluted in the assay medium (AIR-100-ASY, MatTek) before infection. For the control wells, add the assay medium containing DMSO (final DMSO < 0.5%; control) and virus-only control (assay medium only) 1 hour before infection. Add the compounds in triplicate to the apical layer (0.15 mL) and basal layer (0.85 mL) of each insert.

[0191] Viral Infection and Sample Processing :

[0192] After incubating with the compounds for 1 hour, wash the apical side of the cultures, and then infect them with a SARS-CoV-2 clinical isolate (2019-nCoV / USA-WA1 / 2020) at an MOI of 0.1 PFU / cell in the presence of the compounds or assay control medium at 37 °C for 1 hour. After 1 hour of virus incubation, remove the virus from the apical side and wash the cultures once with PBS to remove any unbound virus. Then incubate the cultures with fresh compounds at 37 °C for 72 hours. At 24 hours and 48 hours post-infection, replace the basolateral medium with 1 mL of fresh medium containing the corresponding compound.

[0193] At 72 hours post-infection, collect the tissues and media for processing. Treat the apical layer with 0.4 mL of TEER buffer containing Mg 2+ and Ca 2+washed with PBS) and collected for virus titer assessment via TCID50 (50% tissue culture infective dose) assay. Samples of apical layer supernatant at eight-fold serial dilutions were added to 96-well assay plates (20,000 / well) containing Vero E6 cells. 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 the cytopathic effect (CPE). Virus titers were calculated using the method of Reed and Muench (Reed et al., 1938). TCID50 values were determined from triplicate samples. To confirm the results of the TCID50 assay, a plaque reduction assay was performed. Briefly, samples of apical layer supernatant at 10-fold serial dilutions were added to 24-well assay plates (100,000 cells / well) containing Vero E6 cells for the plaque reduction assay. The plates were incubated at 37 °C, 5% CO2, and 95% relative humidity. After incubation for 3 days (72 ± 4 hours), the plates were fixed with 5% neutral buffered formalin and stained with crystal violet to visualize the plaques. Titers were calculated in PFU / mL using the following formula: Titer (PFU / mL) = plaque count × 10^ 稀释度计数 × 10 (to reach mL as we added 100 μL of diluted sample). The assay was performed twice, with the second assay conducted on virus + DMSO and 0.2 μg / ml ulinastatin to evaluate additional sample dilutions.

[0194] To evaluate the health status of HBEC cells after exposure to olaparib, control compound, and viral infection, a lactate dehydrogenase (LDH) release assay was performed. At 72 hours post-infection, the medium was removed from the basolateral layer of the tissue culture inserts and diluted in LDH storage buffer according to the manufacturer's instructions (Promega). The samples were further diluted with LDH buffer and incubated with an equal volume of LDH detection reagent. Luminescence was recorded after incubation at room temperature for 60 minutes. A cell-free control was included as a negative control to determine the medium background, and bleomycin was included as a positive cytotoxicity control. Luminescence was reported, with the background level found to be within an acceptable luminescence range (range 1,000 - 10,000).

[0195] Additionally, the apical layer of HBEC tissue was collected by adding Trizol LS (Invitrogen) to each culture insert and pipetting up and down several times to lyse and collect the cells, which were stored at -80 °C for future RNA and protein expression analysis.

[0196] Result :

[0197] Ulinastatin and WX - UK1 are Potent Antiviral Inhibitors of SARS - CoV - 2 in Human Bronchial Epithelial Tissue Cultures Agent .

[0198] In this study, normal human bronchial epithelial cells (HBEC) were pretreated in triplicate on the apical and basolateral sides of each culture with 6 different concentrations of ulinastatin (0.12 μg / ml to 30.0 μg / ml) and 3 different concentrations of WX-UK1 (3.33 μg / ml to 30.0 μg / ml). Once pretreated, the HBEC were exposed to SARS-CoV-2 (2019-nCoV / USA-WA1 / 2020) and incubated with the compounds for 3 days. At 3 days post-infection, the apical layer was washed and viral load was assessed by TCID50 assay. The basolateral medium was collected and the presence of lactate dehydrogenase (LDH), released from damaged cells and used as an indicator of cell death / vitality, was evaluated. For comparison, 3 concentrations of camostat (ranging from 0.5 μg / ml to 25.0 μg / ml), an established TMPRSS2 inhibitor, were included.

[0199] Both ulinastatin and WX-UK1 demonstrated potent antiviral activity, where replication was inhibited in a dose-dependent manner without significantly compromising cell viability (except at the highest dose of each compound). A 3-log and 4-log reduction in viral load was observed by TCID50 at the lowest concentrations of ulinastatin (0.12 μg / ml) and WX-UK1 (3.33 μg / ml), respectively. At 3 days post-infection, a similar decrease in viral titre was found for both ulinastatin and WX-UK1. As evaluated in the LDH release assay, cell viability was reported to be completely uncompromised except at the maximum concentrations evaluated for ulinastatin and WX-UK1.

[0200] To demonstrate the antiviral activity of ulinastatin and WX-UK1 against SARS-CoV-2 in a human primary epithelial culture system, we performed antiviral assays in HBEC cultures grown at the air-liquid interface and recapitulating the cellular complexity and physiology of the human conducting airways. In ulinastatin- and WX-UK1-treated SARS-CoV-2-infected HBEC cultures, after 3 days of incubation, a dose-dependent reduction in infectious virus production was confirmed at pharmacologically relevant concentrations by TCID50 and plaque reduction assays ( Figure 4A and Figure 4B ). These results were favourable compared to the known TMPRSS2 inhibitor camostat.

[0201] We calculated the EC50 estimate using plaque reduction assay results. At the highest tested concentration, the inhibition produced by the virus exceeded 50%. The EC50 was estimated using the available data with GraphPad. The estimated EC50 was 0.02 ug / ml. After converting the TCID value to an estimated PFU value as described below, we calculated the percent inhibition using the following formula:

[0202] Percent inhibition = ((Value - Mean virus control) / (Mean cell control - Mean virus control) * 100)

[0203] The percent inhibition values were then analyzed via GraphPad following these instructions:

[0204] - The X value is the concentration of ulinastatin.

[0205] - The Y value is the response.

[0206] - Select "Dose - Response Curve"

[0207] - Select Analyze, Nonlinear Regression, and Dose - Response (Stimulation) Equation Set and select [Dose]

[0208] Vs Response - Variable slope. Accept all other default values. The calculated EC50 was 0.02 ug / ml.

[0209] Measured by LDH release, ulinastatin, WX - UK1, and camostat inhibited virus replication without significantly impairing cell viability (except at the highest tested concentration). To measure the LDH released from non - viable cells, the medium was removed from the basolateral layer of the tissue culture insert 72 hours post - infection and diluted in LDH storage buffer according to the manufacturer's instructions (Promega). The samples were further diluted with 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 the medium background and a positive cytotoxicity control bleomycin (151 ug / ml). The data was plotted using the luminescence value minus the cell - free control (mean luminescence reading of 6936). Using a physiologically relevant human respiratory tissue model, this data demonstrated the potential of ulinastatin to strongly inhibit SARS - CoV - 2 virus replication with limited cytotoxicity in the dose range where potent antiviral effects were observed in HBEC cultures ( Figure 5), thus further demonstrating that ulinastatin has promising potential for the treatment of COVID-19 patients. The cytotoxic concentration (CC50) of 50% cell culture was generated with the available data to determine the compound concentration required to reduce the absorbance of treated cells by 50% compared to control cells. The CC50 value of ulinastatin calculated using the luminescence data generated via MTT assay was 46.37 uM (or 29.2 ug / ml). At this CC50 concentration, an EC50 concentration below 4.6 uM (or 2.9 ug / ml) would result in an SI value (CC50 / EC50) > 10.

[0210] Example 3 : Evaluation of the in vivo efficacy of ABC294640 against ARDS-induced thrombosis This study used a rat venous stasis model to evaluate the efficacy of ABC294640 in reducing the incidence of in situ adverse thromboembolic events in the context of acute respiratory distress syndrome (ARDS). This assay was designed to measure the thrombosis 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 as mimicking the neutrophilic inflammatory response observed in ARDS patients. The mechanism of LPS-induced ARDS is based on damage to endothelial cells and systemic inflammatory responses.

[0211] The venous stasis (Wessler) test in animals has been widely used as a laboratory measurement of in vivo hypercoagulable states for over 40 years. It has been shown to be very valuable for evaluating the thrombogenicity of various blood products.

[0212] The test compound was administered at a dose of 250 mg / kg by oral gavage at 3 hours after infusion and at 24, 48, and 72 hours after infusion. An appropriate amount of LPS from Escherichia coli (E. coli) (O55:B5) was diluted in saline to obtain a final concentration of 400 μg / mL. This solution was administered by intratracheal instillation (0.5 mL / kg). The vehicle consisted of PBS at 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).

[0213] This study used Sprague-dawley rats (male) weighing between 275 g and 400 g. The animals were randomly assigned to the treatment groups by the study director. Food and water were provided ad libitum. Behavior and general health status were observed until sacrifice. Body weights were recorded before infusion and at 24, 48, and 72 hours after infusion.

[0214] Before and 24, 48, and 72 hours after instillation, arterial oxygen saturation (SpO2) and heart rate were recorded using a mouse pulse oximeter collar probe (MouseOx Plus system, Starr Life Sciences) mounted on awake mice. Rats were also introduced into the plethysmography chamber environment, with the same schedule for SpO2. Functional respiratory parameters were evaluated by a whole-body plethysmograph (VivoFlow, SCIREQ). The functional respiratory parameters analyzed included; respiratory rate, PenH (lung congestion index), and inspiratory / expiratory time measurements. Blood samples were also collected before the terminal protocol for complete blood count and cytokine level evaluation.

[0215] In this study, ARDS was induced by intratracheal instillation of LPS. Throughout the ARDS induction and development, animals were administered vehicle or ABC294640 by oral gavage (3 hours, 24 hours, 48 hours, and 72 hours after instillation). After 72 hours of awake measurements, the rats were anesthetized and venous stasis was induced in the inferior vena cava (4 hours after oral gavage at the 72-hour time point). The stasis was maintained for 30 minutes. Then the segment was excised and its contents were scored. Subsequently, the animals were euthanized by exsanguination.

[0216] After exsanguination, a tracheostomy was performed and the chest was opened to expose the lungs. Then the trachea was connected to the cannula of the perfusion system. The left lung was clamped while cold PBS1X, protease inhibitor 1X solution was injected through the trachea to perform bronchoalveolar lavage fluid (BALF) on the right lobe of the lung and collected for further analysis. Total cell count and differential cell count as well as total protein content were evaluated in the BALF samples. Aliquots of BALF were retained for quantification of the levels of chemokines / cytokines in the BALF.

[0217] 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 wet / dry ratio of the left lung, which is an indicator of pulmonary edema. The remaining part of the left lung was homogenized and aliquoted for quantification of the protein content.

[0218] Induction of LPS Lung Injury :

[0219] 1. Before LPS or saline instillation, functional respiratory parameters of all rats were evaluated by whole-body plethysmography, and SpO2 of awake rats was evaluated using a collar probe pulse oximeter. Rats were first acclimated to the plethysmography chamber before physiological assessment. Respiratory rate, Penh, and inspiratory / expiratory time measurements were analyzed.

[0220] 2. Anesthetize rats with 2.5% isoflurane USP (Abbot Laboratories, Montreal, Canada) in oxygen. Then intubate the rats and deliver LPS or saline by intratracheal instillation.

[0221] 3. The rats recover from anesthesia and return to their respective cages.

[0222] 4. Three hours after instillation, administer the first dose of ABC294640 (see Table 1 below) by oral gavage.

[0223] Table 1: Experimental Progress / Steps in Rats

[0224]

[0225]

[0226] 5. Evaluate the rats regularly to ensure animal health (general behavior and daily body weight).

[0227] 6. Also administer drugs to the animals by oral gavage at 24, 48, and 72 hours after instillation.

[0228] 7. Also evaluate SpO2 and whole body plethysmography at 24, 48, and 72 hours after instillation.

[0229] 8. Draw blood samples from the jugular vein for complete blood count and cytokine level measurement before the venous stasis protocol.

[0230] 9. Four hours after the last drug administration, anesthetize the rats with 2.5% isoflurane USP (Abbot Laboratories, Montreal, Canada) in oxygen. Conduct this protocol on a warming blanket to control body temperature.

[0231] 10. Expose the inferior vena cava of the rats and place two (2) loose sutures at 1 cm intervals. Ligate any collateral vessels in the isolated segment.

[0232] 11. Maintain stasis in situ for a period of 30 minutes.

[0233] 12. Remove the venous stasis segment, open it longitudinally, empty it on filter paper, and take a photo. Remove any existing thrombus and blot it dry on filter paper. Measure the thrombus, weigh it, and score it on a scale of 0 to 4 (see Table 2).

[0234] Table 2: Quantitative Evaluation of Thrombogenicity

[0235] Quantitative Evaluation of Thrombogenicity Score No Clot 0 Virtually no Visible Fibrin Strands 0.5 Few Visible Fibrin Strands 1.0 One or several thrombi of <1.5mm 1.5 One Thrombus > 1.5 mm 2.0 Two or More Thrombi > 1.5 mm 2.5 One Large Thrombus > 3 mm 3.0 Two or More Large Thrombi > 3 mm 3.5 Single Thrombus Forming Entire Segment 4.0

[0236] 13. After venous stasis, the animals were euthanized by bloodletting, and bronchoalveolar lavage fluid was collected from the right lung. For this purpose, the muscles on the trachea were incised before performing tracheotomy. The chest cavity was opened to expose the lungs, and the trachea was connected to the cannula of the perfusion system. The left lung was clamped, and 15 mL (3 × 5 mL) of cold PBS 1X, protease inhibitor 1X solution was injected through the trachea to perform bronchoalveolar lavage fluid (BALF) on the right lobe of the lung. The BALF was collected for further analysis. The total cell count and differential cell count were evaluated in the BALF samples. An aliquot of the BALF was retained for quantifying the chemokine / cytokine levels in the BALF.

[0237] 14. Then the left lung was harvested and weighed for calculating the left lung weight and left lung index. The wet / dry ratio calculation was used to evaluate lung tissue edema. 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).

[0238] Each parameter (listed below) for each group was compiled and presented in a bar graph using appropriate statistical analysis.

[0239] 1 - Body weight change

[0240] 2 - Saturation (SpO2) and heart rate (bpm)

[0241] 3 - Respiratory parameters: Inspiratory / expiratory time

[0242] Tidal volume and expiratory volume

[0243] Respiratory rate

[0244] PenH

[0245] 4 - BALF total cell count and differential

[0246] 5 - BALF cytokine / chemokine levels

[0247] 6 - Left lung weight and index

[0248] 7 - Lung wet / dry ratio

[0249] 8 - Total lung protein content in lung homogenate

[0250] LPS induced a significant increase in lung weight associated with inflammation and lethargy. This increase was associated with severe edema, as indicated by a significant increase in the W / D ratio. The lung weight gain in the LPS - vehicle group was greater 4 hours after gavage at the 72 - hour time point compared to the LPS - vehicle group. ABC294640 administered at 250 mg / kg demonstrated a reduction in thrombosis - evidenced by a reduction in clot length, weight, and total thrombus score.

[0251] Example 4 : Evaluation of TMPRSS2 and TMPRSS11(A) as targets for WX-UK1 inhibition

[0252] Several enzymes belonging to different protease families can be hijacked by the CoV S protein for priming. The pH-dependent cysteine protease cathepsin L, TMPRSS2, TMPRSS11A, and the serine protease furin can prime the S protein during viral entry into target cells. We performed an analysis, including structural modeling / prediction, structural analysis, and a review of relevant literature, to determine whether any TTSPs are relevant targets for ulinastatin inhibition.

[0253] The mammalian expression systems for TMPRSS2 and TMPRS11A were purchased from MyBioSource (MBS1193731 and MBS1345824, respectively). The proteins were refolded to 1 mg / ml according to the manufacturer, and we ran gels with the refolded proteins. A fresh stock solution of WX-UK1 (100 mM WX-UK1 in 100% DMSO) was prepared. The concentrated stock solution was diluted to 1 mM in HBS buffer and then further diluted in the assay.

[0254] Enzyme inhibitors can interact with the enzyme and / or the enzyme-substrate complex in several different ways to reduce the rate of the enzyme-catalyzed reaction. For each inhibition mode, the dissociation constant Ki of the inhibitor can be calculated, which reflects the strength of the interaction between the enzyme and the inhibitor. The Ki of an inhibitor is analogous to the Km of a substrate; small Ki values reflect tight binding of the inhibitor to the enzyme, while larger Ki values reflect weaker binding. The exact formula for calculating Ki depends on the inhibition mode, which can be determined experimentally by comparing the "apparent" values of V max and Km of the enzyme in the presence of the inhibitor with the V max and Km values in the absence of any inhibitor (Equation 2 below).

[0255] The chromogenic substrate selected for these studies was the S-2288 substrate. The Ki value was determined by measuring the effect of WX-UK1 on the cleavage of the chromogenic substrate by human serine proteases. To determine the K i value, a concentration series of WX-UK1 was pre-incubated with the target human serine protease before adding the chromogenic substrate to initiate the reaction. The reaction rate was determined from the slope using linear regression and normalized to the rate of the non-inhibited reaction. The normalized activity was plotted against the WX-UK1 concentration before obtaining the K i value by non-linear regression using Equation 1.

[0256] Equation 1:

[0257]

[0258] vi / v0 is the ratio of the initial velocity with and without inhibitor, which is described as a function of inhibitor concentration [I] and substrate concentration [S].

[0259] K M The parameters were obtained by standard Michaelis-Menten kinetics. Serine protease was added to a series of substrates at appropriate concentrations, which were high enough to yield an experimental Vmax value. The subsequent reaction velocities were plotted against substrate concentration, and then the KM value was obtained using the Michaelis equation (2).

[0260] Equation 2:

[0261]

[0262] All experiments were performed at least three times in HBS (30 mM Hepes, pH = 7.4; 150 mM NaCl; 0.5% BSA) at 37 °C. The reaction was monitored at 405 nm at a rate of 2 readings / min for at least 45 minutes. Since WX-UK1 was maintained in 100% DMSO, all experiments included an uninhibited DMSO control to exclude the effect of unwanted DMSO on protease activity.

[0263] WX - UK1 Inhibits Human TMPRSS2

[0264] Figure 6 is the curve-fitting equation 1, with fractional velocity on the y-axis and WX-UK1 concentration on the x-axis. This figure shows how WX-UK1 inhibits the activity of TMPRSS2. K i was determined to be 2.9 ± 0.04 (3) μM.

[0265] WX - UK1 Inhibits Human TMPRSS11a

[0266] Figure 7 is the curve-fitting equation 1, with fractional velocity on the y-axis and WX-UK1 concentration on the x-axis. This figure shows how WX-UK1 inhibits the activity of TMPRSS11A. K i was determined to be 0.39 ± 0.01 (3) μM.

[0267] Table 3 Lists the inhibition constant Ki values of WX-UK1 against a panel of proteases:

[0268] Table 3: K i Value

[0269]

[0270]

[0271] Example 5 : Evaluation of the effects of ulinastatin and WX-UK1 on SARS-CoV-2 spike protein-mediated entry

[0272] Studies were conducted to evaluate the inhibitory effects of both ulinastatin and WX-UK1 on the cellular entry of replication-defective, single-cycle vesicular stomatitis virus (VSV) particles pseudotyped with the SARS-CoV-2 spike protein (VSVpp+SARS-2-SΔ18) or, as a control, the glycoprotein of vesicular stomatitis virus (VSV). Δ18 refers to the deletion of 18 C-terminal amino acids of the S protein, which increases pseudotyping efficiency without affecting the use of ACE2 or TMPRSS2. Pseudotyped entry and its inhibition were evaluated in Calu-3 and Vero-E6 cells. Calu-3 cells are a lung-derived human cancer cell line that permits SARS-CoV-2 S-driven entry in a TMPRSS2-dependent manner. Agents that inhibit TMPRSS2, including camostat, a known TMPRSS2 inhibitor that has been shown to inhibit SARS-CoV-2 infection of cultured lung cells (Hoffmann et al., 2020), were expected to inhibit S-driven entry in this model. Vero cells are a green monkey kidney cell line that permits SARS-CoV-2 spike-driven entry in a TMPRSS2-independent, cathepsin L-dependent manner. Agents that raise the pH of acidic intracellular endosomes, including chloroquine, were expected to inhibit entry in this model. Entry driven by the G protein of vesicular stomatitis virus (VSV) was used as a specific control (VSV-G-driven entry depends on low pH and is thus sensitive to chloroquine but not camostat).

[0273] Method :

[0274] For pseudotyping, vesicular stomatitis virus pseudotypes (VSVpp) were generated according to a published protocol (Berger Rentsch and Zimmer, 2011). Briefly, VSV*DG-fLuc, which contains an expression cassette for eGFP (enhanced green fluorescent protein) and firefly luciferase instead of the VSV-G open reading frame, was used by Gert Zimmer, Institute of Virology and Immunology, 293T cells transfected to express the viral surface glycoprotein under study were inoculated with a replication-deficient VSV vector (kindly provided by / Switzerland). After an incubation period of 1 hour at 37 °C, the inoculum was removed and the cells were washed with PBS, and then medium supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added to neutralize residual input virus (no antibody was added to cells expressing VSV-G). Pseudotyped particles were harvested 16 hours post-inoculation, clarified from cell debris by centrifugation and used for experiments.

[0275] For transduction, target cells were grown in 96-well plates until they reached 50%-75% confluence and then inoculated with the respective pseudotypes. For experiments involving protease inhibitors, target cells were treated with the corresponding chemical 2 hours prior to transduction. Sixteen hours post-transduction, transduction efficiency was quantified by measuring the activity of firefly luciferase in cell lysates using a commercial substrate (Beetle-Juice, PJK) and a Hidex Sense plate reader (Hidex). The transduction assay measures the entry of a single-cycle vesicular stomatitis virus (VSV) carrying the SARS-CoV-2 spike.

[0276] Result :

[0277] The ability of ulipristal and WX-UK1 to inhibit the entry of pseudotypes carrying SARS-2-S and VSV-G was evaluated in Calu-3 (human lung carcinoma cells) and Vero-E6 cells. Calu-3 cells are a lung-derived human cancer cell line that permits SARS-CoV-2 spike-driven entry in a TMPRSS2-dependent and thus camostat-sensitive manner. Vero cells are an African green monkey-derived kidney cell line that permits 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) was used as a specific control (VSV-G-driven entry depends on low pH and is thus sensitive to chloroquine rather than camostat).

[0278] Ulinastatin and WX - UK1 Inhibit SARS - CoV - 2 S Protein - Mediated Entry with Moderate Efficiency in Human Lung Cancer Cells (Calu - 3) and Green Monkey Kidney Cells (Vero E6) Figure 8A 。

[0279] When tested in Calu-3 cells against VSVpp+SARS-2-SΔ18, both WX-UK1 and upamostat showed moderate inhibitory activity, but lower than that of another serine protease inhibitor, camostat ( Figure 8B)。When tested in Vero-E6 cells that do not have surface TMPRSS2, moderate inhibitory activity was still noted for ulmoctastat and WX-UK1; camostat was inactive in this context, while the highest concentration of chloroquine potently inhibited S protein-driven entry( Figure 9 )。WX-UK1 and ulmoctastat moderately inhibited VSV-G entry in calu-3 cells, indicating a broader activity spectrum for ulmoctastat( Example 6 )。When tested against VSV-G in Vero76 cells, all three compounds were inactive except chloroquine. In summary, these results demonstrate that WX-UK1 and ulmoctastat inhibit SARS-CoV-2 spike-driven entry into Calu-3 and Vero cells with moderate efficiency. Due to the nature of the model, it is not possible to specifically extrapolate to actual in vitro or in vivo inhibitory concentrations.

[0280] Primary Objective : Treatment of COVID-19 associated pneumonia with opaganib

[0281] Patients diagnosed with COVID-19 infection who have developed pneumonia and do not require mechanical ventilation or who have been mechanically ventilated for no more than 24 hours are evaluated from this inpatient study.

[0282] Evaluate Viral Clearance during Olaparib Treatment :

[0283] 1) Evaluate the safety and tolerability of opaganib administered at 500 mg Q12 hours in patients hospitalized due to COVID-19 infection

[0284] Secondary / Exploratory May Include One or More of the Following

[0285] Study Design :

[0286] 1) Evaluate the vital signs of patients hospitalized due to COVID-19 infection during opaganib treatment

[0287] 2) Evaluate the clinical improvement of patients hospitalized due to COVID-19 infection during opaganib treatment

[0288] 3) For patients who are not mechanically ventilated at baseline, evaluate the need for mechanical ventilation during opaganib treatment

[0289] 4) Evaluate the improvement of hypoxia via the SpO2 / FiO2 or PaO2 / FiO2 ratio and the SpO2 in room air. Return to room air or a specific SpO2 oxygen saturation in room air.

[0290] Evaluate the changes in viral load during treatment, as well as the changes in D-dimer, cardiac troponin, LDH, and ferritin levels.

[0291] Key Inclusion Criteria :

[0292] This study included an active treatment group; all eligible patients admitted with COVID-19 pneumonia who did not require mechanical ventilation or had been on mechanical ventilation for <24 hours received open-label opaganib 500 mg Q12 hours, twice daily. 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 up for 2 weeks after their last dose of study drug, at the end of the 2-week treatment period, or once they had two consecutive negative daily viral swabs for COVID-19 virus or after premature discontinuation of the drug before day 14. The maximum duration of study participation was 35 days (7 weeks). Study participants received opaganib, 2 × 250 mg capsules (500 mg) Q12 hours, administered for a total of up to 14 days (2 weeks) or until two consecutive daily nasopharyngeal viral swabs were negative for COVID-19, whichever occurred first. Opaganib was administered with food (after a light to moderate meal), followed by 240 mL (8 fluid ounces) of water. If a patient could only take opaganib via a nasogastric tube, the capsule contents were suspended in 20 cc of normal saline solution and pushed through the nasogastric tube, followed by a thorough rinse with sterile water. If a patient was on tube feeding, opaganib was administered shortly after tube feeding (about 15 - 30 minutes).

[0293] Key Exclusion Criteria :

[0294] 1. Adult males or females aged ≥18 years to ≤75 years, including the endpoints

[0295] 2. Confirmed COVID-19 infection and pneumonia that did not require mechanical ventilation or had been on mechanical ventilation for no more than 24 hours at the time of informed consent

[0296] 3. The patient, guardian, or legally acceptable representative had signed a written IRB-approved informed consent form.

[0297] Study Assessment :

[0298] 1. New York Heart Association class III or IV heart disease, myocardial infarction within the past 6 months, unstable arrhythmias, or evidence of ischemia on ECG

[0299] 2. Any comorbidities that, in the investigator's judgment, might increase the risk of treatment.

[0300] 3. Pregnant (serum test positive) or lactating females

[0301] 4. Unwilling or unable to comply with the procedures required by this protocol.

[0302] 5. AST (SGOT) or ALT (SGPT) > 2.5 × upper limit of normal (ULN)

[0303] 6. Bilirubin > 1.5 × ULN (except for cases where bilirubin increase is due to Gilbert syndrome)

[0304] 7. Serum creatinine > 2.0 × ULN

[0305] 8. Absolute neutrophil count < 1000 cells / mm3

[0306] 9. Platelet count < 75,000 / mm3

[0307] 10. Hemoglobin < 8.0 g / dL

[0308] 11. Currently taking warfarin, apixaban, argatroban, or rivaroxaban

[0309] 12. Current drug or alcohol abuse

[0310] Monitor the Following Daily (See Table 4) :

[0311] Table 4 :

[0312] · Concomitant medication review

[0313] · Adverse events

[0314] · Physical examination

[0315] · Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation measured by pulse oximeter)

[0316] · Clinical symptoms (cough, dyspnea, nausea, vomiting, diarrhea)

[0317] · Nasopharyngeal viral swab

[0318] · Serum chemistry

[0319] · CBC with differential

[0320] · Chest X-ray

[0321] · Urinalysis

[0322] Dosage Form and Administration Mode :

[0323] Assessment schedule; other possible assessments not mentioned below include collecting blood and stool samples and determining SARS-CoV-2 RNA levels using quantitative RT-PCR assays. Viral load is assessed daily as long as the subject continues to shed viral RNA.

[0324]

[0325]

[0326] 1 Daily assessment until day 14 or earlier if two consecutive SARS-CoV-2 virus swabs are negative daily

[0327] 2 Assess body temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation by pulse oximeter

[0328] 3 Assess cough, dyspnea, nausea, vomiting, diarrhea

[0329] 4 Fertile females; serum pregnancy test must be negative within 3 days before randomization

[0330] Study Endpoint :

[0331] Olaparib is provided as 250 mg capsules containing 250 mg olaparib and excipients in white opaque hard gelatin capsules

[0332] Olaparib is administered orally as two capsules (500 mg) every 12 hours (or via nasogastric tube if appropriate) for up to 2 weeks. If appropriate, each dose is administered with food or 15 - 30 minutes after tube feeding

[0333] Primary Safety Endpoint :

[0334] Secondary / Exploratory Endpoint :

[0335] 1) Adverse events, laboratory tests, physical examinations, and vital signs

[0336] 2) Percentage of patients with two consecutive negative daily nasopharyngeal virus swabs by day 14 of olaparib treatment

[0337] Result :

[0338] 1) Percentage of patients showing improvement in vital signs (based on improvement in one or more of the following: body temperature, heart rate, respiratory rate, oxygen saturation)

[0339] 2) Percentage of patients showing clinical improvement (based on improvement in one or more of the following symptoms: cough, dyspnea, nausea, vomiting, diarrhea)

[0340] Percentage of patients who were not mechanically ventilated at baseline and did not require mechanical ventilation by the end of the 2 - week off - treatment follow - up

[0341] Table 5: Study Results :

[0342] · Results have been obtained from seven patients who were approved for compassionate use. These patients had moderate to severe COVID-19 related pneumonia with hypoxia requiring assisted oxygenation. Six of the seven patients were given opaganib plus standard care, including hydroxychloroquine (HCQ) as background therapy.

[0343] · As can be seen from Table 5, all six moderate to severe patients had significant improvement except for Patient #7 who was treated for only 1 day due to diarrhea which may or may not be related to opaganib (this patient was also given HCQ and azithromycin), and 5 of the patients returned to breathing room air and 3 patients were discharged.

[0344] · All six patients showed a decrease in C-reactive protein (CRP), and all six patients also demonstrated measurable clinical improvement, including decreased supplemental oxygenation and higher lymphocyte counts.

[0345] · All patients started with 250 mg of opaganib Q12 hours for the first 3 days and then increased to 500 mg of opaganib Q12 hours.

[0346] · Although there were only six patients, these preliminary findings showed clinical improvement in the first COVID-19 patients treated with opaganib and provided preliminary support for the tolerability of opaganib in COVID-19 patients.

[0347] RA - Room Air, D / C - Discharge, SOB - Shortness of Breath

[0348]

[0349] * Improvement one day after treatment start, discharged in room air without repeat blood tests.

[0350] Example 7

[0351] Five patients were included in the analysis, and for comparison purposes, we used a control group (baseline characteristics) of patients of the same gender and same severity. When appropriate, univariate comparisons between groups were performed using the chi-square test for categorical variables and the t-test or Mann-Whitney U test for continuous variables. The time variable was compared with Cox proportional hazards regression, adjusted for age and background diseases. Changes in CRP and lymphocytes were compared using repeated measures general linear model and Bonferroni correction for multiple comparisons.

[0352] Patients treated with opaganib had a significantly faster increase in lymphocyte count. All other clinical outcomes trended non-significantly in favor of the treatment group: the median time to discontinue high-flow nasal cannula (HFNC) was 10 days and 15 days for cases and controls, respectively (HR = 0.3, 95% CI: 0.07 - 1.7, p = 0.2), time to ambient air was 13 days vs. 14.5 days (HR = 0.4, 95% CI: 0.15 - 1.5), and no cases required mechanical ventilation compared with 33% of controls. In this small cohort of severely ill COVID-19 patients, opaganib was safe and well tolerated, with improvement in clinical and laboratory parameters in all treated patients. The efficacy of opaganib in COVID-19 infection should be further tested in a randomized placebo-controlled trial.

[0353] Primary Objective : A Randomized, Double-Blind, Placebo-Controlled Phase 2a Study of Opaganib in COVID-19 Pneumonia

[0354] Secondary Objectives :

[0355] Evaluate the total oxygen requirement (area under the curve) using the daily assisted oxygen flow rate (L / min) over 14 days (day 1 to day 14)

[0356] Exploratory Objectives :

[0357] 1) Evaluate the time to a 50% reduction in assisted oxygen supply based on the oxygen flow rate in L / min from baseline

[0358] 2) Evaluate the proportion of patients who no longer require assisted oxygen supply for at least 24 hours by day 14

[0359] 3) Evaluate the proportion of patients without fever on day 14

[0360] 4) Evaluate the time to a negative SARS-CoV-2 swab by PCR

[0361] 5) Evaluate the proportion of patients with a negative SARS-CoV-2 swab by PCR on day 14

[0362] 6) Evaluate the need for intubation and mechanical ventilation by day 14

[0363] 7) Evaluate the time to mechanical ventilation

[0364] 8) Evaluate the proportion of patients who had at least one fever measurement at baseline (defined as body temperature > 38.0°C [100.4°F]) and no fever on day 14 (defined as body temperature < 37.2°C [99°F])

[0365] 9) Evaluate the mortality rate 30 days after baseline

[0366] Safety Objectives :

[0367] Evaluate changes in systemic inflammation markers (D-dimer, cardiac troponin, C-reactive protein [CRP], lactate dehydrogenase [LDH], and ferritin)

[0368] Study Population :

[0369] Evaluate the safety and tolerability of oral administration of opaganib at 500 mg Q12 hours for up to 14 days in patients with COVID-19 pneumonia

[0370] Study Design and Description :

[0371] The study population consisted of patients diagnosed with COVID-19 infection who developed pneumonia defined as chest radiographic opacities and requiring supplementary oxygen. Patients had to be hospitalized at least during the screening period and at baseline (Day 1).

[0372] Stratification :

[0373] This was a Phase 2a, proof-of-concept, multicenter, randomized, double-blind, parallel-group, placebo-controlled study. After obtaining informed consent, patients entered a screening period of up to 3 days to determine eligibility. Forty-two eligible patients were randomized at a 1:1 randomization ratio to receive opaganib added to standard of care or a matched placebo added to standard of care. Treatment assignment was blinded to patients, investigators, hospital staff, and the sponsor. Since there is no consensus on a definitive treatment specifically targeting the SARS-CoV-2 virus that causes COVID-19 (Wilson, 2020), the standard of care implemented during the COVID-19 pandemic refers to regionally, institutionally, or physician-directed treatment.

[0374] Study participants received 2 × 250 mg capsules of opaganib (500 mg) or a matched placebo every 12 hours in addition to standard of care (pharmacological and / or supportive). The study drug was administered daily for 14 days (Day 1 to Day 14), unless the patient had been discharged without the need for supplementary oxygen, in which case the study drug would only be administered until Day 10.

[0375] All participants were followed up for 4 weeks after the last study drug administration, as determined by the patient or the physician, which could occur at the end of the 2-week double-blind treatment period or after premature discontinuation of the study drug. The maximum duration of study participation was up to 45 days (including up to 3 days of screening; 2-week DB treatment period and 4-week off-treatment follow-up)

[0376] Eligibility Criteria :

[0377] Patients are stratified using a minimization algorithm that takes into account the following three parameters: age ≥ 70 years at screening (yes or no); HbA1c ≥ 6.5 at screening (yes or no); oxygen requirement at baseline, requiring non-invasive positive pressure ventilation (e.g., via BIPAP, CPAP) (yes or no).

[0378] Inclusion :

[0379] Exclusion :

[0380] 1. Adult male or female aged ≥ 18 years to ≤ 80 years

[0381] 2. COVID-19 infection confirmed by RT-PCR assay of a pharyngeal sample (nasopharyngeal or oropharyngeal), and pneumonia defined as a chest X-ray radiographic opacity

[0382] 3. Patient requires assisted oxygenation at baseline

[0383] 4. Patient, guardian, or legal representative has signed a written IRB-approved informed consent form

[0384] Number of Subjects :

[0385] 1. Any comorbidities that the investigator judges may increase the risk of treatment.

[0386] 2. Requiring intubation and mechanical ventilation

[0387] 3. Indoor air oxygen saturation > 95%

[0388] 4. Any pre-existing respiratory disease requiring intermittent or continuous dynamic oxygen before hospitalization

[0389] 5. The patient is judged by the investigator to be unlikely to survive > 72 hours

[0390] 6. Pregnancy (serum test positive within 3 days before randomization) or lactating women

[0391] 7. Unwilling or unable to comply with the procedures required by this protocol.

[0392] 8. Corrected QT (QTc) interval on electrocardiogram (ECG) calculated using the Friedericia formula (QTcF) > 470 ms for women or > 450 ms for men

[0393] 9. AST (SGOT) or ALT (SGPT) > 2.5 × upper limit of normal (ULN)

[0394] 10. Bilirubin > 1.5 × ULN (except for cases of increased bilirubin due to Gilbert syndrome)

[0395] 11. Serum creatinine > 2.0 × ULN

[0396] 12. Absolute neutrophil count < 1000 cells / mm 3

[0397] 13. Platelet count < 75,000 / mm 3

[0398] 14. Hemoglobin < 8.0 g / dL

[0399] 15. Currently taking drugs that are sensitive to CYP3A4, CYP2C9 or CYP2C19 substrates and have a narrow therapeutic index

[0400] 16. Currently taking drugs that are strong inducers or inhibitors of CYP2D6 and CYP3A4.

[0401] 17. Currently taking warfarin, apixaban, argatroban or rivaroxaban

[0402] 18. Current drug or alcohol abuse

[0403] 19. Currently participating in a clinical study evaluating pharmacological treatment, including antiviral research

[0404] Screening / Baseline Assessment :

[0405] A total of 49 patients were screened in this study, of which 42 patients were randomized (23 received opalib, 19 received placebo), while 7 screening failures occurred. Two patients were randomized in each group but not treated. 19 opalib patients and 16 placebo patients completed the treatment (day 14). Three opalib patients and two placebo patients prematurely discontinued treatment. Two patients in the opalib group experienced adverse events, resulting in the discontinuation of the study drug, while one placebo patient was discontinued due to adverse events.

[0406] Study Assessment :

[0407] · Sign the informed consent form

[0408] · Eligibility determination

[0409] · Complete medical history (including onset of COVID-19 symptoms)

[0410] · Concomitant medication assessment

[0411] · Systemic baseline review

[0412] · Physical examination

[0413] ·Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation measured by pulse oximeter)

[0414] ·Weight while the patient is ambulatory

[0415] ·Oxygen demand (L / min)

[0416] ·12-lead electrocardiogram

[0417] ·Chest X-ray

[0418] ·SARS-CoV-2 PCR test of nasopharyngeal or oropharyngeal swab

[0419] ·Serum chemistry

[0420] ·CRP, D-dimer, LDH, ferritin, cardiac troponin

[0421] ·HbA1c

[0422] ·CBC with differential

[0423] ·Urinalysis

[0424] ·Serum pregnancy test within 3 days prior to treatment (for women of childbearing potential)

[0425] Study Endpoint :

[0426] As part of standard care, the following will be monitored and recorded daily:

[0427] ·Concomitant medications

[0428] ·Adverse events

[0429] ·Interim physical examinations

[0430] ·Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation measured by pulse oximeter)

[0431] ·Oxygen demand (L / min)

[0432] As part of standard care and when possible, the following will be monitored less frequently:

[0433] ·For patients receiving concomitant hydroxychloroquine, a 12-lead electrocardiogram will be performed at approximately 3 hours after the first administration of the study drug on Day 1, at any time on Days 2 and 4, and again at the end of treatment (Day 10, Day 14, or at the premature discontinuation of the study drug) if the hospital treatment guidelines for COVID-19 permit. If the patient is using a monitor (including telemetry or Holter monitor), the investigator is encouraged to collect QT interval data

[0434] · SARS-CoV-2 PCR test of nasopharyngeal or oropharyngeal viral swabs every 1 - 3 days

[0435] · Serum chemistry once a week

[0436] · Serum CRP, D-dimer, LDH, ferritin, cardiac troponin once a week

[0437] · CBC with differential once a week

[0438] · Chest X-ray according to the doctor's decision

[0439] Primary :

[0440] Secondary

[0441] Total oxygen demand (area under the curve) of daily assisted oxygen supply flow rate (L / min) within 14 days (day 1 to day 14)

[0442] Exploratory

[0443] 1) Time when the assisted oxygen supply based on oxygen flow rate in L / min decreases by 50% from baseline

[0444] 2) Percentage of patients who no longer receive assisted oxygen supply for at least 24 hours by day 14

[0445] 3) Time when two consecutive SARS-CoV-2 swabs are negative by PCR with an interval of at least 24 hours

[0446] 4) Percentage of patients with at least two consecutive negative SARS-CoV-2 swabs by PCR on day 14 and subsequent consecutive negative swabs

[0447] 5) Percentage of patients who require intubation and mechanical ventilation by the end of the 2-week drug withdrawal follow-up

[0448] 6) Time to intubation and mechanical ventilation

[0449] 7) Percentage of patients with at least one fever measurement at baseline (defined as body temperature > 38.0°C [100.4°F]) and no fever on day 14 (defined as body temperature < 37.2°C [99°F]) 8) Mortality rate due to any cause on day 30

[0450] Safety

[0451] 1) Mean change of systemic inflammatory markers (D-dimer, cardiac troponin, C-reactive protein [CRP], procalcitonin [PCT], lactate dehydrogenase [LDH], and ferritin) on day 14 relative to baseline

[0452] Statistical Method

[0453] 1) Incidence of all treatment-emergent AEs (TEAEs) and SAEs

[0454] 2) Vital sign assessment

[0455] 3) Laboratory parameter assessment (chemistry and hematology)

[0456] 4) Electrocardiogram (ECG) assessment

[0457] Result :

[0458] The primary efficacy objective of this study was to evaluate the effect of opaganib on total supplemental oxygen requirement (area under the curve) using the daily oxygen flow rate (L / min) measurements over 14 days (Day 1 to Day 14). After subtracting the daily baseline oxygen requirement, the primary efficacy endpoint of the area under the curve of the supplemental oxygen requirement for each patient at Day 14 was calculated using the trapezoidal rule. The number of days without supplemental oxygen requirement was recorded as 0. If several oxygen requirement values (L / min) were recorded on a given day, the highest of these values was taken for the primary analysis. In the primary analysis, for patients who died or required intubation and mechanical ventilation before Day 14, the missing daily values were assigned as the maximum supplemental oxygen flow requirement of 8 L / min. For patients who were discharged from supplemental oxygen before Day 14, if no values were collected at the trial site after discharge, the oxygen requirement (L / min) on the discharge day was assigned for each subsequent day until Day 14.

[0459] The primary analysis was based on the modified intention-to-treat population (mITT), which consisted of all patients randomized and treated with at least one dose of the study drug (this population included a total of 40 subjects, 22 in the opaganib group and 18 in the placebo group). Descriptive statistics of the AUC adjusted for baseline were presented by group, with 95% confidence intervals for the mean differences within each group and between groups. The supplemental oxygen requirement was collected up to Day 14, even if the patient discontinued treatment before Day 14 but remained in the study until Day 14. In addition, it was assumed that it was unlikely that loss to follow-up would result in loss of vital status up to Day 14. Therefore, the primary analysis assumed that in the case of all missing supplemental oxygen values after treatment discontinuation, the last value was carried forward until Day 14, or death (if it occurred earlier). A sensitivity analysis of the above missing data handling method was performed using the AUC summary statistics method, where the group AUC was calculated from the estimated parameters of the repeated measures model.

[0460] In the mITT cohort, two subjects withdrew their consent due to grade 1 gastrointestinal AEs. Additionally, one subject did not require any supplemental oxygen at baseline prior to starting treatment and was removed from several analyses as required by the Statistical Analysis Plan (SAP). Therefore, the post-hoc active analysis population (“mITT sensitivity”) excluded these three patients from the analysis and included 37 subjects, 19 on opaganib and 18 on placebo. The results in the mITT population (including these 3 subjects) and the mITT sensitivity population showed similar trends in activity.

[0461] Figure 10 :

[0462] Top-line results from the study found that opaganib was safe and there were no substantial safety differences between the opaganib and placebo treatment groups. In summary, fewer patients experienced serious adverse events (SAEs) in the opaganib treatment group compared to the placebo group. In this small sample size, intubations or deaths were rare and were balanced between the two groups.

[0463] The opaganib treatment group demonstrated a consistent trend of greater improvement in reducing oxygen demand at the end of treatment on day 14 in key primary and secondary efficacy outcomes, associated with clinical improvement as defined by the World Health Organization (WHO) ordinal scale:

[0464] There was greater improvement in the proportion of patients who achieved room air and no longer required oxygen support by day 14 compared to the control group (52.6% vs. 22.2%). Figure 11 Kaplan-Meier curves (mITT sensitivity) showing the time to no longer receive supplemental oxygen for at least 24 hours are presented.

[0465] There was greater improvement in the proportion of patients with a 50% reduction in supplemental oxygen by day 14 compared to the control group (89.5% vs. 66.7%). Figure 12 Kaplan-Meier curves (mITT sensitivity) showing the cumulative incidence of time to a 50% reduction in supplemental oxygen from baseline based on oxygen flow in L / min are presented.

[0466] There was a higher proportion of patients discharged by day 14 compared to the control group (73.7% vs. 55.6%).

[0467] There was a greater reduction in the mean total oxygen demand (AUC) from baseline over 14 days (68.0% vs. 46.7%). Example 8 Dot plots showing the percentage change in total supplemental oxygen demand (area under the curve) relative to baseline using daily oxygen flow measurements (L / min) over 14 days (day 1 to day 14) are presented.

[0468] Primary Objective:Randomized, Double-Blind, Placebo-Controlled International Phase 2 / 3 Study of Opaganib in COVID-19 Pneumonia

[0469] Secondary Objectives :

[0470] Evaluate the proportion of patients requiring intubation and mechanical ventilation by day 14.

[0471] Exploratory Objectives :

[0472] 1) Evaluate changes in the WHO Ordinal Scale for Clinical Improvement

[0473] 2) Evaluate time to intubation and mechanical ventilation

[0474] 3) Evaluate time to low oxygen flow via nasal cannula, e.g., time from high oxygen flow to low oxygen flow via nasal cannula or CPAP if high oxygen flow is not an available option

[0475] 4) Evaluate the proportion of patients no longer requiring supplementary oxygen for at least 24 hours by day 14

[0476] 5) Evaluate total oxygen requirement (area under the curve) in liters per minute of daily supplementary oxygen flow over 14 days (day 1 to day 14)

[0477] 6) Evaluate time to two consecutive negative SARS-CoV-2 swabs by PCR

[0478] 7) Evaluate the proportion of patients with two consecutive negative SARS-CoV-2 swabs by PCR on day 14

[0479] 8) Evaluate the proportion of patients with at least one fever measurement (defined as body temperature > 38.0°C [100.4°F]) at baseline and no fever (defined as body temperature < 37.2°C [99°F]) on day 14

[0480] 9) Evaluate mortality at 30 days after baseline

[0481] Safety Objectives :

[0482] Evaluate changes in systemic inflammation markers (D-dimer, cardiac troponin, C-reactive protein [CRP], lactate dehydrogenase [LDH], and ferritin) during the 14-day treatment period.

[0483] Study Population :

[0484] Evaluate the safety and tolerability of oral administration of opaganib at 500 mg Q12 hours for up to 14 days in patients with severe COVID-19 pneumonia.

[0485] Study Design and Description :

[0486] The study population will consist of patients diagnosed with COVID-19 infection, which is defined as severe based on eligibility criteria in line with current regional-specific diagnostic guidelines. Specifically, patients will have at least pneumonia secondary to SARS-CoV-2, radiographic evidence of pneumonia on chest X-ray or CT scan, and require high-flow oxygen supplementation via nasal cannula or CPAP if high oxygen flow is not an available option. Patients must be hospitalized at least during the screening period and at baseline (Day 1).

[0487] Randomization Strategy :

[0488] This is a Phase 2 / 3 multicenter, randomized, double-blind, parallel-group, placebo-controlled study with an adaptive design that will utilize futility assessment. This study is planned to be conducted at up to approximately 40 clinical trial sites in Italy, other EU countries, Russia, Brazil, Mexico, and the United States.

[0489] After obtaining informed consent, patients will enter a screening period of up to 3 days to determine eligibility. Approximately 270 eligible patients will be randomized and receive opaganib added to standard of care or a matching placebo added to standard of care at a 1:1 randomization ratio. Treatment assignment will be blinded to patients, investigators, hospital staff, and the sponsor. Since the approvals and / or guidelines for treating COVID-19 are evolving, for this protocol, the standard of care will be determined based on the severity of the disease, in line with local diagnostic and guideline documents such as the Interim Methodological Recommendations: Prevention, Diagnosis, and Treatment of Coronavirus Disease (COVID-19); the European Commission, European Medicines Agency (EMA), Heads of Medicines Agencies (HMA), and FDA, and as updated to the most recent version of the recommendations, via the recommended treatment regimens.

[0490] Study participants will receive 2 x 250 mg capsules (500 mg) of opaganib or a matching placebo every 12 hours at any given institution in addition to standard of care (pharmacological and / or supportive as defined above). The study drug will be administered daily for 14 days (Day 1 to Day 14). All participants will be followed up for 28 days after the last study drug administration, which may occur on Day 14 or after premature discontinuation of the study drug, at the discretion of the patient or the physician.

[0491] Adaptive Interim Analysis for Futility :

[0492] Since the treatments in the treatment regimens recommended according to disease severity may differ, based on local diagnoses and guideline documents, such as the interim approach recommendations: Prevention, Diagnosis, and Treatment of Coronavirus Disease (COVID-19); European Commission, European Medicines Agency (EMA), Heads of Medicines Agencies (HMA), and FDA, the standard of care given to patients may vary by institution. To ensure balance in the standard treatment regimens across the two treatment groups, randomization will be determined at the level of individual trial centers.

[0493] Data Safety Monitoring Board :

[0494] After the primary endpoint has been evaluated in approximately 100 subjects (approximately 50 subjects per group), a non-blind futility interim analysis will be conducted to determine the probability of rejecting the futility null hypothesis and whether it is futile to continue the study. The criteria will be determined prospectively and documented in the final version of the Statistical Analysis Plan (SAP) prior to the interim analysis.

[0495] Stratification :

[0496] A Data Safety Monitoring Board (DSMB) will be convened for safety oversight of the study to ensure the safety of trial participants. DSMB meetings are planned at 25%, 50%, and 75% respectively or when approximately 70, 135, and 200 randomized patients reach Day 7 and then Day 14 to review safety data. The DSMB is also responsible for communicating the results (futility / non-futility) of the futility analysis conducted by an independent non-blind statistician to the sponsor.

[0497] Treatment and Administration :

[0498] Patients will be stratified based on three or more high-risk clinical parameters (yes or no) that are consistent with COVID-19 outcomes at baseline. The parameters are: 1) age ≥60 years at screening (yes or no); 2) male (yes or no); 3) HbA1c ≥6.5 at screening (yes or no); 4) hypoxemia without a corresponding increase in work of breathing (defined as an increased respiratory rate, nasal flaring, and / or increased use of respiratory muscles including the diaphragm) [yes or no]; 5) known underlying chronic lung disease (yes or no); 6) known cardiovascular disease or hypertension (yes or no); 7) BMI ≥28.0 kg / m 2 (yes or no); 8) known kidney disease (yes or no).

[0499] Study Duration :

[0500] Olaparib 500 mg Q12 hours or matching placebo. Olaparib or placebo in suspension form can be administered to the patient's stomach via a nasogastric tube.

[0501] Eligibility Criteria :

[0502] The maximum duration of participation in the study will be up to 45 days (including up to 3 days of screening period; up to 14 days of double-blind treatment and 28 days of follow-up after drug withdrawal).

[0503] Inclusion :

[0504] Exclusion :

[0505] 1. Adult males or females aged ≥18 years to ≤80 years

[0506] 2. COVID-19 infection confirmed by RT-PCR assay of pharyngeal samples (nasopharyngeal or oropharyngeal), and pneumonia defined as radiographic opacities on chest X-ray or CT scan.

[0507] 3. If high oxygen flow is not an available option, the patient requires high-flow assisted oxygenation or CPAP at baseline.

[0508] 4. The patient agrees to use appropriate contraceptive methods during the study period and for 3 months after the last study drug administration

[0509] 5. The patient or legal representative has signed an IRB / ethics committee-approved written informed consent form Screening / Baseline Assessment :

[0510] 1. Any comorbidities that the investigator judges may increase the risk of treatment.

[0511] 2. Requiring intubation and mechanical ventilation

[0512] 3. Indoor air oxygen saturation >95%

[0513] 4. Any pre-existing respiratory disease requiring intermittent or continuous dynamic oxygen before hospitalization

[0514] 5. The patient is unlikely to survive >72 hours according to the investigator's clinical judgment

[0515] 6. Pregnancy (serum or urine test positive within 3 days before randomization) or lactating females.

[0516] 7. Unwilling or unable to comply with the procedures required by this protocol.

[0517] 8. Corrected QT (QTc) interval on electrocardiogram (ECG) calculated using the Friedericia formula (QTcF) >470 ms for females or >450 ms for males

[0518] 9. AST (SGOT) or ALT (SGPT) >2.5 × upper limit of normal (ULN)

[0519] 10. Total bilirubin > 1.5 × ULN (except for cases of increased bilirubin due to Gilbert syndrome)

[0520] 11. Serum creatinine > 2.0 × ULN

[0521] 12. Absolute neutrophil count < 1000 cells / mm 3

[0522] 13. Platelet count < 75,000 / mm 3

[0523] 14. Hemoglobin < 8.0 g / dL

[0524] 15. Currently taking a drug that is sensitive to CYP3A4, CYP2C9, or CYP2C19 substrates and has a narrow therapeutic index

[0525] 16. Currently taking a drug that is a strong inducer or inhibitor of CYP2D6 and CYP3A4

[0526] 17. Currently taking warfarin, apixaban, argatroban, or rivaroxaban due to drug-drug interactions based on CYP450 metabolism

[0527] 18. Current drug or alcohol abuse

[0528] 19. Currently participating in a clinical study evaluating pharmacological treatment, including antiviral studies Study Assessment :

[0529] · Informed consent form signed by the patient or legal representative

[0530] · Eligibility determination

[0531] · Complete medical history (including onset of COVID-19 symptoms)

[0532] · Concomitant medication assessment

[0533] · Systemic baseline review

[0534] · Physical examination

[0535] · Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation measured by pulse oximeter)

[0536] · Weight while the patient is ambulatory

[0537] · Oxygen demand (L / min)

[0538] · FiO2 (estimated value)

[0539] · 12-lead electrocardiogram

[0540] · Chest X-ray or CT scan

[0541] · SARS-CoV-2 PCR test of nasopharyngeal or oropharyngeal swab

[0542] · Serum chemistry

[0543] · CRP, D-dimer, LDH, ferritin, cardiac troponin

[0544] · HbA1c

[0545] · CBC with differential

[0546] · Urinalysis

[0547] · Serum or urine pregnancy test within 3 days before treatment (for women of childbearing potential)

[0548] Study Endpoint :

[0549] As part of standard care, the following will be monitored and recorded daily:

[0550] · Concomitant medications

[0551] · Adverse events

[0552] · Interim physical examinations

[0553] · Vital signs (temperature, blood pressure, pulse rate, respiratory rate, and oxygen saturation measured by pulse oximeter)

[0554] · Oxygen flow rate setting (L / min)

[0555] · FiO2 (estimated or known if the patient is ventilated)

[0556] As part of standard care and where possible, the following will be monitored less frequently:

[0557] · For patients taking chloroquine / hydroxychloroquine / mefloquine concomitantly, a 12-lead electrocardiogram will be performed at approximately 3 hours after the first administration of the study drug on Day 1, at any time on Days 2 and 4, and again at the end of treatment (Day 14 or at premature discontinuation of the study drug) if permitted by the hospital treatment guidelines for COVID-19. If the patient is using a monitor (including telemetry or Holter monitor), the investigator is encouraged to collect QT interval data

[0558] · SARS-CoV-2 PCR test of nasopharyngeal or oropharyngeal viral swabs every 3 days

[0559] · Serum chemistry once a week

[0560] · Serum CRP, D-dimer, LDH, ferritin, cardiac troponin once a week

[0561] · CBC with differential once a week

[0562] · Chest X-ray or CT scan as per doctor's decision

[0563] Primary :

[0564] Secondary

[0565] Percentage of patients requiring intubation and mechanical ventilation by day 14

[0566] Exploratory

[0567] 1) Percentage of patients with ≥ 2 categories of improvement on the WHO Clinical Improvement Ordinal Scale

[0568] 2) Duration of intubation and mechanical ventilation

[0569] 3) Time to low oxygen flow via nasal intubation, e.g., time from nasal intubation or CPAP from high to low oxygen flow if high oxygen flow is not an available option

[0570] 4) Percentage of patients no longer receiving supplementary oxygen for at least 24 hours by day 14

[0571] 5) Total oxygen requirement (area under the curve) in litres per minute of supplementary oxygen used per day over 14 days (day 1 to day 14)

[0572] 6) Time to two consecutive negative SARS-CoV-2 swabs at least 24 hours apart by PCR

[0573] 7) Percentage of patients with at least two consecutive negative SARS-CoV-2 swabs by PCR on day 14

[0574] 8) Percentage of patients with at least one fever measurement at baseline (defined as body temperature > 38.0°C [100.4°F]) and afebrile on day 14 (defined as body temperature < 37.2°C [99°F])

[0575] 9) Mortality rate due to any cause on day 30 after baseline

[0576] Safety

[0577] Mean change in systemic inflammation markers (D-dimer, cardiac troponin, C-reactive protein [CRP], procalcitonin [PCT], lactate dehydrogenase [LDH], and ferritin) on day 14 relative to baseline

[0578] Medications Disabled during the Study

[0579] 1) Incidence of all treatment-emergent AEs (TEAEs) and SAEs

[0580] 2) Vital sign assessment

[0581] 3) Laboratory parameter assessment (chemistry and hematology)

[0582] 4) Electrocardiogram (ECG) assessment

[0583] Stopping Rules :

[0584] The following drugs are prohibited during the study period (including the 28-day follow-up period):

[0585] · Drugs sensitive to CYP3A4, CYP2C9 or CYP2C19 substrates with a narrow therapeutic index are prohibited

[0586] · Strong inducers or inhibitors of CYP2D6 and 3A4 are prohibited

[0587] Warfarin, apixaban, argatroban and rivaroxaban are prohibited due to drug-drug interactions based on CYP450 metabolism

[0588] Statistical Method :

[0589] At any time during the study period, if it is determined that a participant has experienced any of the following adverse events, they will discontinue the study drug (using the rating criteria defined in the revised NCI Common Terminology Criteria for Adverse Events [CTCAE v.5.0])

[0590] · Any neuropsychiatric adverse event of grade 3 severity

[0591] · Hallucinations of any severity (any grade)

[0592] · Nausea of grade 3 severity

[0593] · Vomiting of grade 3 severity

[0594] · Elevated creatinine of grade 2 severity

[0595] DSMB Futility Review :

[0596] The primary analysis will be based on a composite failure (yes / no) variable indicating whether the subject requires intubation and mechanical ventilation or death by day 14 of the study

[0597] In rare cases where patient outcomes are unknown (patient lost to follow-up), for the primary analysis, this will be counted as treatment failure. If a patient starts a new investigational treatment for COVID-19 within 14 days, this will also be considered treatment failure in the primary analysis.

[0598] The number and percentage of subjects with failure events in each treatment group will be tabulated. A 95% confidence interval will be constructed for each proportion. The Cochran Mantel-Haenzel (CMH) test will compare the failure proportions between the two groups using the study stratification factors used for randomization, and the corresponding risk difference estimate will be presented with a 95% confidence interval. Exact confidence intervals will be used as needed.

[0599] The significance level of this test will be 5% two-sided. In cases where there are a small number of events (less than 5 events in any study group), Fisher's exact test will be used.

[0600] The number and percentage of each type of failure (intubation and mechanical ventilation) will be described by group.

[0601] The primary analysis will be based on the modified intention-to-treat population (mITT), which consists of all patients who were randomized and treated with at least one dose of the study drug.

[0602] Example 9

[0603] In January 2021, the independent Data Safety Monitoring Board (DSMB) unanimously recommended continuing the study after a pre-planned futility review of the unblinded efficacy data for the first 135 patients treated in the study and the safety data for the first 175 patients.

[0604] Primary Objective : A 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

[0605] This study will evaluate the activity of opaganib compared to placebo in treating COVID-19 patients who, according to the investigator's judgment, do not require hospitalization.

[0606] Secondary Objectives :

[0607] Opaganib and placebo will be compared over the time to sustained recovery from the disease. Once a patient meets the following criteria, they will be considered cured:

[0608] 1) Afebrile (core temperature < 38.0°C) for at least 48 hours without the use of antipyretics;

[0609] 2) All symptoms have resolved or returned to pre-illness levels, except in the following cases (e.g., if the patient had baseline respiratory impairment prior to the onset of COVID-19):

[0610] a. Fatigue, anosmia, ageusia, or dysgeusia, which may persist at a level similar to that during the acute illness, i.e., at the same level on the symptom questionnaire;

[0611] b. Chest pain, cough, or dyspnea, if persistent, must be at least one grade lower than at the start of treatment and no worse than grade 1 (mild).

[0612] Persistent recovery, as defined above, means recovery maintained for at least 28 days or until the end of the study (whichever occurs first).

[0613] Exploratory :

[0614] Comparison between the active treatment group and the placebo group:

[0615] 1) The proportion of patients who are PCR-negative on days 8, 15, 29, and 57 after the start of treatment (landmark analysis);

[0616] 2) The time to resolution of individual disease-related symptoms present at baseline;

[0617] 3) The development of new disease-related symptoms during the study;

[0618] 4) The incidence of pneumonia in patients without baseline pneumonia during the study (clinical diagnosis);

[0619] 5) Changes in laboratory markers of disease severity (i.e., oxygen saturation, CRP, lymphocyte count, cardiac troponin, and D-dimer levels) from baseline to the time points during the study when these are measured;

[0620] 6) Adverse events;

[0621] 7) Hospitalization within 8 weeks after the first dose of the study medication, overall and for COVID-19-related indications;

[0622] 8) Mortality at 30 days after the first dose of the study medication;

[0623] Safety:

[0624] 1) The percentage of patients whose household contacts reported having developed symptomatic, PCR-confirmed COVID-19 by day 57;

[0625] 2) The levels of serum IgM and IgG antibodies against SARS-CoV-2 on day 57 after the start of treatment.

[0626] Population

[0627] Throughout the study, adverse events in the patients will be followed up, including clinical and laboratory events.

[0628] In particular, toxicities leading to dose reduction or treatment discontinuation will be tracked and tabulated.

[0629] Inclusion Criteria :

[0630] Exclusion Criteria :

[0631] 1. Based on RT-PCR assay of respiratory samples, the patient is symptomatic and is diagnosed as COVID-19.

[0632] 2. The patient must be symptomatic within 3 days of randomization or positive by RT-PCR, whichever is greater.

[0633] 3. Males and females are aged ≥ 18 years.

[0634] 4. At baseline, the following laboratory parameters are no worse than grade 2 of NCI CTCAE v5.0, with the following exceptions:

[0635] - Bilirubin ≤ 1.5 times the upper limit of normal (ULN; only grade 1)

[0636] - AST (SGOT), ALT (SGPT) ≤ 5.0 × ULN

[0637] - Serum creatinine ≤ 1.5 × ULN (grade 1)

[0638] - Albumin ≥ 2.0 g / dL

[0639] - Albumin ≥ 2.0 g / dL

[0640] 5. Acceptable hematological status:

[0641] - Absolute neutrophil count ≥ 1000 cells / mm 3

[0642] - Platelet count ≥ 50,000 plt / mm 3

[0643] - Hemoglobin ≥ 8.0 g / dL

[0644] 6. Clinically acceptable blood glucose control as considered by the investigator.

[0645] 7. INR and partial thromboplastin time (PTT) are each ≤ ± 1.5 × ULN (i.e., grade 1), unless the patient is taking dabigatran or heparin.

[0646] 8. Indoor air oxygen saturation measured by pulse oximeter ≥ 92%

[0647] 9. Negative pregnancy test (if the female is fertile).

[0648] 10. Females of reproductive potential and males with female partners of reproductive potential must agree to use acceptable contraceptive methods during the study period and for at least two months after the last study drug administration.

[0649] 11. Able to complete the daily diary independently.

[0650] 12. The patient must give informed consent.

[0651] Design :

[0652] 1. According to the clinician's assessment, the patient requires acute hospitalization.

[0653] 2. Pregnant or lactating females.

[0654] 3. Unwilling or unable to comply with the procedures required by this protocol.

[0655] 4. The patient requires supplementary oxygen

[0656] 5. The patient is currently receiving, has received within the past 7 days, or is expected to receive remdesivir, chloroquine, hydroxychloroquine, azithromycin, or other specific antiviral therapies for COVID-19 or systemic corticosteroids equivalent to ≥ 20 mg prednisone / day / 3 mg dexamethasone / day during the course of the study.

[0657] 6. The patient is currently receiving or has received within 30 days prior to screening for any indication of any other investigational agent, including approved drugs administered for investigational indications (e.g., anti-cytokine therapy).

[0658] 7. The patient is currently taking or is expected to start taking warfarin, apixaban (Eliquis), or rivaroxaban (Xarelto). The patient may be taking or starting to take study dabigatran (Pradaxa), standard or low molecular weight heparin.

[0659] ​ :

[0660] This is a randomized, double-blind, placebo-controlled, parallel-group study of opaganib compared to placebo in patients with symptomatic COVID-19 who do not require hospitalization. The study will include an interim analysis for early termination for futility or sample size increase, as shown by the preliminary results.

[0661] Methodology :

[0662] Part B: Study participants will receive Opaganib 2 x 250 mg capsules (500 mg) every 12 hours, or a matching placebo. Patients will be stratified by the number of the following conditions (none, one, or more than one): age ≥ 65, presence of the following medical conditions: hypertension, chronic lung disease, obesity [BMI ≥ 30], diabetes, heart failure, coronary artery disease, thromboembolic events (current or history), kidney disease. Patients will also be stratified by the region in which they are being treated (US vs non-US). They will then be randomly assigned 1:1 to active drug or placebo. Patients will complete a daily questionnaire on symptoms (including adverse events), vital signs (including temperature and pulse oximetry), and a record of the medications taken, once daily for the first 4 weeks of the study and then three times a week thereafter. Viral swabs and blood for safety laboratories and pharmacodynamic markers will be obtained by medical staff during home visits. After treatment completion, patients will be followed for 57 days from randomization.

[0663] Medications Disabled During the Study :

[0664] The following medications are prohibited during the study (including the 28-day follow-up period):

[0665] · Medications that are sensitive to CYP3A4, CYP2C9, or CYP2C19 substrates and have a narrow therapeutic index are prohibited

[0666] · Strong inducers or inhibitors of CYP2D6 and 3A4 are prohibited

[0667] Warfarin, apixaban, argatroban, and rivaroxaban are prohibited due to drug-drug interactions based on CYP450 metabolism

[0668] Example 10 : A randomized, double-blind, placebo-controlled, phase 2 / 3 study of ulimorelin (a serine protease inhibitor) or placebo for the treatment of COVID-19 disease

[0669] This study will evaluate the activity of ulimorelin compared to placebo in treating COVID-19 patients who, in the investigator's judgment, do not require hospitalization.

[0670] Primary Objective :

[0671] Part A of the study: Determine the safety and tolerability of two dose levels and decide on the ulimorelin dose for Part B. Changes in the severity of disease markers will be evaluated, but are not the primary factor in deciding which dose to pursue. Recovery time will also be calculated, although given the small sample size and expected outcome variability, clinically meaningful differences may not be seen.

[0672] Part B of the study: Compare ulimorelin and placebo during the period of sustained recovery from the disease. Once a patient meets the following criteria, they will be considered cured:

[0673] 1) Absence of fever (core temperature < 38.0°C) for at least 48 hours without the use of antipyretics;

[0674] 2) All symptoms have resolved or returned to pre-illness levels, except for the following (e.g., if the patient had baseline respiratory impairment before the onset of COVID-19):

[0675] c. Fatigue, anosmia, ageusia, or dysgeusia, which may persist at a level similar to that during the acute illness, i.e., at the same level as on the symptom questionnaire;

[0676] d. Chest pain, cough, or dyspnea, if persistent, must be at least one grade lower than at the start of treatment and no worse than grade 1 (mild).

[0677] According to the above definition, sustained recovery refers to recovery maintained for at least 28 days or until the end of the study (whichever occurs first).

[0678] Secondary Objective :

[0679] Comparison between the Active Treatment Group and the Placebo Group :

[0680] 1) The proportion of patients who are PCR-negative on days 8, 15, 29, and 57 after the start of treatment (landmark analysis);

[0681] 2) The time to resolution of individual disease-related symptoms present at baseline;

[0682] 3) The development of new disease-related symptoms during the study;

[0683] 4) The incidence of pneumonia in patients without baseline pneumonia during the study (clinical diagnosis);

[0684] 5) Changes in laboratory markers of disease severity (i.e., oxygen saturation, CRP, lymphocyte count, cardiac troponin, and D-dimer levels) from baseline to the time points during the study when these are measured;

[0685] 6) Adverse events;

[0686] 7) Hospitalization within 8 weeks after the first administration of the study drug, overall and for COVID-19-related indications;

[0687] 8) Mortality within 30 days after the first administration of the study drug;

[0688] Exploratory

[0689] 1) Percentage of patients reporting symptomatic, PCR-confirmed COVID-19 among household contacts by day 57;

[0690] 2) Levels of serum IgM and IgG antibodies against SARS-CoV-2 at day 57 after treatment initiation.

[0691] Safety

[0692] Adverse events, including clinical and laboratory events, will be followed up in patients throughout the study.

[0693] In particular, toxicities leading to dose reduction or treatment discontinuation will be tracked and tabulated.

[0694] Population :

[0695] Inclusion Criteria :

[0696] 1. The patient is symptomatic and diagnosed with COVID-19 based on RT-PCR assay of respiratory samples.

[0697] 2. The patient must be symptomatic within 3 days of randomization or found positive by RT-PCR, whichever is greater.

[0698] 3. Males and females aged ≥ 18 years.

[0699] 4. At baseline, the following laboratory parameters are no worse than grade 2 of NCI CTCAE v5.0, with the following exceptions:

[0700] - Bilirubin ≤ 1.5 times the upper limit of normal (ULN; grade 1 only)

[0701] - AST (SGOT), ALT (SGPT) ≤ 5.0 × ULN

[0702] - Serum creatinine ≤ 1.5 × ULN (grade 1)

[0703] - Albumin ≥ 2.0 g / dL

[0704] 5. Acceptable hematological status:

[0705] - Absolute neutrophil count ≥ 1000 cells / mm 3

[0706] - Platelet count ≥ 50,000 plt / mm 3

[0707] - Hemoglobin ≥ 8.0 g / dL

[0708] 6. Clinically acceptable blood glucose control as determined by the investigator.

[0709] 7. INR and partial thromboplastin time (PTT) each ≤ ±1.5 × ULN (i.e., Grade 1), unless the patient is taking dabigatran or heparin.

[0710] 8. Indoor air oxygen saturation measured by pulse oximetry ≥ 92%.

[0711] 9. Negative pregnancy test (if female is fertile).

[0712] 10. Fertile females and males with fertile female partners must agree to use acceptable contraceptive methods during the study and for at least two months after the last study drug administration.

[0713] 11. Able to complete a daily diary independently.

[0714] 12. The patient must give informed consent.

[0715] Exclusion Criteria :

[0716] 1. The patient requires acute hospitalization as evaluated by the clinician.

[0717] 2. Pregnant or lactating females.

[0718] 3. Unwilling or unable to comply with the procedures required by this protocol.

[0719] 4. The patient requires assisted oxygenation

[0720] 5. The patient is currently receiving, has received within the past 7 days, or is expected to receive during the course of the study remdesivir, chloroquine, hydroxychloroquine, azithromycin, or other specific antiviral therapies for COVID-19 or systemic corticosteroids equivalent to ≥ 20 mg prednisone / day / 3 mg dexamethasone / day.

[0721] 6. The patient is currently receiving or has received within 30 days prior to screening for any indication of any other investigational agent, including approved drugs administered for investigational indications (e.g., anti-cytokine therapy).

[0722] 7. The patient is currently taking or is expected to start taking warfarin, apixaban (Eliquis), or rivaroxaban (Xarelto). The patient may be taking or starting to take investigational dabigatran (Pradaxa), standard or low molecular weight heparin.

[0723] Design :

[0724] This is a randomized, double-blind, placebo-controlled, parallel-group study of ulimorelin compared to placebo in patients with symptomatic COVID-19 who do not require hospitalization. This study will use a seamless Phase 2 / 3 operational design approach for dose selection (Part A) and an inference-independent confirmatory Phase 3 study (Part B). The Phase 3 portion will include interim analyses for early termination for futility or sample size increase as shown in the preliminary results.

[0725] Methodology :

[0726] Part A: After study eligibility, patients will be stratified by age <65 or ≥65. They will then be randomly assigned 1:1:1 to one of the following treatment groups:

[0727] 1. Two capsules of ulimorelin 200 mg qd (n = 20);

[0728] 2. One capsule of ulimorelin 200 mg and one matching placebo capsule qd (n = 20)

[0729] 3. Two placebo capsules qd (n = 20).

[0730] To maintain blinding, patients will be given two bottles of medication and instructed to take one pill from each bottle daily. The two pills will be taken simultaneously.

[0731] The medication should be taken with water and may be taken with or without food.

[0732] Patients will take the medication for 14 days or until one of the following occurs:

[0733] · An adverse event that requires treatment discontinuation according to the investigator's judgment, whether related or not related to the study medication;

[0734] · The patient or investigator decides to stop treatment in the best interest of the patient.

[0735] After a total of 60 patients complete Part A, an interim analysis will be conducted by the Data Safety Monitoring Board (DSMB).

[0736] · If the DSMB determines that the safety of the two regimens is similar, the 400 mg qd dose will continue to be accrued in Part B.

[0737] · If the 200 mg qd regimen is more favorable in terms of safety, the 200 mg qd dose will continue to be accrued in Part B.

[0738] PART B: Based on the safety results of PART A, a treatment regimen of 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 more): age ≥ 65, presence of the following medical conditions: hypertension, chronic lung disease, obesity [BMI ≥ 30], diabetes, heart failure, coronary artery disease, thromboembolic events (current or history), kidney disease. Patients will also be stratified by the region in which they are treated (United States vs. non-United States). They will then be randomly assigned to the active drug or placebo in a 3:2 ratio according to the schedule selected based on PART A. In PART B of this study, a total of approximately 250 additional patients will be enrolled, of whom 150 will receive the active drug and 100 will receive the placebo. Thus, combining the two parts of the study, a total of 170 patients will receive the active agent at the dose selected in PART A, and 120 will receive the placebo. However, the analyses of PART A and PART B will be performed independently.

[0739] Patients will complete a daily questionnaire on symptoms (including adverse events), vital signs (including body temperature and pulse oximetry), and a record of the medications taken, once daily for the first 4 weeks of the study and then three times a week. Viral swabs and blood for safety laboratories and pharmacodynamic markers will be obtained by medical staff during home visits. After treatment completion, patients will be followed up for 57 days from randomization.

[0740] Statistics :

[0741] In PART A of this study, two dose levels of the active drug and placebo will be tested. Based on the incidence and severity of toxicity in each active group and the overall assessment of safety by the DSMB, the protocol for PART B of this study will be selected. In the case of no significant difference in toxicity between the two active groups, the default selection for proceeding to PART B is the 400 mg daily regimen.

[0742] The efficacy data from PART A and PART B will be analyzed separately.

[0743] The overall sample size may be expanded based on the results of the interim study.

[0744] The sample size is determined based on the primary endpoint, i.e., the time to sustained recovery from COVID-19 disease, as defined in the primary objective. It is calculated that in order to detect a hazard ratio = 1.5, comparing the active group with the placebo group at a 3:2 allocation ratio, a total of 201 recovery events are required to provide 80% power using a log-rank test with a two-sided significance level of 0.05. Assuming a sustained recovery rate of 80% at the end of the follow-up (assuming equal follow-up rates for all enrolled patients), the minimum number of patients to be enrolled in PART B will be a total of 250, of whom 150 will be in the active group of the regimen adopted in PART B of the study and 100 will be in the placebo group.

[0745] Industrial Applicability

[0746] The present invention provides an anti-coronavirus agent, which contains a compound represented by the following formula as an active ingredient: As a free base or its salt, an anti-SARS agent containing the anti-coronavirus agent; and a method for treating SARS using the anti-coronavirus agent. The present invention can treat diseases caused by coronaviruses, especially SARS-related coronaviruses.

[0747] The present invention provides an anti-coronavirus agent, which contains a compound represented by the following formula as an active ingredient:

[0748] As the (L)- or (D)-enantiomer and as the E- or Z-isomer or (E / Z)-mixture and as a free base or as its salt; an anti-SARS agent containing the anti-coronavirus agent; and a method for treating SARS using the anti-coronavirus agent. The present invention can treat diseases caused by coronaviruses, especially SARS-related coronaviruses.

[0749] All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. Various modifications and variations of the compositions and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific embodiments, it should be understood that the present invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the present invention that are obvious to those skilled in the fields of molecular biology, medicine, immunology, pharmacology, virology, or related fields are intended to be within the scope of the present invention.

Claims

1. Use of WX-671.1 in the preparation of a medicament for treating coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus, the structure of said WX-671 being 2. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier material and is in a unit dosage form suitable for oral administration.

3. The use according to claim 2, wherein the unit dosage form is a solid dosage form.

4. The use according to claim 3, wherein the solid dosage form is a capsule.

5. The use according to claim 1, wherein the SARS-CoV-2 virus is wild-type.

6. The use according to claim 1, wherein the SARS-CoV-2 virus is a naturally occurring coronavirus variant.

7. The use according to claim 1, wherein the medicament is a capsule having 231 mg of WX-671.1, and wherein a single capsule is administered once a day for at least 10 days, with a total daily dose of 231 mg of WX-671.

1.

8. The use according to claim 1, wherein the medicament is a capsule having 231 mg of WX-671.1, and wherein two capsules are administered once a day for at least 10 days, with a total daily dose of 463 mg of WX-671.1.

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