COMPOSITIONS FOR AND METHODS OF INHIBITING SARS-COV-2 INFECTION

BACKGROUND

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a novel RNA betacoronavirus, is the causative agent for coronavirus disease 2019 (COVID-19), which has emerged as an ongoing global pandemic.¹⁰⁴Worldwide, SARS-CoV-2 has spread rampantly to more than 188 countries/regions and has resulted in over 200 million confirmed cases, including over 4.35 million deaths. In the United States, there have been more As of August 2021, there has been more than 37 million cases and 635,000 deaths, in the United States alone. About 80% of people infected with SARS-CoV-2 experience mild symptoms or are asymptomatic.¹⁰³A majority of symptomatic patients with moderate to severe symptoms have shown a broad range of clinical manifestation and/or significant complications, including severe pneumonia, multi-organ failure, acute cardiac injury, neurological damage, septic shock, acute respiratory distress syndrome (ARDS).^{38, 55, 61, 116,}Case tracking has revealed that individuals with pre-existing medical conditions have increased risk of COVID-19 related morbidity and mortality.³⁰

Currently, the has approved only one drug for the treatment of COVID-19, remdesivir. Although there are several studies are investigating the potential utility of repurposing clinically approved drugs as treatment options for COVID-19,^{36, 79, 83, 88, 95}the U.S. Food and Drug Administration (FDA) has approved only Remdesivir, an inhibitor of RNA dependent RNA Polymerase, and has granted emergency use authorization (EUA) for the rheumatoid arthritis drug baricitinib (Olumiant) for the treatment of hospitalized patients with severe cases of COVID-19.¹⁹

Despite advances in the understanding of the pathology of coronaviruses including SARS-CoV-2, there is still a need for compositions and methods that efficiently treat or prevent the development, progression, and reoccurrence of coronavirus infections including SARS-CoV-2 infections.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1C shows the efficacy of clioquinol (CLQ) and analogues against SARS-CoV-2 induced cytopathic effect (CPE) in Vero E6 cells: (A) CLBQ14, (B). CLCQ, and (C) CLQ.

FIG. 2A-2E shows the efficacy of reference inhibitors against SARS-CoV-2 induced cytopathic effect (CPE) in Vero E6 cells: (A) Calpain Inhibitor IV, (B) Chloroquine, (C) Remdesivir, (D) Hydroxychloroquine, and (E) E64d (Aloxistatin).

FIG. 3 shows the effect of clioquinol (CLQ) and analogues against ACE2 exopeptidase activity: (A) CLBQ14 (circles—red), (B) CLQ (squares—green), and (C) ZnCl₂(triangle—blue), and (D) CLBQ14 and ZnCl₂(inverted triangles—magenta).

FIG. 4A-4C shows the inhibition of ACE2 and SARS-CoV-2 Spike (RBD) protein interaction by clioquinol (CLQ) and analogues: (A) CLBQ14, (B) CLCQ, and (C) CLQ.

FIG. 5A-5B shows the effect of (A) Bromhexine Hydrochloride (BHH) and (B) Ambroxol Hydrochloride (AMB) on the interaction of rhACE2 with SARS-CoV-2 Spike (RBD) Glycoprotein Interaction.

FIG. 6 shows the chemical structure and activity of Ambroxol Hydrochloride (AMB) and Bromhexine Hydrochloride (BHH) against SARS-CoV-2 induced Cytopathic Effect (CPR) and Vero E6 Cells.

FIG. 7 shows chemical structure and activity of reference inhibitors against SARS-CoV-2 induced Cytopathic Effect (CPE) in vero E6 cells.

FIG. 8 shows cytotoxicity of Ambroxol Hydrochloride (AMB) and Bromhexine Hydrochloride (BHH) in vero E6 cells, in comparison to reference inhibitors of SARS-CoV-2.

FIG. 9 shows activity of Ambroxol Hydrochloride (AMB) and Bromhexine Hydrochloride (BHH) against rhACE2 and SARS-CoV-2 spike (RBD) glycoprotein interaction.

FIGS. 10A & 10B depict inhibition of the interaction between rhACE2 and SARS-CoV-2 Spike (RBD) protein by AMB (FIG. 10A) and BHH (FIG. 10B).

FIGS. 11A & 11B depict inhibition of exopeptidase activity of ACE (FIG. 11A) and ACE2 (FIG. 11B) where BHH (OJT009) inhibited the exopeptidase activity of ACE and ACE2 at high concentration while AMB (OJT010) did not inhibit either ACE or ACE2.

BRIEF SUMMARY

Disclosed herein is a method comprising inhibiting or ameliorating a SARS-CoV-2 infection by administering a composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class.

Disclosed herein is a method comprising inhibiting or ameliorating a SARS-CoV-2 infection by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; and inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising prophylactically administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method comprising inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in the subject.

Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in the subject by administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or reducing exopeptidase activity of an enzyme comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

Disclosed herein is a method of inhibiting or reducing exopeptidase activity of an enzyme comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; administering a composition comprising an effective amount of zinc chloride; and inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

Disclosed herein is a method of inhibiting or reducing exopeptidase activity of an enzyme comprising inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is method of inhibiting or reducing exopeptidase activity of an enzyme comprising inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof and by administering a composition comprising an effective amount of zinc chloride.

Disclosed herein is a method comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2.

Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject.

Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing viral entry into cells of the subject.

Disclosed herein is composition comprising a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection.

Disclosed herein is composition comprising a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; an effective amount of one or more clinically approved active agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection.

Disclosed herein is a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more clinically approved active agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection.

Disclosed herein is a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects.

Disclosed herein is a composition for inhibiting or ameliorating cytopathic effects in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject in need thereof.

Disclosed herein is a composition for inhibiting or ameliorating cytopathic effects in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject in need thereof.

Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject diagnosed with or suspected of having a SARS-CoV-2 infection.

Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer; wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject diagnosed with or suspected of having a SARS-CoV-2 infection.

Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and an effective amount of zinc chloride; wherein the composition inhibits or reduces the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and an effective amount of zinc chloride, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

Disclosed herein is a composition for inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a composition for inhibiting or reducing viral infectivity in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a composition for inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a composition for inhibiting or reducing viral entry into cells of a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and wherein the composition inhibits or disrupts they physical interactions of angiotensin converting enzyme 2 (ACE2) and the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject.

Disclosed herein is a method comprising administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; administering one or more clinically approved active agents; and inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method comprising inhibiting or ameliorating a SARS-CoV-2 infection by administering a composition comprising one or more compounds belonging to the benzylamine structural class.

Disclosed herein is a method comprising inhibiting or ameliorating a SARS-CoV-2 infection by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection.

A method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; and inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising prophylactically administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method comprising inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in the subject.

Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in the subject by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or reducing exopeptidase activity of an enzyme comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

Disclosed herein is a method of inhibiting or reducing exopeptidase activity of an enzyme comprising inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2.

Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject.

Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing viral entry into cells of the subject.

Disclosed herein is a method comprising administering a composition comprising an effective amount AMB or BHH, or analogs or derivatives thereof, or a combination thereof; inhibiting or reducing the activity of a type II transmembrane serine protease; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2.

Disclosed herein is a method comprising inhibiting or reducing the activity of a type II transmembrane serine protease and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or reducing the activity of a type II transmembrane serine protease, thereby inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2).

Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, or a combination thereof; inhibiting or reducing the activity of a type II transmembrane serine protease; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising inhibiting or reducing the activity of a type II transmembrane serine protease by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a method of inhibiting or reducing a SARS-CoV-2 infection in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; inhibiting or reducing the activity of a type II transmembrane serine protease; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or reducing a SARS-CoV-2 infection in a subject comprising inhibiting or reducing the activity of a type II transmembrane serine protease and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing a SARS-CoV-2 infection.

Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; inhibiting or reducing the activity of a type II transmembrane serine protease; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject.

Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, and inhibiting or reducing the activity of a type II transmembrane serine protease, thereby inhibiting or reducing viral entry into cells of the subject.

Disclosed herein is a composition comprising administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection.

Disclosed herein is a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more clinically approved active agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection.

Disclosed herein is a composition comprising administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; an effective amount of one or more clinically approved active agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection.

Disclosed herein is a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects.

Disclosed herein is a composition for inhibiting or ameliorating cytopathic effects in a subject comprising an effective amount of AMB, BHH, analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject in need thereof.

Disclosed herein is a composition for inhibiting or ameliorating cytopathic effects in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject in need thereof.

Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject diagnosed with or suspected of having a SARS-CoV-2 infection.

Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer; wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject diagnosed with or suspected of having a SARS-CoV-2 infection.

Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and wherein the composition disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

Disclosed herein is a composition for inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

Disclosed herein is a composition for inhibiting or reducing viral infectivity in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a composition for inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a composition for inhibiting or reducing viral entry into cells of a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and wherein the composition inhibits or disrupts they physical interactions of angiotensin converting enzyme 2 (ACE2) and the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject.

Disclosed herein is a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or reduces the activity of a type II transmembrane serine protease, and wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2.

Disclosed herein is a composition for inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, whereby the composition inhibits or reduces the activity of a type II transmembrane serine protease.

Disclosed herein is a composition for inhibiting or reducing viral infectivity in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or reduces the activity of a type II transmembrane serine protease, and wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity.

Disclosed herein is a composition for inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or reduces the activity of a type II transmembrane serine protease, and wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection.

Disclosed herein is a composition for inhibiting or reducing viral entry into cells of a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof and wherein the composition inhibits or reduces the activity of a type II transmembrane serine protease, and wherein the composition inhibits or disrupts they physical interactions of angiotensin converting enzyme 2 (ACE2) and the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject.

DETAILED DESCRIPTION

SARS-CoV-2 enters the host cells through two main pathways, both of which comprise key interactions between viral envelope-anchored Spike (S) glycoprotein of the novel coronavirus and the host angiotensin-converting enzyme 2 (ACE2) receptor, which is a membrane-bound metalloprotease.^{98, 100, 105, 107}ACE2 is a zinc metalloprotease and essential cellular receptor for SARS-CoV-2 entry into host cells. ACE2 is mainly expressed in alveolar epithelial cells of the lungs, heart, kidney, and gastrointestinal tract. ACE2 primarily functions as a carboxypeptidase that catalyzes the conversion of a single residue from angiotensin (Ang II), generating L-phenylalanine and Ang (1-7), a potent vasodilator, thus playing a role in controlling hypertension, renal disease, cardiac function, and lung injury. The crystalline structure of the ACE2 shows two domains: (i) a N-terminal zinc metallopeptidase domain (MPD) capable of binding the viral envelope-anchored Spike (S) glycoprotein of coronaviruses, and (ii) a C terminal “collectrin-like” domain.

The first pathway involves receptor mediated endocytosis. The second pathway involves cell fusion consisting of host receptor recognition and attachment of surface unit S1 to the peptidase domain of ACE2. The interaction of the MPD of ACE2 and S glycoprotein of SARS-CoV-2 is the initial and important step in viral infection by receptor recognition and fusion of host and viral cellular membranes.^{98, 100, 105, 108}In addition, viral entry requires priming of S protein by a host protease into S1 and S2 subunits, which are responsible for receptor attachment and membrane fusion, respectively.^{35, 41, 89}A receptor-binding domain (RBD) of the S1 subunit specifically recognizes ACE2 on human cells.^{98, 100, 105, 108}, Binding of the S1 subunit to ACE2 receptor triggers a conformational change in S glycoprotein from metastable pre-fusion state to stable post-fusion conformation, resulting in shedding of S1 and transition of the S2 subunit to expose a hydrophobic fusion peptide.^{31, 42, 100}, The initial priming at S1/S2 boundary by a plasma membrane-associated type II transmembrane serine protease (TMPRSS2) promotes subsequent cleavage at the S2 site by host proteases, which is important for membrane fusion and viral infectivity.^{7, 35-56, 66, 114}Therefore, targeting the interaction between human ACE2 receptor and the RBD in S protein of SARS-CoV-2 can be a promising approach for the development of effective entry inhibitors for potential prevention and/or treatment of COVID-19, thus providing possible countermeasures against viral entry, pathogenesis, and survival.

Clioquinol (5-chloro-7-iodo-8-quinolinol (CLQ)) and its derivatives belonging to the 8-hydroxyquinoline structural class have shown potent broad-spectrum activity against clinically relevant pathogens.^{4, 6, 8, 21, 75, 91, 111,}CLQ and its analogues have been extensively investigated as potential treatments for cancer and neurodegenerative diseases.^{1, 5, 14, 46, 81, 84, 87, 92}Additional studies have also shown the involvement of CLQ in the efflux mechanisms of ATP binding cassette (ABC) transporters^{64, 77}and the cellular autophagic pathway,^{24, 112}an important process in the host defense machinery against viral infections.¹⁷Furthermore, using a high-throughput screen (HTS) and chemical genomics approach, Olaleye et al. have identified and characterized CLQ and certain analogues as potent inhibitors of methionine aminopeptidase,⁷⁵a universally conserved metalloprotease important for N-terminal methionine excision.^{29, 52}As an established metal chelator and zinc ionophore, CLQ modulates underlying molecular and physiologic machinery involved in metal homeostatis.^{2, 24, 25, 32, 50, 112}Altogether, these pharmacologic properties make CLQ an attractive drug for potential targeting of ACE2.

As described herein, the data evaluated the effect of CLQ and two of its analogues (7-bromo-5-chloro-8-hydroxyquinoline (CLBQ14) and 5, 7-Dichloro-8-hydroxyquinoline (CLCQ)) on SARS-CoV-2 infection induced cytopathic effect (CPE) in vitro. The cytotoxicity of these compounds was also assessed. Furthermore, the impact of the three compounds on recombinant human ACE2 (rhACE2) interaction with the RBD on Spike protein of SARS-CoV-2 was examined. The effects of these three compounds on the exopeptidase activity of rhACE2 was also independently examined. These data show, for the first time, that CLQ, CLBQ14 and CLCQ effectively inhibits the novel SARS-CoV-2 infection induced CPE in vitro, inhibited rhACE2 and its interaction with Spike protein, and inhibited rhACE2 exopeptidase activity in the low micromolar range.

Belonging to the benzylamine structural class, Ambroxol hydrochloride ((AMB) 4-[(2-amino-3,5-dibromophenyl) methylamino]cyclohexan-1-ol; hydrochloride)⁷⁰is a demethylated active metabolite of Bromhexine hydrochloride (BHH).⁶⁰Both AMB and its progenitor BHH are used to treat respiratory tract infections and disorders^{28, 73, 76, 113}clinically indicated for their secretolytic activity for treatment of acute and chronic bronchopulmonary diseases associated with abnormal mucus secretion and impaired mucus transport^{60, 73, 113}AMB and BHH have been available, affordable, and used as over the counter drugs with no significant adverse effects.^{15, 113}Furthermore, AMB and BHH have been investigated in translational studies because of their multiple activities including mucociliary clearance activity, mucokinetic properties, stimulation of surfactant production, anti-inflammatory and antioxidative actions, and the local anesthetic effect.^{28, 45, 69, 76, 97,}AMB and BHH have also been shown to induce cellular autophagic-lysosome pathway,^{16, 23, 58}which are processes in the host defense machinery against viral infections.¹⁷AMB is reportedly involved in modulation of the homeostasis of ions such as hydrogen, calcium and sodium.²⁷Due to its potential to act as a chaperone, pH-dependent, mixed-type inhibitor of glucocerebrosidase (GCase) and its involvement in mechanisms for mitochondria, lysosomal biogenesis, and secretory pathway,^{27, 57, 58,}AMB is being considered for the clinical development of therapeutics for neurodegenerative diseases.⁵⁷Reports have also shown that AMB can inhibit viruses that cause influenza virus and rhinovirus infections.^{106, 109}in addition, AMB's progenitor BHH is a potent inhibitor of TMPRSS2⁵³, one of the proteases for viral fusion into host cells. BHH's activity against TMPRSS2 and lung protective properties makes it an attractive drug for the prevention and treatment of coronavirus infections.^{59, 86}

The effects of AMB and its progenitor BHH on the interaction between recombinant human ACE2 (ACE2) and the RBD on the S glycoprotein of SARS-CoV-2 are described herein. These data show the effect of both AMB and BHH on SARS-CoV-2 infection-induced cytopathic effect (CPE) in vitro. The cytotoxicity of AMB and BHH (as well as other clinically approved drugs) was also evaluated. AMB and BHH effectively modulated the ACE2's interaction with the Spike (RBD) protein in the micromolar range. At certain concentrations, both AMB and BHH inhibited SARS-CoV-2 infection-induced CPE. These data represent the first report that the AMB and the BHH pharmacophore have the capacity to target and modulate a protein-protein interaction involved in two known SARS-CoV-2 entry pathways. Altogether, the potent efficacy, stellar safety and pharmacologic profile of both drugs along with their affordability and availability, makes them promising candidates for drug repurposing as possible prophylactic and/or treatment options against SARS-CoV-2 infection.

The present disclosure describes dry formulations, compounded compositions, kits, capsules, containers, and/or methods thereof. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

A. Definitions

Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

The phrase “consisting essentially of” limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase “consisting of” excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps.

As used herein, when referring to any numerical value, the term “about” means a value falling within a range that is ±10% of the stated value.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

As used herein, the term “subject” refers to the target of administration, e.g., a human being. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have a coronavirus infection, be suspected of having a coronavirus infection, or be at risk of developing a coronavirus infection. In an aspect, a coronavirus infection can comprise a SARS-CoV-2 infection. A subject can have a SARS-CoV-2 infection, be suspected of having a SARS-CoV-2 infection, or be at risk of developing a SARS-CoV-2 infection. For example, a subject at risk of developing a coronavirus infection can have, for example, risk factors for developing a coronavirus infection. Risk factors include, but are not limited to the following: cancer, chronic kidney disease, chronic obstructive pulmonary disease, an immunocompromised state (weakened immune system) from solid organ transplant, obesity (body mass index [BMI] of 30 or higher), serious heart conditions (e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, diabetes mellitus, asthma (moderate-to-severe), cerebrovascular disease (i.e., disease that affects blood vessels and blood supply to the brain), cystic fibrosis, hypertension or high blood pressure, immunocompromised state (weakened immune system) from blood or bone marrow transplant, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions (e.g. dementia, Alzheimer's), liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), tobacco use, smoking, thalassemia. A subject at risk for developing a coronavirus infection can be exposed to a coronavirus due to employment (e.g., a health care worker), attendance at a specific location (e.g., school), attendance at social events (e.g., sporting events, concerns, religious services, political rallies and events, social justice rallies, marches, and events, etc.), and/or by use of public transportation or public services. Exposure can happen in a subject's home as well.

As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by one or more of the disclosed compositions, a pharmaceutical preparation comprising one or more disclosed compositions to a subject, and/or disclosed methods. For example, “diagnosed with a coronavirus infection” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be treated by one or more of the disclosed compositions, a pharmaceutical preparation comprising one or more disclosed compositions to a subject, and/or disclosed methods. For example, “suspected of having a coronavirus infection” can mean having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can likely be treated by one or more of the disclosed compositions, a pharmaceutical preparation comprising one or more disclosed compositions to a subject, and/or disclosed methods.

The words “treat” or “treating” or “treatment” refer to therapeutic or medical treatment wherein the object is to slow down (lessen), ameliorate, and/or diminish an undesired physiological change, disease, pathological condition, or disorder (for example, a SARS-CoV-2 infection or SAR-CoV-2 re-infection or a suspected SARS-CoV-2 infection or suspected SARS-CoV-2 re-infection) in a subject. As used herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment may not necessarily result in the complete clearance of an infection but may reduce or minimize complications and side effects of infection and the progression of infection (such as, for example, a SARS-CoV-2 infection or re-infection). The success or otherwise of treatment may be monitored by physical examination of the subject as well as cytopathological, DNA, and/or mRNA detection techniques. The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating an infection can reduce the severity of an established infection in a subject by 1%-100% as compared to a control (such as, for example, a non-infected subject or a subject pre-SARS-CoV-2 infection). In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established coronavirus infection. For example, treating an infection can reduce one or more symptoms of an infection (including induced cytopathic effects) in a subject by 1%-100% as compared to a control (such as, for example, a non-infected subject or a subject pre-SARS-CoV-2 infection). In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms (induced cytopathic effects) of an established coronavirus infection. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of the coronavirus infection. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of a coronavirus infection or re-infection.

A “patient” refers to a subject afflicted with a coronavirus. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a coronavirus infection. In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a coronavirus infection and is seeking treatment or receiving treatment for the coronavirus infection.

As used herein, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a coronavirus infection (e.g., a SARS-CoV-2 infection) is intended. The words “prevent” and “preventing” and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., an individual) not having a given infection related complication from progressing to that complication. Individuals in which prevention is required include those who have an infection.

As used herein, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions to a subject to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.

In various aspects, one or more of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, one or more of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compounds can be administered prophylactically; that is, administered for prevention of a disease or condition (e.g., a SARS-CoV-2 infection). In an aspect, the skilled person can determine an efficacious dose, an efficacious schedule, and an efficacious route of administration for one or more of the disclosed compounds and/or a pharmaceutical preparation comprising one or more disclosed compositions so as to treat or prevent an infection. In an aspect, the skilled person can also alter, change, or modify an aspect of an administering step to improve efficacy of one or more of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compounds.

As used herein, “modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions administered to a subject, or by changing the frequency of administration of one or more of the disclosed compounds and/or a pharmaceutical preparation comprising one or more disclosed compositions to a subject, or by changing the duration of time one or more of the disclosed compounds and/or a pharmaceutical preparation comprising one or more disclosed compositions are administered to a subject.

As used herein, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

The term “contacting” as used herein refers to bringing one or more of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions together with a target area or intended target area in such a manner that the one or more of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions can exert an effect on the intended target or targeted area either directly or indirectly.

A target area or intended target area can be one or more of a subject's organs (e.g., lungs, heart, liver, kidney, etc.) In an aspect, a target area or intended target area can be any cell or any organ infected by SARS-CoV-2 or any cell or organ demonstrating one or more CPEs due to SARS-CoV-2.

As used herein, “determining” can refer to measuring or ascertaining the presence and severity of an infection, such as, for example, a coronavirus infection (e.g., a SARS-CoV-2). Methods and techniques used to determining the presence and/or severity of an infection are typically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a coronavirus infection such as SARS-CoV-2.

As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein such as, for example, CLQ, CLBQ14, CLCQ, AMB, and BHH) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

As used herein, the term “analog” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein such as, for example, CLQ, CLBQ14, CLCQ, AMB, and BHH) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.

As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a coronavirus infection (e.g., a SARS-CoV-2 infection) or a suspected coronavirus infection (e.g., a SARS-CoV-2 infection). As used herein, the terms “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired an effect on an undesired condition (e.g., a coronavirus infection). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount of a disclosed composition that (i) treats the particular disease, condition, or disorder (e.g., a coronavirus infection like SARS-CoV-2), (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder e.g., a coronavirus infection like SARS-CoV-2), or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein e.g., a coronavirus infection like SARS-CoV-2). The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions, or methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions employed; the duration of the treatment; drugs used in combination or coincidental with a disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a disclosed composition and/or a pharmaceutical preparation comprising one or more disclosed composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of a disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions, or methods can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition, such as, for example, a coronavirus infection (e.g., a SARS-CoV-2 infection).

Disclosed are the components to be used to prepare disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions as well as the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

B. Agents

i. Biologically Active Agents

As used herein, the term “biologically active agent” or “biologic active agent” or “bioactive agent” means an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied. For example, the bioactive agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions. Other suitable bioactive agents can include anti-viral agents, vaccines, hormones, antibodies (including active antibody fragments sFv, Fv, and Fab fragments), aptamers, peptide mimetics, functional nucleic acids, therapeutic proteins, peptides, or nucleic acids. Other bioactive agents include prodrugs, which are agents that are not biologically active when administered but, upon administration to a subject are converted to bioactive agents through metabolism or some other mechanism. Additionally, any of the compositions of the invention can contain combinations of two or more bioactive agents. It is understood that a biologically active agent can be used in connection with administration to various subjects, for example, to humans (i.e., medical administration) or to animals (i.e., veterinary administration). As used herein, the recitation of a biologically active agent inherently encompasses the pharmaceutically acceptable salts thereof.

ii. Pharmaceutically Active Agents

As used herein, the term “pharmaceutically active agent” includes a “drug” or a “vaccine” and means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. This term include externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like. This term may also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a bioactive effect, for example deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Pharmaceutically active agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the invention. Examples include a radiosensitizer, the combination of a radiosensitizer and a chemotherapeutic, a steroid, a xanthine, a beta-2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha-agonist, an alpha-1-antagonist, carbonic anhydrase inhibitors, prostaglandin analogs, a combination of an alpha agonist and a beta blocker, a combination of a carbonic anhydrase inhibitor and a beta blocker, an anticholinergic/antispasmodic agent, a vasopressin analogue, an antiarrhythmic agent, an antiparkinsonian agent, an antiangina/antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, or a vaccine. In a further aspect, the pharmaceutically active agent can be coumarin, albumin, bromolidine, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2-agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal antiinflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxene, acetominophen, ibuprofen, ketoprofen and piroxicam; analgesic agents such as salicylates; calcium channel blockers such as nifedipine, amlodipine, and nicardipine; angiotensin-converting enzyme inhibitors such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; beta-blockers (i.e., beta adrenergic blocking agents) such as sotalol hydrochloride, timolol maleate, timol hemihydrate, levobunolol hydrochloride, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally active alpha-2-agonists (i.e., alpha adrenergic receptor agonist) such as clonidine, brimonidine tartrate, and apraclonidine hydrochloride; alpha-1-antagonists such as doxazosin and prazosin; anticholinergic/antispasmodic agents such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; prostaglandin analogs such as latanoprost, travoprost, and bimatoprost; cholinergics (i.e., acetylcholine receptor agonists) such as pilocarpine hydrochloride and carbachol; glutamate receptor agonists such as the N-methyl D-aspartate receptor agonist memantine; anti-Vascular endothelial growth factor (VEGF) aptamers such as pegaptanib; anti-VEGF antibodies (including but not limited to anti-VEGF-A antibodies) such as ranibizumab and bevacizumab; carbonic anhydrase inhibitors such as methazolamide, brinzolamide, dorzolamide hydrochloride, and acetazolamide; antiarrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecaimide acetate, procainamide hydrochloride, moricizine hydrochloride, and diisopyramide phosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, and bromocryptine; antiangina agents and antihypertensive agents such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulant and antiplatelet agents such as coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives such as benzodiazapines and barbiturates; ansiolytic agents such as lorazepam, bromazepam, and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte/macrophage-CSF, and heparin; antineoplastic agents such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, and procarbazine hydrochloride; laxatives such as senna concentrate, casanthranol, bisacodyl, and sodium picosulphate; antidiarrheal agents such as difenoxine hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride, and microorganisms; vaccines such as bacterial and viral vaccines; antimicrobial agents such as penicillins, cephalosporins, and macrolides, antifungal agents such as imidazolic and triazolic derivatives; and nucleic acids such as DNA sequences encoding for biological proteins, and antisense oligonucleotides. It is understood that a pharmaceutically active agent can be used in connection with administration to various subjects, for example, to humans (i.e., medical administration) or to animals (i.e., veterinary administration). As used herein, the recitation of a pharmaceutically active agent inherently encompasses the pharmaceutically acceptable salts thereof.

iii. Anti-Bacterial Agents

As used herein, anti-bacterial agents are known to the art. For example, the art generally recognizes several categories of anti-bacterial agents including (1) penicillins, (2) cephalosporins, (3) quinolones, (4) aminoglycosides, (5) monobactams, (6) carbapenems, (7) macrolides, and (8) other agents. For example, as used herein, an anti-bacterial agent can comprise Afenide, Amikacin, Amoxicillin, Ampicillin, Arsphenamine, Augmentin, Azithromycin, Azlocillin, Aztreonam, Bacampicillin, Bacitracin, Balofloxacin, Besifloxacin, Capreomycin, Carbacephem (loracarbef), Carbenicillin, Cefacetrile (cephacetrile), Cefaclomezine, Cefaclor, Cefadroxil (cefadroxyl), Cefalexin (cephalexin), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium), Cefaloram, Cefaloridine (cephaloradine), Cefalotin (cephalothin), Cefamandole, Cefaparole, Cefapirin (cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (cephazolin), Cefcanel, Cefcapene, Cefclidine, Cefdaloxime, Cefdinir, Cefditoren, Cefedrolor, Cefempidone, Cefepime, Cefetamet, Cefetrizole, Cefivitril, Cefixime, Cefluprenam, Cefmatilen, Cefmenoxime, Cefmepidium, Cefmetazole, Cefodizime, Cefonicid, Cefoperazone, Cefoselis, Cefotaxime, Cefotetan, Cefovecin, Cefoxazole, Cefoxitin, Cefozopran, Cefpimizole, Cefpirome, Cefpodoxime, Cefprozil (cefproxil), Cefquinome, Cefradine (cephradine), Cefrotil, Cefroxadine, Cefsumide, Ceftaroline, Ceftazidime, Ceftazidime/Avibactam, Cefteram, Ceftezole, Ceftibuten, Ceftiofur, Ceftiolene, Ceftioxide, Ceftizoxime, Ceftobiprole, Ceftriaxone, Cefuracetime, Cefuroxime, Cefuzonam, Cephalexin, Chloramphenicol, Chlorhexidine, Ciprofloxacin, Clarithromycin, Clavulanic Acid, Clinafloxacin, Clindamycin, Cloxacillin, Colimycin, Colistimethate, Colistin, Crysticillin, Cycloserine 2, Demeclocycline, Dicloxacillin, Dirithromycin, Doripenem, Doxycycline, Efprozil, Enoxacin, Ertapenem, Erythromycin, Ethambutol, Flucloxacillin, Flumequine, Fosfomycin, Furazolidone, Gatifloxacin, Geldanamycin, Gemifloxacin, Gentamicin, Glycopeptides, Grepafloxacin, Herbimycin, Imipenem, Isoniazid, Kanamycin, Levofloxacin, Lincomycin, Linezolid, Lipoglycopeptides, Lomefloxacin, Meropenem, Meticillin, Metronidazole, Mezlocillin, Minocycline, Mitomycin, Moxifloxacin, Mupirocin, Nadifloxacin, Nafcillin, Nalidixic Acid, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Oxazolidinones, Oxolinic Acid, Oxytetracycline, Oxytetracycline, Paromomycin, Pazufloxacin, Pefloxacin, Penicillin G, Penicillin V, Pipemidic Acid, Piperacillin, Piromidic Acid, Pivampicillin, Pivmecillinam, Platensimycin, Polymyxin B, Pristinamycin, Prontosil, Prulifloxacin, Pvampicillin, Pyrazinamide, Quinupristin/dalfopristin, Rifabutin, Rifalazil, Rifampin, Rifamycin, Rifapentine, Rosoxacin, Roxithromycin, Rufloxacin, Sitafloxacin, Sparfloxacin, Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfacetamide, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfisoxazole, Sulphonamides, Sultamicillin, Teicoplanin, Telavancin, Telithromycin, Temafloxacin, Tetracycline, Thiamphenicol, Ticarcillin, Tigecycline, Tinidazole, Tobramycin, Tosufloxacin, Trimethoprim, Trimethoprim-Sulfamethoxazole, Troleandomycin, Trovafloxacin, Tuberactinomycin, Vancomycin, Viomycin, or pharmaceutically acceptable salts thereof (e.g., such as, for example, chloride, bromide, iodide, and periodate), or a combination thereof. As used herein, the recitation of an anti-bacterial agent inherently encompasses the pharmaceutically acceptable salts thereof.

iv. Anti-Fungal Agents

Anti-fungal agents are known to the art. The art generally recognizes several categories of anti-fungal agents including (1) azoles (imidazoles), (2) antimetabolites, (3) allylamines, (4) morpholine, (5) glucan synthesis inhibitors (echinocandins), (6) polyenes, (7) benoxaaborale; (8) other antifungal/onychomycosis agents, and (9) new classes of antifungal/onychomycosis agents. For example, as used herein, an anti-fungal agent can comprise Abafungin, Albaconazole, Amorolfin, Amphotericin B, Anidulafungin, Bifonazole, Butenafine, Butoconazole, Candicidin, Caspofungin, Ciclopirox, Clotrimazole, Econazole, Fenticonazole, Filipin, Fluconazole, Flucytosine, Griseofulvin, Haloprogin, Hamycin, Isavuconazole, Isoconazole, Itraconazole, Ketoconazole, Micafungin, Miconazole, Naftifine, Natamycin, Nystatin, Omoconazole, Oxiconazole, Polygodial, Posaconazole, Ravuconazole, Rimocidin, Sertaconazole, Sulconazole, Terbinafine, Terconazole, Tioconazole, Tolnaftate, Undecylenic Acid, Voriconazole, or pharmaceutically acceptable salts thereof, or a combination thereof. In an aspect, an anti-fungal agent can be an azole. Azoles include, but are not limited to, the following: clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, oxiconazole, sulconazole, and voriconazole. As used herein, the recitation of an anti-fungal agent inherently encompasses the pharmaceutically acceptable salts thereof.

v. Anti-Viral Agents

Anti-viral agents are known to the art. As used herein, for example, an anti-viral can comprise Abacavir, Acyclovir (Aciclovir), Adefovir, Amantadine, Ampligen, Amprenavir (Agenerase), Umifenovir (Arbidol), Atazanavir, Atripla, Baloxavir marboxil (Xofluza), Biktarvy, Boceprevir, Bulevirtide, Cidofovir, Cobicistat (Tybost), Combivir, Daclatasvir (Daklinza), Darunavir, Delavirdine, Descovy, Didanosine, Docosanol, Dolutegravir, Doravirine (Pifeltro), Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Entecavir, Etravirine (Intelence), Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Ganciclovir (Cytovene), Ibacitabine, Ibalizumab (Trogarzo), Idoxuridine, Imiquimod, Imunovir, Indinavir, Lamivudine, Letermovir (Prevymis), Lopinavir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfinavir, Nevirapine, Nexavir (formerly Kutapressin), Nitazoxanide, Norvir, Oseltamivir (Tamiflu), Penciclovir, Peramivir, Penciclovir, Peramivir (Rapivab), Pleconaril, Podophyllotoxin, Raltegravir, Remdesivir, Ribavirin, Rilpivirine (Edurant), Rilpivirine, Rimantadine, Ritonavir, Saquinavir, Simeprevir (Olysio), Sofosbuvir, Stavudine, Taribavirin (Viramidine), Telaprevir, Telbivudine (Tyzeka), Tenofovir alafenamide, Tenofovir disoproxil, Tenofovir, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Umifenovirk, Valaciclovir, Valganciclovir (Valtrex), Vicriviroc, Vidarabine, Zalcitabine, Zanamivir (Relenza), Zidovudine, and combinations thereof. As used herein, the recitation of any anti-viral agent inherently encompasses the pharmaceutically acceptable salts thereof.

vi. Corticosteroids

Corticosteroids are well-known in the art. Corticosteroids mimic the effects of hormones that the body produces naturally in your adrenal glands. Corticosteroids can suppress inflammation and can reduce the signs and symptoms of inflammatory conditions (e.g., arthritis and asthma). Corticosteroids can also suppress the immune system. Corticosteroids can act on a number of different cells (e.g., mast cells, neutrophils, macrophages and lymphocytes) and a number of different mediators (e.g., histamine, leukotriene, and cytokine subtypes).

Steroids include, but are not limited to, the following: triamcinolone and its derivatives (e.g., diacetate, hexacetonide, and acetonide), betamethasone and its derivatives (e.g., dipropionate, benzoate, sodium phosphate, acetate, and valerate), dexamethasone and its derivatives (e.g., dipropionate and valerate), flunisolide, prednisone and its derivatives (e.g., acetate), prednisolone and its derivatives (e.g., acetate, sodium phosphate, and tebutate), methylprednisolone and its derivatives (e.g., acetate and sodium succinate), fluocinolone and its derivatives (e.g., acetonide), diflorasone and its derivatives (e.g., diacetate), halcinonide, desoximetasone (desoxymethasone), diflucortolone and its derivatives (e.g., valerate), flucloronide (fluclorolone acetonide), fluocinonide, fluocortolone, fluprednidene and its derivatives (e.g., acetate), flurandrenolide (flurandrenolone), clobetasol and its derivatives (e.g., propionate), clobetasone and its derivatives (e.g., butyrate), alclometasone, flumethasone and its derivatives (e.g., pivalate), fluocortolone and its derivatives (e.g., hexanoate), amcinonide, beclometasone and its derivatives (e.g., dipropionate), fluticasone and its derivatives (e.g., propionate), difluprednate, prednicarbate, flurandrenolide, mometasone, and desonide. As used herein, the recitation of a corticosteroid inherently encompasses the pharmaceutically acceptable salts thereof.

vii. Analgesics

The compositions of the present disclosure can also be used in combination therapies with opioids and other analgesics, including narcotic analgesics, Mu receptor antagonists, Kappa receptor antagonists, non-narcotic (i.e., non-addictive) analgesics, monoamine uptake inhibitors, adenosine regulating agents, cannabinoid derivatives, Substance P antagonists, neurokinin-1 receptor antagonists and sodium channel blockers, among others. Preferred combination therapies comprise a composition useful in methods described herein with one or more compounds selected from aceclofenac, acemetacin, .alpha.-acetamidocaproic acid, acetaminophen, acetaminosalol, acetanilide, acetylsalicylic acid (aspirin), S-adenosylmethionine, alclofenac, alfentanil, allylprodine, alminoprofen, aloxiprin, alphaprodine, aluminum bis (acetylsalicylate), amfenac, aminochlorthenoxazin, 3-amino-4-hydroxybutyric acid, 2-atnino-4-picoline, aminopropylon, aminopyrine, amixetrine, ammonium salicylate, ampiroxicam, amtolmetin guacil, anileridine, antipyrine, antipyrine salicylate, antrafenine, apazone, bendazac, benorylate, benoxaprofen, benzpiperylon, benzydamine, benzylmorphine, bermoprofen, bezitramide, .alpha.-bisabolol, bromfenac, p-bromoacetanilide, 5-bromosalicylic acid acetate, bromosaligenin, bucetin, bucloxic acid, bucolome, bufexamac, bumadizon, buprenorphine, butacetin, butibufen, butophanol, calcium acetylsalicylate, carbamazepine, carbiphene, carprofen, carsalam, chlorobutanol, chlorthenoxazin, choline salicylate, cinchophen, cinmetacin, ciramadol, clidanac, clometacin, clonitazene, clonixin, clopirac, clove, codeine, codeine methyl bromide, codeine phosphate, codeine sulfate, cropropamide, crotethamide, desomorphine, dexoxadrol, dextromoramide, dezocine, diampromide, diclofenac sodium, difenamizole, difenpiramide, diflunisal, dihydrocodeine, dihydrocodeinone enol acetate, dihydromorphine, dihydroxyalutninum acetylsalicylate, dimenoxadol, dimepheptanol, dimethylthiambutene, dioxaphetyl butyrate, dipipanone, diprocetyl, dipyrone, ditazol, droxicam, emorfazone, enfenamic acid, epirizole, eptazocine, etersalate, ethenzamide, ethoheptazine, ethoxazene, ethylmethylthiambutene, ethylmorphine, etodolac, etofenamate, etonitazene, eugenol, felbinac, fenbufen, fenclozic acid, fendosal, fenoprofen, fentanyl, fentiazac, fepradinol, feprazone, floctafenine, flufenamic acid, flunoxaprofen, fluoresone, flupirtine, fluproquazone, flurbiprofen, fosfosal, gentisic acid, glafenine, glucametacin, glycol salicylate, guaiazulene, hydrocodone, hydromorphone, hydroxypethidine, ibufenac, ibuprofen, ibuproxam, imidazole salicylate, indomethacin, indoprofen, isofezolac, isoladol, isomethadone, isonixin, isoxepac, isoxicam, ketobemidone, ketoprofen, ketorolac, p-lactophenetide, lefetamine, levorphanol, lofentanil, lonazolac, lomoxicam, loxoprofen, lysine acetylsalicylate, magnesium acetylsalicylate, meclofenamic acid, mefenamic acid, meperidine, meptazinol, mesalamine, metazocine, methadone hydrochloride, methotrimeprazine, metiazinic acid, metofoline, metopon, mofebutazone, mofezolac, morazone, morphine, morphine hydrochloride, morphine sulfate, morpholine salicylate, myrophine, nabumetone, nalbuphine, 1-naphthyl salicylate, naproxen, narceine, nefopam, nicomorphine, nifenazone, niflumic acid, nimesulide, 5′-nitro-2′-propoxyacetanilide, norlevorphanol, normethadone, normorphine, norpipanone, olsalazine, opium, oxaceprol, oxametacine, oxaprozin, oxycodone, oxymorphone, oxyphenbutazone, papaveretum, paranyline, parsalmide, pentazocine, perisoxal, phenacetin, phenadoxone, phenazocine, phenazopyridine hydrochloride, phenocoll, phenoperidine, phenopyrazone, phenyl acetylsalicylate, phenylbutazone, phenyl salicylate, phenyramidol, piketoprofen, piminodine, pipebuzone, piperylone, piprofen, pirazolac, piritramide, piroxicam, pranoprofen, proglumetacin, proheptazine, promedol, propacetamol, propiram, propoxyphene, propyphenazone, proquazone, protizinic acid, ramifenazone, remifentanil, rimazolium metilsulfate, salacetamide, salicin, salicylamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalte, salverine, simetride, sodium salicylate, sufentanil, sulfasalazine, sulindac, superoxide dismutase, suprofen, suxibuzone, talniflumate, tenidap, tenoxicam, terofenamate, tetrandrine, thiazolinobutazone, tiaprofenic acid, tiaramide, tilidine, tinoridine, tolfenamic acid, tolmetin, tramadol, tropesin, viminol, xenbucin, ximoprofen, zaltoprofen and zomepirac. Analgesics are well known in the art. See, for example, The Merck Index, 12th Edition (1996), Therapeutic Category and Biological Activity Index, and the lists provided under “Analgesic”, “Anti-inflammatory” and “Antipyretic”. As used herein, the recitation of an analgesic inherently encompasses the pharmaceutically acceptable salts thereof.

viii. Immunostimulants

The term “immunostimulant” is used herein to describe a substance which evokes, increases, and/or prolongs an immune response to an antigen. Immunomodulatory agents modulate the immune system, and, as used herein, immunostimulants are also referred to as immunomodulatory agents, where it is understood that the desired modulation is to stimulate the immune system. There are two main categories of immunostimulants, specific and non-specific. Specific immunostimulants provide antigenic specificity in immune response, such as vaccines or any antigen, and non-specific immunostimulants act irrespective of antigenic specificity to augment immune response of other antigen or stimulate components of the immune system without antigenic specificity, such as adjuvants and non-specific immunostimulators. Immunostimulants can include, but are not limited to, levamisole, thalidomide, erythema nodosum leprosum, BCG, cytokines such as interleukins or interferons, including recombinant cytokines and interleukin 2 (aldeslukin), 3D-MPL, QS21, CpG ODN 7909, miltefosine, anti-PD-1 or PD-1 targeting drugs, and acid (DCA, a macrophage stimulator), imiquimod and resiquimod (which activate immune cells through the toll-like receptor 7), chlorooxygen compounds such as tetrachlorodecaoxide (TCDO), agonistic CD40 antibodies, soluble CD40L, 4-1BB:4-1BBL agonists, OX40 agonists, TLR agonists, moieties that deplete regulatory T cells, arabinitol-ceramide, glycerol-ceramide, 6-deoxy and 6-sulfono-myo-insitolceramide, iNKT agonists, and TLR agonists. As used herein, the recitation of an immunostimulant inherently encompasses the pharmaceutically acceptable salts thereof.

ix. Immune-Based Product

As used herein, immune-based products include, but are not limited to, toll-like receptors modulators such as tlr1, tlr2, tlr3, tlr4, tlr5, tlr6, tlr7, tlr8, tlr9, tir10, tlr11, tlr12, and tlr13; programmed cell death protein 1 (Pd-1) modulators; programmed death-ligand 1 (Pd-L1) modulators; IL-15 agonists; DermaVir; interleukin-7; plaquenil (hydroxychloroquine); proleukin (aldesleukin, IL-2); interferon alfa; interferon alfa-2b; interferon alfa-n3; pegylated interferon alfa; interferon gamma; hydroxyurea; mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF); ribavirin; rintatolimod, polymer polyethyleneimine (PEI); gepon; rintatolimod; IL-12; WF-10; VGV-1; MOR-22; BMS-936559; CYT-107, interleukin-15/Fc fusion protein, normferon, peginterferon alfa-2a, peginterferon alfa-2b, recombinant interleukin-15, RPI-MN, GS-9620, and IR-103. As used herein, the recitation of an immune-based product inherently encompasses the pharmaceutically acceptable salts thereof.

x. Blood Derived Products

As used herein, blood-derived products are obtained from subjects that have recovered from a SARS-CoV-2 infection and include convalescent plasma, immunoglobulin products, and neutralizing monoclonal antibodies.

C. 8-Hydroxyquinoline Structural Class
1. Methods Comprising 8-Hydroxyquinoline Structural Class
General Methods

Disclosed herein is a method comprising administering a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method comprising administering a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; administering one or more clinically approved active agents; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; administering one or more clinically approved active agents; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method comprising inhibiting or ameliorating a SARS-CoV-2 infection by administering a composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class. Disclosed herein is a method comprising inhibiting or ameliorating a SARS-CoV-2 infection by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising prophylactically administering a composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising prophylactically administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection.

The 8-hydroxyquinoline structural class is known to the art. For example, 8-hydroxyquinoline (Quinolin-8-ol) comprises the formula C₁₈H₁₂CuN₂O₂or C₉H₇NO and has a molecular weight of 145.16 g/mol. 8-hydroxyquinoline is a monohydroxyquinoline comprising a quinoline substituted by a hydroxy group at position 8. The 8-hydroxyquinoline structural class comprises at least CLQ, CLBQ14, and CLCQ. 5-chloro-7-iodoquinolin-8-ol (Clioquinol or CLQ) comprises the formula C₉H₅ClINO and has a molecular weight of 305.5 g/mol. 5-chloro-7-iodoquinolin-8-ol is a monohydroxyquinoline that is a quinolin-8-ol in which the hydrogens at positions 5 and 7 are replaced by chlorine and iodine, respectively. 7-bromo-5-chloro-8-hydroxyquinoline (CLBQ14) comprises the formula C₉H₅BrClNO and has a molecular weight of 258.5 g/mol. 7-bromo-5-chloro-8-hydroxyquinoline is a monohydroxyquinoline that is a quinolin-8-ol in which the hydrogens at positions 5 and 7 are replaced by chlorine and bromine, respectively. 5,7-dichloro-8-hydroxyquinoline (CLCQ) comprise the formula C₉H₅Cl₂NO and has a molecular weight of 214.04 g/mol. 5,7-Dichloro-8-hydroxyquinoline (CLCQ) is a monohydroxyquinoline that is a quinolin-8-ol in which the hydrogens at positions 5 and 7 have been substituted by chlorine.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise administering one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent.

In an aspect, a disclosed composition in a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise administering (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof. In an aspect, a disclosed composition in a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a composition in a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection may comprise CLQ, CLBQ14, and/or CLCQ, or analogs or derivatives thereof, or a combination thereof. The composition may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In various embodiments, the composition comprising CLQ, CLBQ14, and/or CLCQ, or analogs or derivatives thereof, or a combination thereof, may be administered via any suitable administration route. Example administration routes include parenteral administration, which may include intramuscular, intraarterial, or intravenous, as examples. Example administration routes include nonparenteral administration, such as oral, rectal, vaginal, nasal, mucosal, percutaneous, transdermal, or ophthalmic, as examples.

The composition comprising CLQ, CLBQ14, and/or CLCQ, or analogs or derivatives thereof, or a combination thereof, may be administered in a suitable dosage format, via a suitable route of administration, such as any of those identified above, and in an effective daily dose to inhibit or ameliorate a SARS-CoV-2 infection, such as by any mechanism identified herein. The daily dose may be between 30 mg up to 2,000 mg. Example daily dosages may include greater than 200 mg, greater than 400 mg, greater than 500 mg, greater than 600 mg, greater than 700 mg, greater than 800 mg, greater than 900 mg, greater than 1000 mg, between 50 mg and 100 mg, between 100 mg and 200 mg, between 150 mg and 300 mg, between 200 mg and 1000 mg, between 500 mg and 1500 mg, between 450 mg and 1200 mg, between 500 mg and 2000 mg, between 1000 mg and 2000 mg, or any range between 30 mg and 2000 mg. The composition will typically be administered once daily, twice daily, or three times daily; however, additional administrations may be used.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise repeating one or more steps.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise identifying a subject having been diagnosed with or suspected of having a SARS-CoV-2 re-infection. As known to the art, a past SARS-CoV-2 infection can be diagnosed and/or confirmed through an antibody test. In an aspect, an antibody test (also known as serology testing) can check for Immunoglobulin G (IgG) antibody. In an aspect, a variety of factors can impact the results from an antibody test in a disclosed method (e.g., the time the test was taken after experiencing symptoms, the absence of or time since exposure to the virus, or the lack of an adequate immune response, which can be due to conditions or treatments that suppress immune function).

In an aspect, the administering step of a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise administering to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise identifying and/or characterizing one or more comorbidities in a subject. In an aspect, a subject can have one or more comorbidities. Comorbidities are known to the art and can comprise cancer, chronic kidney disease, COPD (chronic obstructive pulmonary disease), an immunocompromised state (weakened immune system) from a solid organ transplant or from a blood or bone marrow transplant, obesity (body mass index [BMI] of 30 or higher), heart conditions (such as, e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, type 1 diabetes mellitus or type 2 diabetes mellitus, asthma, cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions, liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), a history of smoking, and thalassemia (a type of blood disorder).

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise treating or ameliorating one or more comorbidities in a subject. For example, in an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise (i) administering one or more active agents to treat or ameliorate one or more comorbidities, (ii) administering one or more active agents to treat or ameliorate the same comorbidity, (iii) administering one or more active agents to treat or ameliorate different comorbidities (e.g., an active agent for type 2 diabetes and a different active agent for hypertension), or (iv) a combination thereof. In an aspect, administering an active agent to treat or ameliorate one or more comorbidities can occur prior to, concurrently with, or after the administering of a disclosed composition. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise repeating the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise modifying or altering one or more steps of a disclosed method. For example, in an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise modifying or altering an administering step. In an aspect, an administering step can be modified or altered, for example, by changing the route of administration, or changing the dose of a disclosed composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

In an aspect, altering or modifying one or more steps of a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can be based on the identification and/or characterization of one or more comorbidities in a subject.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise modifying or altering the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise determining, measuring, and/or ascertaining the presence and/or severity of an infection, such as, for example, a SARS-CoV-2 infection, a bacterial infection, a viral infection, a fungal infection, or a combination thereof. Methods and techniques used to determine, measure, and/or ascertain the presence and/or severity of an infection such as a SARS-CoV-2 infection are typically known to the medical arts.

Methods and techniques to monitor a subject's response to a disclosed method can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, qualitative means (or subjective means) can comprise a subject's own perspective. For example, a subject can report how he/she is feeling, whether he/she has experienced improvements and/or setbacks, whether he/she has experienced an amelioration or an intensification of one or more symptoms, or a combination thereof. In an aspect, quantitative means (or objective means) can comprise methods and techniques that include, but are not limited to, the following: (i) fluid analysis (e.g., tests of a subject's fluids including but not limited to aqueous humor and vitreous humor, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, and urine), (ii) imaging (e.g., ordinary x-rays, ultrasonography, radioisotope (nuclear) scanning, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and angiography), (iii) endoscopy (e.g., laryngoscopy, bronchoscopy, esophagoscopy, gastroscopy, GI endoscopy, coloscopy, cystoscopy, hysteroscopy, arthroscopy, laparoscopy, mediastinoscopy, and thoracoscopy), (iv) analysis of organ activity (e.g., electrocardiography (ECG), electroencephalography (EEG), and pulse oximetry), (v) biopsy (e.g., removal of tissue samples for microscopic evaluation), and (vi) genetic testing.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise obtaining a disclosed compound (e.g., CLQ, CLBQ14, or CLCQ, or an analog or derivative thereof), obtaining a disclosed composition, obtaining a disclosed formulation comprising a disclosed composition, obtaining one or more active agents, one or more biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or obtaining a combination thereof.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise preparing a disclosed compound (e.g., CLQ, CLBQ14, or CLCQ, or an analog or derivative thereof) or preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise (1) preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and (2) preparing (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 can comprise preparing one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise preparing a disclosed composition comprising (i) CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and (ii) one or more active agents, biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof.

i. Method of Inhibiting or Ameliorating One or More SARS-CoV-2 Infection Induced Cytopathic Effects

Disclosed herein is a method comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects. Disclosed herein is a method comprising inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in the subject. Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in the subject by administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise administering one or more active agents, one or more biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent.

In an aspect, a disclosed composition in a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise administering (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof. In an aspect, a disclosed composition in a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a composition in a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects may comprise CLQ, CLBQ14, and/or CLCQ, or analogs or derivatives thereof, or a combination thereof. The composition may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise repeating one or more steps.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise identifying a subject having been diagnosed with or suspected of having a SARS-CoV-2 infection. As known to the art, a SARS-CoV-2 infection can be diagnosed and/or confirmed through various tests (such as, e.g., a PCR test or an antigen test). In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise identifying a subject having been diagnosed with or suspected of having a SARS-CoV-2 re-infection. As known to the art, a past SARS-CoV-2 infection can be diagnosed and/or confirmed through an antibody test. In an aspect, an antibody test (also known as serology testing) can check for Immunoglobulin G (IgG) antibody. In an aspect, a variety of factors can impact the results from an antibody test in a disclosed method (e.g., the time the test was taken after experiencing symptoms, the absence of or time since exposure to the virus, or the lack of an adequate immune response, which can be due to conditions or treatments that suppress immune function).

In an aspect, the administering step of a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise administering to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise identifying and/or characterizing one or more comorbidities in a subject. In an aspect, a subject can have one or more comorbidities. Comorbidities are known to the art and can comprise cancer, chronic kidney disease, COPD (chronic obstructive pulmonary disease), an immunocompromised state (weakened immune system) from a solid organ transplant or from a blood or bone marrow transplant, obesity (body mass index [BMI] of 30 or higher), heart conditions (such as, e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, type 1 diabetes mellitus or type 2 diabetes mellitus, asthma, cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions, liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), a history of smoking, and thalassemia (a type of blood disorder).

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise treating or ameliorating one or more comorbidities in a subject. For example, in an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise (i) administering one or more active agents to treat or ameliorate one or more comorbidities, (ii) administering one or more active agents to treat or ameliorate the same comorbidity, (iii) administering one or more active agents to treat or ameliorate different comorbidities (e.g., an active agent for type 2 diabetes and a different active agent for hypertension), or (iv) a combination thereof. In an aspect, administering an active agent to treat or ameliorate one or more comorbidities can occur prior to, concurrently with, or after the administering of a disclosed composition. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise repeating the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise modifying or altering one or more steps of a disclosed method. For example, in an aspect, a disclosed method can comprise modifying or altering an administering step. In an aspect, an administering step can be modified or altered, for example, by changing the route of administration, or changing the dose of a disclosed composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

In an aspect, altering or modifying one or more steps of a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects, such as, for example, an administering step, can be based on the identification and/or characterization of one or more comorbidities in a subject. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise modifying or altering the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise determining, measuring, and/or ascertaining the presence and/or severity of an infection, such as, for example, a SARS-CoV-2 infection, a bacterial infection, a viral infection, a fungal infection, or a combination thereof. Methods and techniques used to determine, measure, and/or ascertain the presence and/or severity of an infection such as a SARS-CoV-2 infection are typically known to the medical arts.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise monitoring a subject's response to the administration of a disclosed composition comprising (i) CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof and (ii) one or more disclosed active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect of a disclosed method, a monitoring step can be repeated one or more times.

Methods and techniques to monitor a subject's response to a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, qualitative means (or subjective means) can comprise a subject's own perspective. For example, a subject can report how he/she is feeling, whether he/she has experienced improvements and/or setbacks, whether he/she has experienced an amelioration or an intensification of one or more symptoms, or a combination thereof. In an aspect, quantitative means (or objective means) can comprise methods and techniques that include, but are not limited to, the following: (i) fluid analysis (e.g., tests of a subject's fluids including but not limited to aqueous humor and vitreous humor, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, and urine), (ii) imaging (e.g., ordinary x-rays, ultrasonography, radioisotope (nuclear) scanning, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and angiography), (iii) endoscopy (e.g., laryngoscopy, bronchoscopy, esophagoscopy, gastroscopy, GI endoscopy, coloscopy, cystoscopy, hysteroscopy, arthroscopy, laparoscopy, mediastinoscopy, and thoracoscopy), (iv) analysis of organ activity (e.g., electrocardiography (ECG), electroencephalography (EEG), and pulse oximetry), (v) biopsy (e.g., removal of tissue samples for microscopic evaluation), and (vi) genetic testing.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise obtaining a disclosed compound (e.g., CLQ, CLBQ14, or CLCQ, or an analog or derivative thereof), obtaining a disclosed composition, obtaining a disclosed formulation comprising a disclosed composition, obtaining one or more active agents, one or more biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or obtaining a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise preparing a disclosed compound (e.g., CLQ, CLBQ14, or CLCQ, or an analog or derivative thereof) or preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise (1) preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and (2) preparing (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise preparing one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise preparing a disclosed composition comprising (i) CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and (ii) one or more active agents, biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof.

ii. Method of Inhibiting or Reducing the Exopeptidase Activity of ACE2

Disclosed herein is a method comprising inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method comprising inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and by administering a composition comprising an effective amount of zinc chloride. Disclosed herein is a method of inhibiting or reducing exopeptidase activity of an enzyme comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2). Disclosed herein is a method of inhibiting or reducing exopeptidase activity of an enzyme comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; administering a composition comprising an effective amount of zinc chloride; and inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2). Disclosed herein is a method of inhibiting or reducing exopeptidase activity of an enzyme comprising inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is method of inhibiting or reducing exopeptidase activity of an enzyme comprising inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof and by administering a composition comprising an effective amount of zinc chloride.

In an aspect, the receptor binding domain (RBD) of the Spike glycoprotein can bind to the metallopeptidase domain (MPD) of ACE2.

In an aspect, a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme can comprise administering one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent.

In an aspect, a disclosed composition in a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme can comprise administering (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof. In an aspect, a disclosed composition in a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a composition in a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) may comprise CLQ, CLBQ14, and/or CLCQ, or analogs or derivatives thereof, or a combination thereof. The composition may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise repeating one or more steps.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise identifying a subject having been diagnosed with or suspected of having a SARS-CoV-2 re-infection. As known to the art, a past SARS-CoV-2 infection can be diagnosed and/or confirmed through an antibody test. In an aspect, an antibody test (also known as serology testing) can check for Immunoglobulin G (IgG) antibody. In an aspect, a variety of factors can impact the results from an antibody test in a disclosed method (e.g., the time the test was taken after experiencing symptoms, the absence of or time since exposure to the virus, or the lack of an adequate immune response, which can be due to conditions or treatments that suppress immune function).

In an aspect, the administering step of a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise administering to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise identifying and/or characterizing one or more comorbidities in a subject. In an aspect of a disclosed method, a subject can have one or more comorbidities. Comorbidities are known to the art and can comprise cancer, chronic kidney disease, COPD (chronic obstructive pulmonary disease), an immunocompromised state (weakened immune system) from a solid organ transplant or from a blood or bone marrow transplant, obesity (body mass index [BMI] of 30 or higher), heart conditions (such as, e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, type 1 diabetes mellitus or type 2 diabetes mellitus, asthma, cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions, liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), a history of smoking, and thalassemia (a type of blood disorder).

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise treating or ameliorating one or more comorbidities in a subject. For example, in an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise (i) administering one or more active agents to treat or ameliorate one or more comorbidities, (ii) administering one or more active agents to treat or ameliorate the same comorbidity, (iii) administering one or more active agents to treat or ameliorate different comorbidities (e.g., an active agent for type 2 diabetes and a different active agent for hypertension), or (iv) a combination thereof. In an aspect, administering an active agent to treat or ameliorate one or more comorbidities can occur prior to, concurrently with, or after the administering of a disclosed composition. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise repeating the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise modifying or altering one or more steps of a disclosed method. For example, in an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise modifying or altering an administering step. In an aspect, an administering step can be modified or altered, for example, by changing the route of administration, or changing the dose of a disclosed composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

In an aspect, altering or modifying one or more steps of a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can be based on the identification and/or characterization of one or more comorbidities in a subject. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise modifying or altering the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise determining, measuring, and/or ascertaining the presence and/or severity of an infection, such as, for example, a SARS-CoV-2 infection, a bacterial infection, a viral infection, a fungal infection, or a combination thereof. Methods and techniques used to determine, measure, and/or ascertain the presence and/or severity of an infection such as a SARS-CoV-2 infection are typically known to the medical arts.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise monitoring a subject's response to the administration of a disclosed composition comprising (i) CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof and (ii) one or more disclosed active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect of a disclosed method, a monitoring step can be repeated one or more times.

Methods and techniques to monitor a subject's response to a disclosed method can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, qualitative means (or subjective means) can comprise a subject's own perspective. For example, a subject can report how he/she is feeling, whether he/she has experienced improvements and/or setbacks, whether he/she has experienced an amelioration of one or more symptoms, or a combination thereof. In an aspect, quantitative means (or objective means) can comprise methods and techniques that include, but are not limited to, the following: (i) fluid analysis (e.g., tests of a subject's fluids including but not limited to aqueous humor and vitreous humor, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, and urine), (ii) imaging (e.g., ordinary x-rays, ultrasonography, radioisotope (nuclear) scanning, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and angiography), (iii) endoscopy (e.g., laryngoscopy, bronchoscopy, esophagoscopy, gastroscopy, GI endoscopy, coloscopy, cystoscopy, hysteroscopy, arthroscopy, laparoscopy, mediastinoscopy, and thoracoscopy), (iv) analysis of organ activity (e.g., electrocardiography (ECG), electroencephalography (EEG), and pulse oximetry), (v) biopsy (e.g., removal of tissue samples for microscopic evaluation), and (vi) genetic testing.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise obtaining a disclosed compound (e.g., CLQ, CLBQ14, or CLCQ, or an analog or derivative thereof), obtaining a disclosed composition, obtaining a disclosed formulation comprising a disclosed composition, obtaining one or more active agents, one or more biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or obtaining a combination thereof.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise preparing a disclosed compound (e.g., CLQ, CLBQ14, or CLCQ, or an analog or derivative thereof) or preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise (1) preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and (2) preparing (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise preparing one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise preparing a disclosed composition comprising (i) CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and (ii) one or more active agents, biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof.

iii. Method of Inhibiting or Disrupting the Interaction Between ACE2 and Spike Protein

Disclosed herein is a method comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. Disclosed herein is a method comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising administering a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity. Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing viral infectivity. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject. Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing viral entry into cells of the subject.

In an aspect, the receptor binding domain (RBD) of the Spike glycoprotein can bind to the metallopeptidase domain (MPD) of the ACE2 receptor.

In an aspect, a disclosed composition in a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a method of inhibiting or reducing viral entry into cells of a subject can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof.

In an aspect, a disclosed composition in a a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a method of inhibiting or reducing viral entry into cells of a subject can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a composition in a disclosed method or a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a method of inhibiting or reducing viral entry into cells of a subject may comprise CLQ, CLBQ14, and/or CLCQ, or analogs or derivatives thereof, or a combination thereof. The composition may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In an aspect, a disclosed method or a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a method of inhibiting or reducing viral entry into cells of a subject can comprise identifying a subject having been diagnosed with or suspected of having a SARS-CoV-2 re-infection. As known to the art, a past SARS-CoV-2 infection can be diagnosed and/or confirmed through an antibody test. In an aspect, an antibody test (also known as serology testing) can check for Immunoglobulin G (IgG) antibody. In an aspect, a variety of factors can impact the results from an antibody test in a disclosed method (e.g., the time the test was taken after experiencing symptoms, the absence of or time since exposure to the virus, or the lack of an adequate immune response, which can be due to conditions or treatments that suppress immune function).

In an aspect, the administering step of a disclosed method or a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a method of inhibiting or reducing viral entry into cells of a subject can comprise administering to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed method or a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a method of inhibiting or reducing viral entry into cells of a subject can comprise identifying and/or characterizing one or more comorbidities in a subject. In an aspect, a subject can have one or more comorbidities. Comorbidities are known to the art and can comprise cancer, chronic kidney disease, COPD (chronic obstructive pulmonary disease), an immunocompromised state (weakened immune system) from a solid organ transplant or from a blood or bone marrow transplant, obesity (body mass index [BMI] of 30 or higher), heart conditions (such as, e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, type 1 diabetes mellitus or type 2 diabetes mellitus, asthma, cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions, liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), a history of smoking, and thalassemia (a type of blood disorder).

In an aspect, a disclosed method or a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a method of inhibiting or reducing viral entry into cells of a subject can comprise (i) administering one or more active agents to treat or ameliorate one or more comorbidities, (ii) administering one or more active agents to treat or ameliorate the same comorbidity, (iii) administering one or more active agents to treat or ameliorate different comorbidities (e.g., an active agent for type 2 diabetes and a different active agent for hypertension), or (iv) a combination thereof. In an aspect, administering an active agent to treat or ameliorate one or more comorbidities can occur prior to, concurrently with, or after the administering of a disclosed composition. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise repeating the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, altering or modifying one or more steps of a disclosed method or a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a method of inhibiting or reducing viral entry into cells of a subject can be based on the identification and/or characterization of one or more comorbidities in a subject.

2. Compositions Comprising 8-Hydroxyquinoline Structural Class

i. General Composition

Disclosed herein is composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection. Disclosed herein is a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection. Disclosed herein is composition comprising an effective amount of one or more compounds belonging to the 8-hydroxyquinoline structural class; an effective amount of one or more clinically approved active agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection. Disclosed herein is a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more clinically approved active agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection.

The 8-hydroxyquinoline structural class is known to the art and discussed herein. Pharmaceutically acceptable diluents, carriers, excipients, and stabilizers are known to the art and discussed herein.

In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can comprise one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent.

In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof.

A disclosed composition comprising CLQ, CLBQ14, and/or CLCQ, or analogs or derivatives thereof, or a combination thereof, may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can be administered to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can be administered to a subject having one or more comorbidities. In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can comprise one or more active agents to treat or ameliorate one or more comorbidities. Comorbidities are known to the art and are discussed herein.

ii. Compositions for Inhibiting or Ameliorating a SARS-CoV-2 Infection

Disclosed herein is a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects. Disclosed herein is a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects. Disclosed herein is a composition for inhibiting or ameliorating cytopathic effects in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects in a subject in need thereof. Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject in need thereof. Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects in a subject diagnosed with or suspected of having a SARS-CoV-2 infection. Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof; wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects in a subject diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can comprise one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent.

In an aspect, a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can be administered to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can be administered to a subject having one or more comorbidities. In an aspect, a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can comprise one or more active agents to treat or ameliorate one or more comorbidities. Comorbidities are known to the art and are discussed herein.

iii. Compositions for Inhibiting or Reducing the Exopeptidase Activity of ACE2

Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or reduces the exopeptidase activity of angiotensin converting enzyme 2 (ACE2). Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and an effective amount of zinc chloride; wherein the composition inhibits or reduces the exopeptidase activity of angiotensin converting enzyme 2 (ACE2). Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2). Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and an effective amount of zinc chloride, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

CLQ, CLBQ14, CLCQ, and analogs or derivatives thereof are known to the art and are discussed herein. Pharmaceutically acceptable diluents, carriers, excipients, stabilizers, and combinations thereof are known to the art and are discussed herein.

In an aspect, the receptor binding domain (RBD) of the Spike glycoprotein can bind to the metallopeptidase domain (MPD) of the ACE2 receptor.

In an aspect, a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In an aspect, a disclosed composition can be administered to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

iv. Compositions for Inhibiting or Disrupting the Interaction Between ACE2 and Spike Protein

Disclosed herein is a composition comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. Disclosed herein is a composition for inhibiting or disrupting the physical interaction of an angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a composition for inhibiting or reducing viral infectivity in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) receptor with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity. Disclosed herein is a composition for inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a composition for inhibiting or reducing viral entry into cells of a subject comprising an effective amount of CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof; and wherein the composition inhibits or disrupts they physical interactions of angiotensin converting enzyme 2 (ACE2) and the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject.

In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can inhibit or disrupt the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can inhibit or reduce viral infectivity. In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can inhibit or ameliorate a SARS-CoV-2 infection. In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can inhibit or reduce viral entry into cells of the subject.

In an aspect, the receptor binding domain (RBD) of the Spike glycoprotein can bind to the metallopeptidase domain (MPD) of the ACE2 receptor.

In an aspect, a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof can comprise an effective amount of zinc chloride.

D. Benzylamine Structural Class

SARS-CoV-2 enters the host cells through two main pathways, both involving key interactions between viral envelope-anchored spike glycoprotein and the host receptor angiotensin-converting enzyme 2 (ACE2). AMB has been available as an over the counter mucolytic medication since 1970s and has been proven to be safe and well tolerated in adults and children. Through focused search for clinically approved drugs to target the coronavirus pathway for cell entry, we discovered that Ambroxol hydrochloride (AMB) exerts superior pharmacological efficacy at the molecular and cellular level against SARS-CoV-2 viral infection. More excitingly, nano- to micromolar concentrations of AMB effectively: (1) blocks the pathway of SARS-CoV-2 entry into human cells via modulating ACE2's interaction with receptor binding domain protein of SARS-CoV-2; (2) inhibit SARS-CoV-2 infection-induced cytopathic effect; and (3) protect ACE2 exopeptidase function, while modulating its interaction with SARS-CoV-2 Spike glycoprotein, thus avoiding potential non-target cardiac toxicities observed in other ACE2 modulating agents. In various embodiments, one or both of AMB or its progenitor, bromhexine hydrochloride (BHH) may be administered to a subject to (1) block the pathway of SARS-CoV-2 entry into human cells via modulating ACE2's interaction with receptor binding domain protein of SARS-CoV-2; (2) inhibit SARS-CoV-2 infection-induced cytopathic effect; and/or (3) protect ACE2 exopeptidase function, while modulating its interaction with SARS-CoV-2 Spike glycoprotein, thus avoiding potential non-target cardiac toxicities observed in other ACE2 modulating agents. AMB has shown better clinical safety and pharmacologic profile compared to BHH historically. AMB accumulates in the lungs (a key site for SARS-CoV-2 viral replication), increases surfactant production, inhibits autophagy and reduces the production of certain inflammatory cytokines by bronchoalveolar macrophages. As described herein AMB and/or BHH may be administered to a subject for the treatment of moderate COVID-19 to effectively inhibit SARS-CoV-2 pathways, and therefore results in decreased viral load, reduced inflammation, reduced rate of hospitalization and improved clinical outcomes in moderate COVID-19 subjects.

Effective therapeutic interventions against SARS-CoV-2 infection require inhibiting essential viral entry and/or post-entry pathways by targeting viral enzymes or host receptors. The emergence of SARS-CoV-2 variants with mutations on the viral genes have made it more imperative to discover therapeutics that targets the host receptors for COVID-19 treatment. Benzylamine structural class targets two critical host entry receptors: Angiotensin-converting enzyme-2 (ACE2) and tyrosine-protein kinase receptor (AXL) for SARS-CoV-2 entry into the human cells.

According to various embodiments, an effective amount of Ambroxol Hydrochloride (AMB) may be administered to a subject to therein target the interaction between RBD and ACE2, without inactivating the exopeptidase activity of human Angiotensin-Converting Enzyme-2 (ACE2).

ACE2, a membrane-bound metalloprotease is an essential cellular receptor for SARS-CoV-2 entry into host cells. It is an important component in the Renin-Angiotensin system converting Angiotensin II (Ang II) to Angiotensin 1-7, a potent vasopressor. Although ACE2 facilitates viral entry, it provides defense against acute lung damage, indicating that the ACE2/Ang 1-7 pathway must be carefully manipulated to reduce SARS-CoV-2 induced lung injuries.

Applicant has discovered that Ambroxol hydrochloride (AMB) and its progenitor Bromhexine hydrochloride (BHH) inhibits the interaction of SARS-CoV-2 spike protein receptor-binding domain (RBD) with human recombinant ACE2 (rhACE2) in a nano to micro molar range thereby blocking its entry into human cells. Applicant has further discovered that AMB targets the interaction between RBD and rhACE2, without inactivating the exopeptidase activity of rhACE2. Our findings reveal that AMB binding to rhACE2 may preserving its physiological function, unlike BHH which inhibits rhACE2 exopeptidase activity at high concentrations (Tables 1(a), 1(b)). Thus, potentially prevents non-target cardiac toxicities observed in other ACE2 modulating drugs.

According to various embodiments, an effective amount of Ambroxol Hydrochloride (AMB) and/or Bromhexine Hydrochloride may be administered to a subject to therein inhibit the Interaction between Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein's N-Terminal Binding Domain and Tyrosine-Protein kinase Receptor (AXL).

AXL is a plasma membrane associated with the Tyro3/Axl/Mer (TAM) family: a group of tyrosine kinase receptors that mediate apoptotic cells' clearance and regulate innate immunity response. Previously, AXL was identified as a receptor for the Zika virus, allowing viral entry into the human glial cells and facilitating infection by downregulating interferon signaling. It also serves as an entry factor for the dengue virus and facilitates the entry of filoviruses. Studies have identified AXL as an additional critical entry receptor that promotes the entry of SARS-CoV-2 into cells of the respiratory system. The interaction of the N-terminal domain (NTD) of SARS-CoV-2 Spike protein with AXL facilitates the viral entry into the human cells. In this study, we discovered for the first time that AMB and BHH both inhibit the interaction of recombinant AXL with the NTD of the spike protein in the micromolar range (Table 2).

Inhibition of the two critical viral entry pathways into host cells represents a promising therapeutic possibility to combat SARS-CoV-2 infection. Therefore, compounds such as AMB and BHH, with potent efficacy, excellent safety and pharmacologic profile along with their availability and affordability makes this pharmacophore promising candidates for drug repurposing as a possible prophylactical and or treatment options against COVID-19 infection.

TABLE 1(a)

Effect of Bromhexine Hydrochloride (BHH) and Ambroxol Hydrochloride

(AMB) on ACE2 Exopeptidase Activity: Percent Activity

Percent Activity (%)

Concentration (uM)
BHH
AMB

500
−6.6
141.0

250
−6.7
144.7

100
−6.2
156.9

50
2.7
157.1

10
104.6
159.9

1
114.7
152.3

0.1
111.3
148.7

TABLE 1(b)

Effect of Bromhexine Hydrochloride (BHH) and Ambroxol Hydrochloride

(AMB) on the ACE2 Exopeptidase Activity: Percent Inhibition

Percent Inhibition (%)

Concentration (uM)
BHH
AMB

500
106.6
−41.0

250
106.7
−44.7

100
106.2
−56.9

50
97.3
−57.1

10
−4.6
−59.9

1
−14.7
−52.3

0.1
−11.3
−48.7

Effect of Bromhexine Hydrochloride (BHH) and Ambroxol Hydrochloride (AMB) on the interaction between rhAXL and SARS-CoV-2 Spike (NTD) protein Interaction.

According to various embodiments, an effective amount of Ambroxol Hydrochloride (AMB) and/or Bromhexine Hydrochloride may be administered to a subject to therein blocks the entry of SARS-CoV-2 spike pseudotyped lentivirus into human cells.

To date, there are rapidly spreading new variants with mutations on the viral genes of SARS-CoV-2. Successful intervention measures are needed now more than ever to contain the pandemic. Inhibiting critical viral entry and post-entry processes by targeting viral enzymes or host receptors is necessary to develop effective therapeutic interventions against SARS-CoV-2 infection. Therefore, Applicant has also focused research on blocking the first step of viral fusion of SARS-CoV-2 spike protein receptor-binding domain (RBD) with host receptor; Angiotensin-converting enzyme 2 (ACE2) and thereby prevent the cellular entry of SARS-CoV-2 into the human cells.

Applicant tested the ability of Ambroxol Hydrochloride (AMB) and Bromhexine Hydrochloride (BHH) to inhibit the cellular entry of the SARS-CoV-2 spike pseudotyped lentivirus in a low micromolar range. Briefly, Spike (SARS-CoV-2) pseudotyped lentivirus containing luciferase reporter gene was purchased from BPS Bioscience (Catalog no 79942, San Diego, CA). HEK293 cells with stable expression of full-length ACE2 were seeded at a density of 7500 cells per well into a white 96-well cell culture microplate. On day two, pseudotyped lentivirus was preincubated with AMB and BHH at concentrations ranging from 100, 50, 25, and M for 30 minutes at room temperature (RT); the reaction mixture was then added to the cells. After 48 hours on day four, luciferase activity was measured by adding 50 l Luciferase reagent (BPS Bioscience, catalog no. 60690) for 30 min at RT luminescence was measured with Spectra max ID3 (molecular devices). The measured luminescence signal was directly proportional to the amount of pseudotyped lentivirus successfully transduced into the cells. Data are presented as percent inhibition of entry of pseudo-type lentivirus into the HEK293-ACE2 cells. All experiments were done in duplicates and were repeated twice. Taken together, our in vitro data on SARS-CoV-2 spike pseudotyped lentivirus suggest that administration of AMB and BHH may be effective and safe prophylaxis or treatment for SARS-CoV-2 infection (Tables 1(a), 1(b)).

TABLE 3

Effect of Ambroxol Hydrochloride (AMB) and Bromhexine Hydrochloride

(BHH) on the fusion of SARS-CoV-2 spike pseudotyped lentivirus with HEK293-ACE2 Cells

Percent Inhibition %

Concentration (μM)
BHH
BHH
AMB
AMB

100
73
76
41
47

50
57
61
72
59

25
8
27
78
64

10
−195
−81
86
55

According to various embodiments, an effective amount of Ambroxol Hydrochloride (AMB) and/or Bromhexine Hydrochloride may be administered to a subject to block or affect entry of SARS-CoV-2 spike (B.1.617.2 Delta Variant) pseudotyped lentivirus into HEK293-ACE2 Cells.

Applicant Ambroxol Hydrochloride (AMB) and Bromhexine Hydrochloride against the newly emerged delta variant of SARSC0V-2. Briefly, Spike (B.1.617.2 Delta Variant) pseudo typed lentivirus containing luciferase reporter gene was purchased from BPS Bioscience (Catalog no 78215-1, San Diego, CA). HEK293 cells with stable expression of full-length ACE2 were seeded at a density of 7500 cells per well into a white 96-well cell culture microplate. On the same day, pseudo typed lentivirus was preincubated with AMB and BHH at concentrations ranging from 100, 50, 25, and 10 μM for 30 minutes at room temperature (RT); the reaction mixture was then added to the cells. After 48 hours on day four, luciferase activity was measured by adding 100 μl Luciferase reagent (BPS Bioscience, catalog no. 60690) for 30 min at RT luminescence was measured with Spectra max ID3 (molecular devices). The measured luminescence signal is directly proportional to the amount of pseudotyped lentivirus successfully transduced into the cells.

1. Methods Comprising the Benzylamine Structural Class
General Methods

Disclosed herein is a method comprising administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method comprising administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; administering one or more clinically approved active agents; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; administering one or more clinically approved active agents; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method comprising inhibiting or ameliorating a SARS-CoV-2 infection by administering a composition comprising one or more compounds belonging to the benzylamine structural class. Disclosed herein is a method comprising inhibiting or ameliorating a SARS-CoV-2 infection by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising prophylactically administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection comprising prophylactically administering a composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; and inhibiting or ameliorating a SARS-CoV-2 infection.

The benzylamine structural class structural class is known to the art. The benzylamine structural class comprises at least Ambroxol hydrochloride (AMH) and bromhexine hydrochloride (BHH). For example, Ambroxol hydrochloride (AMH) is an aromatic amine that comprises the formula C₁₃H₁₉Br₂ClN₂O and has a molecular weight of 414.56 g/mol. Bromhexine hydrochloride is the hydrochloride salt form of bromhexine. Bromhexine hydrochloride comprises the formula C₁₄H₂₁Br₂ClN₂and has a molecular weight of 412.59 g/mol.

In an aspect, a disclosed composition in a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise administering (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof. In an aspect, a disclosed composition in a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a composition in a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise a AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof. The composition may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In various embodiments, the composition comprising AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof, may be administered via any suitable administration route. Example administration routes include parenteral administration, which may include intramuscular, intraarterial, or intravenous, as examples. Example administration routes include nonparenteral administration, such as oral, rectal, vaginal, nasal, mucosal, percutaneous, transdermal, or ophthalmic, as examples.

The composition comprising AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof, may be administered in a suitable dosage format, via a suitable route of administration, such as any of those identified above, and in an effective daily dose to inhibit or ameliorate a SARS-CoV-2 infection, such as by any mechanism identified herein. The daily dose may be between 30 mg up to 2,000 mg. Example daily dosages may include greater than 200 mg, greater than 400 mg, greater than 500 mg, greater than 600 mg, greater than 700 mg, greater than 800 mg, greater than 900 mg, greater than 1000 mg, between 50 mg and 100 mg, between 100 mg and 200 mg, between 150 mg and 300 mg, between 200 mg and 1000 mg, between 500 mg and 1500 mg, between 450 mg and 1200 mg, between 500 mg and 2000 mg, between 1000 mg and 2000 mg, or any range between 30 mg and 2000 mg. The composition will typically be administered once daily, twice daily, or three times daily; however, additional administrations may be used.

In one example, AMB and/or BHH is administered at a total daily dose of 240 mg, 480 mg or 960 mg, twice daily. The administration route may be oral. In one example, AMB and/or BHH is administered orally in a tablet or capsule.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise repeating one or more steps.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise identifying and/or characterizing one or more comorbidities in a subject. In an aspect of a disclosed method, a subject can have one or more comorbidities. Comorbidities are known to the art and can comprise cancer, chronic kidney disease, COPD (chronic obstructive pulmonary disease), an immunocompromised state (weakened immune system) from a solid organ transplant or from a blood or bone marrow transplant, obesity (body mass index [BMI] of 30 or higher), heart conditions (such as, e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, type 1 diabetes mellitus or type 2 diabetes mellitus, asthma, cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions, liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), a history of smoking, and thalassemia (a type of blood disorder).

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise monitoring a subject's response to the administration of a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof. In an aspect of a disclosed method, a monitoring step can be repeated one or more times. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise monitoring a subject's response to the administration of a disclosed composition comprising (i) AMB or BHH, or analogs or derivatives thereof, or a combination thereof and (ii) one or more disclosed active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect of a disclosed method, a monitoring step can be repeated one or more times.

Methods and techniques to monitor a subject's response to a disclosed method can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, qualitative means (or subjective means) can comprise a subject's own perspective. For example, a subject can report how he/she is feeling, whether he/she has experienced improvements and/or setbacks, whether he/she has experienced an amelioration of one or more symptoms, or a combination thereof. In an aspect, quantitative means (or objective means) can comprise methods and techniques that include, but are not limited to, the following: (i) fluid analysis (e.g., tests of a subject's fluids including but not limited to aqueous humor and vitreous humor, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, and urine), (ii) imaging (e.g., ordinary x-rays, ultrasonography, radioisotope (nuclear) scanning, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and angiography), (iii) endoscopy (e.g., laryngoscopy, bronchoscopy, esophagoscopy, gastroscopy, GI endoscopy, coloscopy, cystoscopy, hysteroscopy, arthroscopy, laparoscopy, mediastinoscopy, and thoracoscopy), (iv) analysis of organ activity (e.g., electrocardiography (ECG), electroencephalography (EEG), and pulse oximetry), (v) biopsy (e.g., removal of tissue samples for microscopic evaluation), and (vi) genetic testing.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise obtaining a disclosed compound (e.g., AMB or BHH, or an analog or derivative thereof), obtaining a disclosed composition, obtaining a disclosed formulation comprising a disclosed composition, obtaining one or more active agents, one or more biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or obtaining a combination thereof.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise preparing a disclosed compound (e.g., AMB or BHH, or an analog or derivative thereof) or preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise (1) preparing a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof, and (2) preparing (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise preparing one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating a SARS-CoV-2 infection can comprise preparing a disclosed composition comprising (i) AMB or BHH, or analogs or derivatives thereof, or a combination thereof, and (ii) one or more active agents, biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof.

i. Method of Inhibiting or Ameliorating One or More SARS-CoV-2 Infection Induced Cytopathic Effects

Disclosed herein is a method comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects. Disclosed herein is a method comprising inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in the subject. Disclosed herein is a method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in the subject by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise administering one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise (i) one or more active agents, (ii) biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise administering (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a composition in a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof. The composition may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can further comprise repeating one or more steps.

In an aspect, the administering step of a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise administering to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise identifying and/or characterizing one or more comorbidities in a subject. In an aspect of a disclosed method, a subject can have one or more comorbidities. Comorbidities are known to the art and can comprise cancer, chronic kidney disease, COPD (chronic obstructive pulmonary disease), an immunocompromised state (weakened immune system) from a solid organ transplant or from a blood or bone marrow transplant, obesity (body mass index [BMI] of 30 or higher), heart conditions (such as, e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, type 1 diabetes mellitus or type 2 diabetes mellitus, asthma, cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions, liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), a history of smoking, and thalassemia (a type of blood disorder).

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise modifying or altering one or more steps of a disclosed method. For example, in an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise modifying or altering an administering step. In an aspect, an administering step can be modified or altered, for example, by changing the route of administration, or changing the dose of a disclosed composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

In an aspect, altering or modifying one or more steps of a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects, such as, for example, an administering step, can be based on the identification and/or characterization of one or more comorbidities in a subject. In an aspect a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise modifying or altering the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise monitoring a subject's response to the administration of a disclosed composition comprising (i) AMB or BHH, or analogs or derivatives thereof, or a combination thereof and (ii) one or more disclosed active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect of a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects, a monitoring step can be repeated one or more times.

Methods and techniques to monitor a subject's response to a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, qualitative means (or subjective means) can comprise a subject's own perspective. For example, a subject can report how he/she is feeling, whether he/she has experienced improvements and/or setbacks, whether he/she has experienced an amelioration or an intensification of one or more symptoms, or a combination thereof. In an aspect, quantitative means (or objective means) can comprise methods and techniques that include, but are not limited to, the following: (i) fluid analysis (e.g., tests of a subject's fluids including but not limited to aqueous humor and vitreous humor, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, and urine), (ii) imaging (e.g., ordinary x-rays, ultrasonography, radioisotope (nuclear) scanning, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and angiography), (iii) endoscopy (e.g., laryngoscopy, bronchoscopy, esophagoscopy, gastroscopy, GI endoscopy, coloscopy, cystoscopy, hysteroscopy, arthroscopy, laparoscopy, mediastinoscopy, and thoracoscopy), (iv) analysis of organ activity (e.g., electrocardiography (ECG), electroencephalography (EEG), and pulse oximetry), (v) biopsy (e.g., removal of tissue samples for microscopic evaluation), and (vi) genetic testing.

In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise obtaining a disclosed compound (e.g., AMB or BHH, or an analog or derivative thereof), obtaining a disclosed composition, obtaining a disclosed formulation comprising a disclosed composition, obtaining one or more active agents, one or more biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or obtaining a combination thereof. In an aspect a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise preparing a disclosed compound (e.g., AMB or BHH, or an analog or derivative thereof) or preparing a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise (1) preparing a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof, and (2) preparing (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise preparing one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects can comprise preparing a disclosed composition comprising (i) AMB or BHH, or analogs or derivatives thereof, or a combination thereof, and (ii) one or more active agents, biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof.

ii. Method of Inhibiting or Reducing the Exopeptidase Activity of ACE2

In an aspect, the receptor binding domain (RBD) of the Spike glycoprotein can bind to the metallopeptidase domain (MPD) of ACE2.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise administering one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise administering (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a composition in a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof. The composition may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise identifying a subject having been diagnosed with or suspected of having a SARS-CoV-2 infection. As known to the art, a SARS-CoV-2 infection can be diagnosed and/or confirmed through various tests (such as, e.g., a PCR test or an antigen test).

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise identifying a subject having been diagnosed with or suspected of having a SARS-CoV-2 re-infection. As known to the art, a past SARS-CoV-2 infection can be diagnosed and/or confirmed through an antibody test. In an aspect, an antibody test (also known as serology testing) can check for immunoglobulin G (IgG) antibody. In an aspect, a variety of factors can impact the results from an antibody test in a disclosed method (e.g., the time the test was taken after experiencing symptoms, the absence of or time since exposure to the virus, or the lack of an adequate immune response, which can be due to conditions or treatments that suppress immune function).

In an aspect, the administering step of a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise administering to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise identifying and/or characterizing one or more comorbidities in a subject. In an aspect of a disclosed method, a subject can have one or more comorbidities. Comorbidities are known to the art and can comprise cancer, chronic kidney disease, COPD (chronic obstructive pulmonary disease), an immunocompromised state (weakened immune system) from a solid organ transplant or from a blood or bone marrow transplant, obesity (body mass index [BMI] of 30 or higher), heart conditions (such as, e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, type 1 diabetes mellitus or type 2 diabetes mellitus, asthma, cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions, liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), a history of smoking, and thalassemia (a type of blood disorder).

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise treating or ameliorating one or more comorbidities in a subject. For example, in an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise (i) administering one or more active agents to treat or ameliorate one or more comorbidities, (ii) administering one or more active agents to treat or ameliorate the same comorbidity, (iii) administering one or more active agents to treat or ameliorate different comorbidities (e.g., an active agent for type 2 diabetes and a different active agent for hypertension), or (iv) a combination thereof. In an aspect, administering an active agent to treat or ameliorate one or more comorbidities can occur prior to, concurrently with, or after the administering of a disclosed composition. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise repeating the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise modifying or altering one or more steps of a disclosed method. For example, in an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise modifying or altering an administering step. In an aspect, an administering step can be modified or altered, for example, by changing the route of administration, or changing the dose of a disclosed composition, or changing the timing of administration, or changing the frequency of the administration, or a combination thereof.

In an aspect, altering or modifying one or more steps of a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can be based on the identification and/or characterization of one or more comorbidities in a subject. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise modifying or altering the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise determining, measuring, and/or ascertaining the presence and/or severity of an infection, such as, for example, a SARS-CoV-2 infection, a bacterial infection, a viral infection, a fungal infection, or a combination thereof. Methods and techniques used to determine, measure, and/or ascertain the presence and/or severity of an infection such as a SARS-CoV-2 infection are typically known to the medical arts.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise monitoring a subject's response to the administration of a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. In an aspect of a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2), a monitoring step can be repeated one or more times.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise monitoring a subject's response to the administration of a disclosed composition comprising (i) CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof and (ii) one or more disclosed active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect of a disclosed method, a monitoring step can be repeated one or more times.

Methods and techniques to monitor a subject's response to a disclosed method can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, qualitative means (or subjective means) can comprise a subject's own perspective. For example, a subject can report how he/she is feeling, whether he/she has experienced improvements and/or setbacks, whether he/she has experienced an amelioration of one or more symptoms, or a combination thereof. In an aspect, quantitative means (or objective means) can comprise methods and techniques that include, but are not limited to, the following: (i) fluid analysis (e.g., tests of a subject's fluids including but not limited to aqueous humor and vitreous humor, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, and urine), (ii) imaging (e.g., ordinary x-rays, ultrasonography, radioisotope (nuclear) scanning, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and angiography), (iii) endoscopy (e.g., laryngoscopy, bronchoscopy, esophagoscopy, gastroscopy, GI endoscopy, coloscopy, cystoscopy, hysteroscopy, arthroscopy, laparoscopy, mediastinoscopy, and thoracoscopy), (iv) analysis of organ activity (e.g., electrocardiography (ECG), electroencephalography (EEG), and pulse oximetry), (v) biopsy (e.g., removal of tissue samples for microscopic evaluation), and (vi) genetic testing.

In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise preparing a disclosed compound (e.g., CLQ, CLBQ14, or CLCQ, or an analog or derivative thereof) or preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise (1) preparing a disclosed composition comprising CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and (2) preparing (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise preparing one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or reducing exopeptidase activity of an enzyme or a disclosed method of inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2) can comprise preparing a disclosed composition comprising (i) CLQ, CLBQ14, or CLCQ, or analogs or derivatives thereof, or a combination thereof, and (ii) one or more active agents, biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof.

iii. Method of Inhibiting or Disrupting the Interaction Between ACE2 and Spike Protein

Disclosed herein is a method comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. Disclosed herein is a method comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or disrupting the physical interaction of an angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity. Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing viral infectivity. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject. Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing viral entry into cells of the subject. Disclosed herein is a method comprising administering a composition comprising an effective amount AMB or BHH, or analogs or derivatives thereof, or a combination thereof; inhibiting or reducing the activity of a type II transmembrane serine protease; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. Disclosed herein is a method comprising inhibiting or reducing the activity of a type II transmembrane serine protease and inhibiting or disrupting the physical interaction of an angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising administering a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or reducing the activity of a type II transmembrane serine protease, thereby inhibiting or disrupting the physical interaction of an angiotensin converting enzyme 2 (ACE). Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, or a combination thereof; inhibiting or reducing the activity of a type II transmembrane serine protease; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity. Disclosed herein is a method of inhibiting or reducing viral infectivity in a subject comprising inhibiting or reducing the activity of a type II transmembrane serine protease by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity. Disclosed herein is a method of inhibiting or reducing a SARS-CoV-2 infection in a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; inhibiting or reducing the activity of a type II transmembrane serine protease; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or reducing a SARS-CoV-2 infection in a subject comprising inhibiting or reducing the activity of a type II transmembrane serine protease and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, thereby inhibiting or reducing a SARS-CoV-2 infection. Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; inhibiting or reducing the activity of a type II transmembrane serine protease; and inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject. Disclosed herein is a method of inhibiting or reducing viral entry into cells of a subject comprising disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 by administering to a subject a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, and inhibiting or reducing the activity of a type II transmembrane serine protease, thereby inhibiting or reducing viral entry into cells of the subject.

In an aspect, the receptor binding domain (RBD) of the Spike glycoprotein can bind to the metallopeptidase domain (MPD) of the ACE2 receptor. In an aspect, the type II transmembrane serine protease is TMPRSS2.

In an aspect, a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise administering (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof. In an aspect, a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In an aspect, a composition in a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise a AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof. The composition may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

In an aspect, the administering step of a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise administering to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise identifying and/or characterizing one or more comorbidities in a subject. In an aspect of a disclosed method, a subject can have one or more comorbidities. Comorbidities are known to the art and can comprise cancer, chronic kidney disease, COPD (chronic obstructive pulmonary disease), an immunocompromised state (weakened immune system) from a solid organ transplant or from a blood or bone marrow transplant, obesity (body mass index [BMI] of 30 or higher), heart conditions (such as, e.g., heart failure, coronary artery disease, or cardiomyopathies), sickle cell disease, type 1 diabetes mellitus or type 2 diabetes mellitus, asthma, cerebrovascular disease, cystic fibrosis, hypertension or high blood pressure, immune deficiencies, HIV, use of corticosteroids, or use of other immune weakening medicines, neurologic conditions, liver disease, pregnancy, pulmonary fibrosis (having damaged or scarred lung tissues), a history of smoking, and thalassemia (a type of blood disorder).

In an aspect, a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise (i) administering one or more active agents to treat or ameliorate one or more comorbidities, (ii) administering one or more active agents to treat or ameliorate the same comorbidity, (iii) administering one or more active agents to treat or ameliorate different comorbidities (e.g., an active agent for type 2 diabetes and a different active agent for hypertension), or (iv) a combination thereof. In an aspect, administering an active agent to treat or ameliorate one or more comorbidities can occur prior to, concurrently with, or after the administering of a disclosed composition. In an aspect, a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise repeating the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect, altering or modifying one or more steps of a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can be based on the identification and/or characterization of one or more comorbidities in a subject.

In an aspect a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise modifying or altering the administering step of an active agent to treat or ameliorate one or more comorbidities.

In an aspect a disclosed method or a disclosed method of inhibiting or disrupting the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 or a disclosed method of inhibiting or reducing viral infectivity in a subject or a method of inhibiting or ameliorating a SARS-CoV-2 infection or a disclosed method of inhibiting or reducing viral entry into cells of a subject or a disclosed method of inhibiting or reducing the activity of a type II transmembrane serine protease can comprise (1) preparing a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof, and (2) preparing (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof.

General Composition

Disclosed herein is composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection. Disclosed herein is a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection. Disclosed herein is composition comprising an effective amount of one or more compounds belonging to the benzylamine structural class; an effective amount of one or more clinically approved active agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection. Disclosed herein is a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more clinically approved active agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates a SARS-CoV-2 infection.

The benzylamine structural class is known to the art and discussed herein. Pharmaceutically acceptable diluents, carriers, excipients, and stabilizers are known to the art and discussed herein.

In an aspect, a disclosed composition comprising one or more compounds belonging to the benzylamine structural class or a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can comprise one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent.

In an aspect, a disclosed composition comprising one or more compounds belonging to the benzylamine structural class or a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed composition comprising one or more compounds belonging to the benzylamine structural class or a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

A disclosed composition comprising AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof, may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administered in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

The composition comprising AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof, may be administered via any suitable administration route. Example administration routes include parenteral administration, which may include intramuscular, intraarterial, or intravenous, as examples. Example administration routes include nonparenteral administration, such as oral, rectal, vaginal, nasal, mucosal, percutaneous, transdermal, or ophthalmic, as examples.

In an aspect, a disclosed composition comprising one or more compounds belonging to the benzylamine structural class or a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can be administered to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a human or a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

i. Compositions for Inhibiting or Ameliorating a SARS-CoV-2 Infection

Disclosed herein is a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects. Disclosed herein is a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects. Disclosed herein is a composition for inhibiting or ameliorating cytopathic effects in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more SARS-CoV-2 infection induced cytopathic effects in a subject in need thereof. Disclosed herein is a composition for inhibiting or ameliorating cytopathic effects in a subject comprising an effective amount AMB or BHH, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject in need thereof. Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject diagnosed with or suspected of having a SARS-CoV-2 infection. Disclosed herein is a composition for inhibiting or ameliorating one or more SARS-CoV-2 infection induced cytopathic effects in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; an effective amount of one or more anti-viral agents; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof; wherein the composition inhibits or ameliorates one or more cytopathic effects in a subject diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can comprise one or more active agents, one or more biologically active agents, one or more pharmaceutically active agents, one or more immune-based therapeutic agents, one or more clinically approved agents, or a combination thereof. Biologically active agents are described herein and are known to the art. Pharmaceutically active agents are described herein and are known to the art. Immune-based therapeutic agents are described herein and are known to the art. Clinically approved active agents can comprise one or more FDA-approved active agents regardless of whether an active agent is a biologically active agent, a pharmaceutically active agent, or an immune-based therapeutic agent. In an aspect, a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can comprise (i) one or more active agents, (ii) one or more biologically active agents, (iii) one or more pharmaceutically active agents, (iv) one or more immune-based therapeutic agents, (v) one or more clinically approved agents, or (vi) a combination thereof. In an aspect, a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can comprise (i) one or more anti-bacterial agents, (ii) one or more anti-fungal agents, (iii) one or more anti-viral agents (such as, for example, remdesivir, favipiravir, merimepodib, etc.), (iv) one or more corticosteroids (such as, e.g., dexamethasone, prednisone, methylprednisolone, hydrocortisone, etc.), or (v) a combination thereof.

In various embodiments, the composition comprising AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof, may be administered in a suitable dosage format, via a suitable route of administration, such as any of those identified above, and in an effective daily dose to inhibit or ameliorate a SARS-CoV-2 infection, such as by any mechanism identified herein. The daily dose may be between 30 mg up to 2,000 mg. Example daily dosages may include greater than 200 mg, greater than 400 mg, greater than 500 mg, greater than 600 mg, greater than 700 mg, greater than 800 mg, greater than 900 mg, greater than 1000 mg, between 50 mg and 100 mg, between 100 mg and 200 mg, between 150 mg and 300 mg, between 200 mg and 1000 mg, between 500 mg and 1500 mg, between 450 mg and 1200 mg, between 500 mg and 2000 mg, between 1000 mg and 2000 mg, or any range between 30 mg and 2000 mg. The composition will typically be administered once daily, twice daily, or three times daily; however, additional administrations may be used.

In an aspect, a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can be administered to a subject. In an aspect, a subject can be a human. In an aspect, a human subject can be a participant in a clinical trial. In an aspect, a subject can be a non-human primate diagnosed with or suspected of having a SARS-CoV-2 infection.

In an aspect, a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can be administered to a subject having one or more comorbidities. In an aspect, a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can comprise one or more active agents to treat or ameliorate one or more comorbidities. Comorbidities are known to the art and are discussed herein.

ii. Compositions for Inhibiting or Reducing the Exopeptidase Activity of ACE2

Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and a pharmaceutically acceptable diluent, carrier, excipient, or stabilizer, or a combination thereof, wherein the composition inhibits or reduces the exopeptidase activity of angiotensin converting enzyme 2 (ACE2). Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and an effective amount of zinc chloride; wherein the composition inhibits or reduces the exopeptidase activity of angiotensin converting enzyme 2 (ACE2). Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2). Disclosed herein is a composition for inhibiting or reducing exopeptidase activity of an enzyme comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and an effective amount of zinc chloride, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing the exopeptidase activity of angiotensin converting enzyme 2 (ACE2).

AMB, BHH, and analogs or derivatives thereof are known to the art and are discussed herein. Pharmaceutically acceptable diluents, carriers, excipients, stabilizers, and combinations thereof are known to the art and are discussed herein.

In an aspect, the receptor binding domain (RBD) of the Spike glycoprotein can bind to the metallopeptidase domain (MPD) of the ACE2 receptor.

A disclosed composition comprising AMB and/or BHH, or analogs or derivatives thereof, or a combination thereof, may be administered in various formats, such as tablet, capsule, syrup, dry powder sachets, inhalation solution/nebulization, drops, ampules, suppository, creams or ointments. For example, the composition may be administered in a tablet format. Tablets may be formulated for controlled release formulation, delayed release formulation, extended release formulation, sustained release formulation, pulsatile release formulation, or mixed immediate release formulation. Tablets may be effervescent. The composition may be administerd in a capsule format. Capsules may be formulated for immediate release or sustained release, as examples. Capsules may be hard capsules or and soft capsules. Solution formats may be oil based, aqueous, or emulsions.

iii. Compositions for Inhibiting or Disrupting the Interaction Between ACE2 and Spike Protein

Disclosed herein is a composition comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. Disclosed herein is a composition for inhibiting or disrupting the physical interaction of an angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2 comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof. Disclosed herein is a composition for inhibiting or reducing viral infectivity in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral infectivity. Disclosed herein is a composition for inhibiting or ameliorating a SARS-CoV-2 infection in a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof, wherein the composition inhibits or disrupts the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or ameliorating a SARS-CoV-2 infection. Disclosed herein is a composition for inhibiting or reducing viral entry into cells of a subject comprising an effective amount of AMB or BHH, or analogs or derivatives thereof, or a combination thereof; and wherein the composition inhibits or disrupts they physical interactions of angiotensin converting enzyme 2 (ACE2) and the Spike (S) glycoprotein of SARS-CoV-2, thereby inhibiting or reducing viral entry into cells of the subject.

In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can inhibit or disrupt the physical interaction of angiotensin converting enzyme 2 (ACE2) with the Spike (S) glycoprotein of SARS-CoV-2. In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can inhibit or reduce viral infectivity. In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can inhibit or ameliorate a SARS-CoV-2 infection. In an aspect, a disclosed composition comprising one or more compounds belonging to the 8-hydroxyquinoline structural class or a disclosed composition comprising AMB or BHH, or analogs or derivatives thereof, or a combination thereof can inhibit or reduce viral entry into cells of the subject.

In an aspect, the receptor binding domain (RBD) of the Spike glycoprotein can bind to the metallopeptidase domain (MPD) of the ACE2 receptor.

E. Kits

Disclosed herein is a kit comprising one or more disclosed compositions. In an aspect, a kit can comprise one or more compositions comprising a member of a 8-hydroxyquinoline structural class. In an aspect, a kit can comprise one or more compositions comprising a member of a benzylamine structural class. In an aspect, a kit can comprise one or more compositions comprising CLQ, CLBQ14, CLCQ, analogs thereof, derivatives thereof, or a combination thereof. In an aspect, a kit can comprise one or more compositions comprising AMB, BHH, analogs thereof, derivatives thereof, or a combination thereof. In an aspect, a kit can comprise one or more compositions comprising CLQ, CLBQ14, CLCQ, analogs thereof, derivatives thereof, or a combination thereof and one or more active agents. In an aspect, a kit can comprise one or more compositions comprising AMB, BHH, analogs thereof, derivatives thereof, or a combination thereof and one or more active agents. In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, treating a subject diagnosed with or suspected of having a coronavirus infection like SARS-CoV-2). Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a kit for use in a disclosed method can comprise one or more containers holding a disclosed composition and a label or package insert with instructions for use. In an aspect, a kit can contain one or more additional agents (e.g., active agents, biologically active agents, pharmaceutically active agents, immune-based therapeutic agents, clinically approved agents, or a combination thereof). In an aspect, one or more active agents can treat, inhibit, and/or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, and/or ameliorate a SARS-CoV-2 related infection and/or complication. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed composition or a pharmaceutical formulation comprising a disclosed composition and can have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

The label or package insert can indicate that a disclosed composition or a pharmaceutical formulation comprising a disclosed composition can be used for treating, preventing, inhibiting, and/or ameliorating a coronavirus infection (e.g., SARS-CoV-2) or complications and/or symptoms associated with a coronavirus infection. A kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes.

As introduced above, in some formulations may include a tablet. According to one embodiment, a method of making an immediate-release tablet containing AMB, BHH, CLQ, CLBQ14, and/or CLCQ includes a wet granulation method wherein the fine powder of containing AMB, BHH, CLQ, CLBQ14, and/or CLCQ is mixed with diluents (such as lactose or microcrystalline cellulose) and disintegrants (croscarmellose (2%), sodium starch gylcolate (5%), sodium carboxymethylcellulose, polyvinylpyrrolidone (PVP)); a binder solution is also prepared (such as aqueous solutions of povidone, cornstarch, methylcellulose, carboxymethylcellulose or glucose); the binder solution is mixed with the powder mixture to form an adhesive mass which can be granulated; the wet massed powder blend is then be screened using 6- to 12-mesh screen to prepare wet granules; the moist granules are dried in an oven at a controlled temperature not exceeding 55C to a consistent weight; the dried granules are mixed with appropriate quantity of lubricant, such as magnesium stearate (1% to 2% of the weight of the granulation); the mixed granules are compressed in a single punch or multi-station tablet press fitted with the appropriate punches and dies.

EXAMPLES
A. Examples Comprising CLQ, CLBQ14, and CLCQ
Materials and Methods

African Green Monkey Kidney Vero E6 cells (ATCC #CRL-1586, American Tissue Culture Type) were maintained using medium purchased from Gibco (modified eagle's medium (MEM) Gibco (#11095), 10% fetal bovine serum (HI FBS) Gibco (#14000), and Penicillin/Streptomycin (PS) Gibco (#15140); 10 U/mL penicillin and 10 μg/mL streptomycin (only in assay media)). For the SARS-CoV-2 infection induced cytopathic effect (CPE) assay, cells were grown in MEM/10% HI FBS and harvested in MEM/1% PS/supplemented with 2% HI FBS. Cells were batch inoculated with SARS-CoV-2 USA_WA1/2020 (M.O.I.˜0.002), which resulted in 5-10% cell viability 72 hours post infection.

The small molecule inhibitors 5-chloro-7-iodo-8-quinolinol (Clioquinol, CLQ; C0187-Lot JJ01 SPGN) and 7-bromo-5-chloro-8-hydroxyquinoline (CLBQ14; B1190-P61JD-FD)) were purchased from TCI America whereas 5, 7-dichloro-8-hydroxyquinoline (CLCQ; D64600-Lot #STBH7389) and Zinc Chloride (ZnCl_{2; 208086}-Lot #MKCL1763) were purchased from Sigma Aldrich. 10 mM stocks solutions of the inhibitors were prepared in dimethylsulfoxide (DMSO; D8418-Lot #SHBL5613) purchased from Sigma Aldrich. For the CPE assay, compound samples were serially diluted 2-fold in DMSO nine times and screened in duplicates. Assay Ready Plates (ARPs; Corning 3764BC) pre-drugged with test compounds (90 nL sample in 100% DMSO per well dispensed using a Labcyte (ECHO 550) are prepared in the Biosafety Level-2 (BSL-2) laboratory by adding 5 μL assay media to each well.

Compound cytotoxicity was assessed in a BSL-2 counter screen as follows using the Cell Titer-Glo Luminescent Cell Viability Assay.⁶⁰⁸⁵Host cells in media were added in 25 μL aliquots (4000 cells/well) to each well of assay ready plates prepared with test compounds as above. Cells only (100% viability) and cells treated with hyamine at 100 μM final concentration (0% viability) serve as the high and low signal controls, respectively, for cytotoxic effect in the assay. DMSO was maintained at a constant concentration for all wells (0.3%) as dictated by the dilution factor of stock test compound concentrations. After incubating plates at 37° C./5% CO₂and 90% humidity for 72 hours, 30 μL CellTiter Glo (CTG) (G7573, Promega) was added to each well. Luminescence was read using a BMG CLARIOstar plate reader following incubation at room temperature for 10 minutes to measure cell viability.

The SARS-CoV-2 infection induced cytopathic effect (CPE) assay and cytotoxicity assays were generated and performed through a sub-contract to Southern Research Institute (SRI) (Birmingham, Alabama) from Texas Southern University (Houston, Texas). The CPE reduction assay was conducted at SRI to screen for antiviral agents in high throughput screening (HTS) format as previously described.^{54, 85}Briefly, Vero E6 cells selected for expression of the SARS-CoV-2 receptor (ACE2; angiotensin-converting enzyme 2) were used for the CPE assay. Cells were grown in MEM/10% HI FBS supplemented and harvested in MEM/1% PS/supplemented with 2% HI FBS. Cells were batch inoculated with SARS-CoV-2 (M.O.I.˜0.002), which resulted in 5% cell viability 72 hours post infection. Compound samples were serially diluted 2-fold in DMSO nine times and screened in duplicates. Assay Ready Plates (ARPs; Corning 3764 BC black-walled, clear bottom plates) pre-drugged with test compounds (90 nL sample in 100% DMSO per well dispensed using a Labcyte (ECHO 550) were prepared in the BSL-2 lab by adding 5 μL assay media to each well. The plates were passed into the BSL-3 facility where a 25 μL aliquot of virus inoculated cells (4000 Vero E6 cells/well) was added to each well in Columns 3-22. The wells in Columns 23-24 contained virus infected cells only (no compound treatment). Prior to virus infection, a 25 μL aliquot of cells was added to Columns 1-2 of each plate for the cell only (no virus) controls. After incubating plates at 37° C./5% CO₂and 90% humidity for 72 hours, 30 μL of Cell Titer-Glo (Promega) was added to each well. Luminescence was read using a Perkin Elmer Envision or BMG CLARIOstar plate reader following incubation at room temperature for 10 minutes to measure cell viability. Raw data from each test well was normalized to the average (Avg.) signal of non-infected cells (Avg. Cells; 100% inhibition) and virus infected cells only (Avg. Virus; 0% inhibition) to calculate % inhibition of CPE using the following formula: % inhibition=100*(Test Cmpd−Avg. Virus)/(Avg. Cells−Avg. Virus). The SARS CPE assay was conducted in BSL-3 containment with plates being sealed with a clear cover and surface decontaminated prior to luminescence reading. Reference compounds for CPE assay were made available by SRI.

ACE2 Inhibitor Screening Assay

An ACE2 inhibitor screening assay kit with fluorogenic substrate (Catalogue #79923) was purchased from BPS Bioscience (San Diego, CA) and adapted to measure the exopeptidase activity of ACE2 in the presence and absence of inhibitors. The fluorescence assay was performed using a black flat-bottom 96-well plate with a final reaction volume of 50 μL following the manufacturer's instructions. 10 mM stock solutions of the compounds were prepared in Dimethyl sulfoxide (DMSO). Next, the compounds were serially diluted in DMSO as follows: 100 μM, 50 μM, 10 μM, 1 μM, 0.5 μM, and 0.1 μM for CLQ and CLBQ14 as well as 10 μM and 1 μM for CLCQ. All experiments were performed in triplicates. Each plate contained a positive control of enzyme-treated with vehicle alone (2% DMSO) and a blank control with no enzyme. Briefly, each reaction contained 24 μL of purified recombinant human ACE2 protein (0.42 ng/L) in ACE2 buffer, 1 μL of compound at serially diluted concentrations, and 25 μL ACE2 fluorogenic substrate. The total reaction volume was 50 μL. The reaction mixtures were protected from light and incubated for 2.5 hours at room temperature (22° C.). Thereafter, the fluorescence intensities (λ_Excitation=535 nm, λ_Emission=595 nm) were measured using a Beckman Coulter DTX880 multimode plate reader. A similar experiment was conducted to measure and compare the exopeptidase activity of ACE2 in the presence and absence of Zinc Chloride (ZnCl₂) alone, CLBQ14 alone, and ZnCl₂in combination with CLBQ14 at concentrations ranging from 100 μM to 100 nM. ZnCl₂was serially diluted in water and a positive control of enzyme-treated with vehicle alone (water for ZnCl₂only; DMSO for CLBQ14 alone; and water plus DMSO for ZnCl₂and CLBQ14) was carried out for this experiment. The background hydrolysis was subtracted and the data was fitted to a four-parameter logistic (variable slope) equation using GraphPad prism software 8.4.3.

Ace2-Spike (Rbd) Protein Interaction Assay

A Spike-ACE2 binding assay kit (Cat #CoV-SACE2-1, Lot #062320 7066) was purchased from RayBiotech (Norcross, GA). The in vitro enzyme-linked immunoabsorbent assay (ELISA) was adapted and performed in a transparent flat-bottom 96-well plate. 10 mM stock solutions of the compounds were prepared in Dimethyl sulfoxide (DMSO), with serially diluted the compounds in DMSO as follows: 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, and 0.1 μM for CLQ, CLBQ14, and CLCQ. All experiments were performed in triplicates. Each plate contained positive controls (1% DMSO) and blank controls with no ACE2. Briefly, 1 μL of serially diluted compounds were incubated with recombinant SARS-CoV-2 Spike receptor binding domain (RBD) protein, pre-coated on the 96 well plates in 49 μL of 1× assay diluent buffer for 31 minutes at room temperature (22° C.) with shaking at 180 rpm. Next, 50 μL of ACE2 protein in 1× assay diluent buffer was added into the 96 well plate and incubated for 2.5 hours at room temperature (22° C.) with shaking at 180 rpm. Thereafter, the solution was discarded and the plate was washed consecutively four times with 300 μL 1× wash buffer followed by the addition of the detection antibody (anti-ACE2 goat antibody). The reaction was allowed to go on for 1 hour at room temperature (22° C.) with shaking at 180 rpm. Then, the solution was discarded and the wash step was repeated as described above. Next, the HRP-conjugated anti-goat IgG was added to each well and the reaction plate was further incubated for 1 hour at room temperature (22° C.) with shaking at 180 rpm. Again, the solution was discarded and the wash step was repeated as described above. Then, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) one-step substrate was added to each well. The reaction mixtures were incubated in the dark at room temperature (22° C.) with shaking at 180 rpm for an additional 30 minutes and then stopped by the addition of 50 μL stop solution. The absorbance was read at 405 nm using a Beckman Coulter DTX880 multimode plate reader. The background hydrolysis was subtracted and the data was fitted to a special bell-shaped dose-response curve equation using GraphPad prism software 8.4.3.

Results and Discussion
Cytotoxicity Effects of CLQ and Analogues in Vero E6 Cells

The preliminary cytotoxicity of CLQ and its analogues (CLBQ14 and CLCQ) was determined using a Cell Titer-Glo Luminescent Cell Viability Assay.⁸⁵The cytotoxic effects of the various compounds in Vero E6 cells measured at the 50% cytotoxic concentration (CC₅₀) of CLQ and its derivatives were all greater than 30 μM. When compared to the other reference compounds tested, CLQ and its analogues displayed lower percent minimum viability at higher concentrations. Similar percent maximum viability for CLQ pharmacophore and the other reference compounds at lower concentrations was observed (FIG. 8). This indicates that the cytotoxic effects may not be a concern at lower concentrations of CLQ and its analogues. FIG. 8 shows the cytotoxicity of clioquinol (CLQ) and analogues in Vero E6 Cells compared to reference inhibitors of SARS-CoV-2.

Efficacy of Clioquinol (CLQ) and Analogues Against SARS-CoV-2 Infection Induced Cytopathic Effect (CPE) in Vero E6 Cells

To identify inhibitors of SARS-CoV-2 infection for potential treatment of COVID-19, the in vitro antiviral activity of CLQ and two of its derivatives, CLBQ14 and CLCQ, were examined using a standard luminescent-based high-throughput screening (HTS) platform^{59, 6054, 85}for SARS-CoV-2 infection induced CPE in African Green Monkey Kidney Vero E6 cells. The three compounds inhibited SARS-CoV-2 infection induced CPE in vitro with 50% Inhibitory Concentration (IC₅₀) values in the low micromolar concentration (FIGS. 1A-1C). Amongst the three analogues tested, CLQ displayed the most potent antiviral activity in the CPE assay. Compared to its counterparts, CLBQ14 exhibited the highest maximum inhibition at about 102.96% inhibition at 30 μM. FIG. 6 shows the chemical structure and activity of Clioquinol (CLQ) and analogues against SARS-CoV-2 induced cytopathic effect (CPE) in Vero E6 cells.

The antiviral effects of CLBQ14 and its analogues were also compared with five other known inhibitors of SARS-CoV-2 in vitro: Chloroquine, Hydroxychloroquine, Remdesivir, Aloxistatin, and Calpain Inhibitor IV. The dose-response curves of the CLQ, CLBQ14, CLCQ and the reference compounds mentioned above were determined at multiplicities of infection (MOI) of about 0.002. The IC₅₀for CLQ (12.62 μM) and its analogues [(CLBQ14, 14.69 μM) and (CLCQ, 16.30 μM)] were slightly lower than the IC₅₀of Aloxistatin (16.72 μM), but moderately higher than Chloroquine (1.10 μM), Hydroxychloroquine (5.04 μM), Remdesivir (4.42 μM), and Calpain Inhibitor IV (0.41 μM) (FIGS. 2A-2E). FIG. 7 shows the chemical structure and activity of reference inhibitors against SARS-CoV-2 induced cytopathic effect (CPE) in Vero E6 cells. This is the first report that CLQ and its analogues effectively inhibit the novel SARS-CoV-2 infection induced CPE.

Effects of Clq and its Analogues on Ace2 Exopeptidase Activity

The effects of CLQ, CLBQ14, and CLCQ on the exopeptidase activity of rhACE2 were determined using a fluorometric assay provided by BPS Bioscience and adapted accordingly. BPS Bioscience's method for the ACE2 Inhibitor Screening Assay Kit can be found at https://bpsbioscience.com/pub/media/wysiwyg/79923.pdf.

The three compounds inhibited rhACE2 activity with similar IC₅₀values in the low micromolar concentration with CLQ being the most potent amongst the three analogues tested, having an IC₅₀of 5.36 μM. FIG. 9 shows the activity of clioquinol (CLQ) and analogues against ACE2 exopeptidase activity and ACE2 and SARS-CoV-2 spike (RBD) protein interaction. This is the first report that rhACE2 is a biochemical target of CLQ and its analogues.

Using the same fluorometric assay described above, the exopeptidase activity of rhACE2 was assessed in the presence of zinc chloride (ZnCl₂) alone, CLBQ14 alone, and ZnCl₂in combination with CLBQ14 at concentrations ranging from 100 μM to 100 nM. In the presence of ZnCl₂alone, rhACE2 displayed increasing exopeptidase activity. In the presence of ZnCl₂in combination with CLBQ14, there was an increased shift in IC₅₀value by over 28-fold compared to CLBQ14 alone (FIG. 3). The increasing concentrations of ZnCl₂titrates the inhibitory effect of CLBQ14 on rhACE2 from concentrations ranging from above 5 μM-10 PM, which is consistent with the required optimal concentration range of Zinc for the exopeptidase activity of ACE2.⁶¹

Effects of CLQ and its Analogues on ACE2 and Spike (RBD) Protein Interaction

The interaction of human ACE2 receptor with SARS-CoV-2's Spike protein receptor binding domain is an important first step in the process of viral entry into host cells.^{98, 100, 105, 108}Using an adapted in vitro enzyme-linked immunoabsorbent assay (ELISA) (see User Manual for RayBio® COVID-19 Spike-ACE2 binding assay kit available at https://doc.raybiotech.com/pdf/Manual/CoV-SACE2_2020.07.09.pdf), the effect of CLQ, CLBQ14, and CLCQ on the binding affinity of rhACE2 and RBD of S protein was examined at concentrations ranging from 100 μM to 100 nM. A unique bell shaped dose-response curve for the three compounds was observed, which showed higher inhibition of ACE2-Spike (RBD) protein interaction at lower compound concentrations compared to higher concentrations (FIGS. 4A-4C). The bell shaped curve generated two IC₅₀values (IC_{50_1}and IC_{50_2}). These three compounds had similar IC₅₀values in the low micromolar concentration ranging from 0.85 μM to 2.76 μM for IC₅₀_1; however, CLQ displayed a higher IC_{50_2}at 18.15 μM. The unconventional dose response curve observed in this interaction assay can indicate one or more additional binding sites or one or more additional targets for the CLQ pharmacophore, such as, for example, other sites on ACE2 or the Spike (RBD) protein. These data represent the first report that CLQ and its analogues inhibited and interfered with the binding between human ACE2 receptor and SARS-CoV-2 Spike RBD protein.

Summary of Experiments Comprising CLQ, CLBQ14, and CLCQ

Given the ongoing COVID-19 pandemic and the emerging virulence of novel SARS-CoV-2 strains, there is an urgent need to accelerate the development of effective therapeutic agents as countermeasures against this pathogen. Here, three independent approaches were applied to investigate the possibility of CLQ and its analogues as potential inhibitors of the SARS-CoV-2 infection in vitro. These data represent the first report that CLQ and its analogues target rhACE2. CLQ significantly inhibited binding of rhACE2 receptor with SARS-CoV-2 Spike (RBD) protein and SARS-CoV-2 infection induced CPE.

CLQ, a known metal chelator and zinc ionophore, was successfully identified and characterized as an inhibitor of SARS-CoV-2 infection induced CPE. CLQ and two structural analogues of CLQ (CLBQ14 and CLCQ) displayed similar potent inhibition in the low micromolar range against SARS-CoV-2 infection induced CPE, rhACE2 activity, and its interaction with Spike Protein. The dose-response curves of antiviral effects of CLQ and its analogues was compared with five other known inhibitors of SARS-CoV-2 in vitro: Chloroquine, Hydroxychloroquine, Remdesivir, Aloxistatin, and Calpain Inhibitor IV. CLQ's potency was better than Aloxistatin, but lower than the other reference inhibitors FIGS. 2A-2E. Because the Vero E6 cells used for the SARS-CoV-2 infection induced CPE assay were first sorted by flow cytometry by SRI for selection of cells that had higher levels of ACE2 expression to increase the efficiency of infection, the observed IC₅₀values may be higher than the actual values in cells that are does not have high levels of ACE2 expression. The IC₅₀values of the compounds in the biochemical assays were much lower than the IC₅₀in the cellular antiviral assay.

The cytotoxic effects of the compounds in Vero E6 cells were assessed and CLQ and its analogues displayed lower percent minimum viability at higher concentrations compared to the other reference compounds tested. Similar percent maximum viability for CLQ pharmacophore and the other reference compounds were measured at lower concentrations (FIG. 8). The date indicate that cytotoxic effects may not be a concern at lower concentrations of CLQ and its analogues. In addition, the observed IC₅₀values for inhibition of rhACE exopeptidase activity and rhACE2-RBD interaction were in the low micromolar range, which can indicate the need for lower concentrations for in vivo activity.

A correlation between the high potency of CLQ compared to its other two analogues in the antiviral screen, inhibition of rhACE2 metalloprotease activity, and its ability to disrupt the binding of rhACE2 with SARS-CoV-2 Spike (RBD) protein was consistently observed during these studies. Amongst the three compounds, CLQ displayed the highest potency in the three independent assays; except for IC_{50_2}. Clioquinol and its derivatives can act as metal chelators and zinc ionophores, which can be capable of modulating underlying molecular and physiologic switches in metal homeostatis in vivo.^{1-2, 4-6, 8, 14, 21, 24-25, 32, 46, 50, 64, 75, 77, 81, 84, 87, 91-92, 111-112,}These data demonstrate that CLQ and its analogs can target ACE2, a zinc metalloenzyme and essential cellular receptor for SARS-CoV-2 entry into host cells.^{98, 100, 105, 108}

ACE2, a carboxypeptidase, is a type I integral membrane protein made up of about 805 amino acids belonging to the large family of zinc metalloproteases with high level of structural homology for a catalytic motif, containing one characteristic HEXXH+E zinc-binding consensus sequence and binding sites for inhibitor or specific substrates respectively.⁹⁴The first crystalline structures of the metallopeptidase domain of ACE2 revealed a large inhibitor-dependent hinge bending movement of one catalytic subdomain relative to the other that brings important amino acid residues into position for catalysis,⁹⁴which was similar to observed subdomains on other zinc metalloproteases. The residues critical for coordinating the binding of zinc to ACE2 are His³74 His³⁷⁸and Glu⁴⁰², according to earlier x-ray structures.⁹⁴Moreover, ACE2 is activated by monovalent anions and also known to contain an inhibitor-specific anion binding site.^94,96The reported optimal/stabilized metalloprotease activity of recombinant soluble human ACE2 was found to be in the presence of 10 μM ZnCl₂.⁹⁶

As described herein, CLQ or CLBQ14 alone significantly decreased exopeptidase activity for ACE2 in the low micromolar concentrations. An increased shift in IC₅₀values was identified in exopeptidase activity in the presence of ZnCl₂in combination with CLBQ14 by over 28 fold compared to CLBQ14 alone, indicating that CLBQ14 can be working through zinc chelation, interaction, and/or coordination. The data provided herein demonstrate that rhACE2 is a target for the CLQ pharmacophore. These data also demonstrate a potential reversibility of inhibition as well as one or more avenues of inhibition. For example, the concentration of CLBQ14 can be titrated with excess ZnCl₂, thus rendering it pre-occupied and unavailable to inhibit rhACE2 exopeptidase activity. Or, there can be potential competition for the similar binding sites on rhACE2.

Targeting ACE2 requires caution as it is imprudent to permanently inactivated its exopeptidase activity or other activities. CLQ is a weak metal chelator and zinc ionophore that can shuttle free zinc across the membrane.^{18, 112}Because of these properties, CLQ may temporarily or reversibly affect ACE2 function and prevent its interaction with SARS-CoV-2 RBD protein without permanently inhibiting its exopeptidase function. The crystal structure of full length human ACE2 revealed that the RBD on SARS-CoV-2 S1 binds directly to the metallopeptidase domain (MPD) of ACE2 receptor,^{105, 108}which consists of amino acid residues that coordinates zinc. Using a sensitive ELISA, CLQ and its analogues potently disrupted the interaction of ACE2 and Spike (RBD) protein (with CLQ being the most potent). CLQ and its derivatives bound to ACE2 and surprisingly inhibited exopeptidase activity. Unlike the CLQ pharmacopore, other studies revealed that MLN-4760, which is also known as (S,S)-2-{1-Carboxy-2-[3-(3,5-dichloro-benzyl)-3H-imidazol-4-yl]-ethylamino}-4-methyl-pentanoi c acid, is a potent inhibitor of ACE2 exopeptidase activity. But, MLN-4760 did not disrupt ACE2-Spike interaction in several coronaviruses, including SARS-CoV, SARS-CoV-2, and NL63S.^{53, 67}Studies have shown that the MLN-4760 binding site on ACE2 is different than the site where RBD interacts with ACE2.^{49, 67, 94}. Moreover, mutations in the catalytic site required for exopeptidase activity of ACE2 had no effect on Spike RBD binding to ACE2.⁹⁴

However, as shown in the work described herein, CLQ can affect ACE2 by reversibly chelating its zinc ion (which is involved in its activity) as well as interfere with the ACE2-RBD interaction. Although CLQ was the most potent amongst the 3 analogs, except for IC_{50_2}, preliminary structure activity relationship studies (SAR) revealed that the other two derivatives are comparable to CLQ as both show potent inhibition of rhACE2-RBD interaction as well as inhibition of antiviral and anti-rhACE2 activity. Alternative analogues can avoid the same adverse effects experienced with CLQ in the past.

The impact of the COVID-19 pandemic on human health, healthcare systems, and the global economy has imposed an urgent call and a pressing need for the development of novel antivirals.⁷⁴Using a multi-prong approach, CLQ and two of its analogues (CLBQ14 and CLCQ) were examined and characterized as potent inhibitors of SARS-CoV-2 infection induced CPE in vitro, rhACE2 metalloprotease activity, and the binding of rhACE2 with SARS-CoV-2 spike (RBD) protein necessary for viral entry. These data provide strong cellular and biochemical evidence that CLQ, CLBQ14, and CLCQ can serve as new anti-COVID19 treatments.

B. Examples Comprising AMB and BHH
Materials and Methods

African Green Monkey Kidney Vero E6 cells (ATCC #CRL-1586, American Tissue Culture Type) were maintained using medium purchased from Gibco (modified eagle's medium (MEM) Gibco (#11095), 10% fetal bovine serum (HI FBS) Gibco (#14000), and Penicillin/Streptomycin (PS) Gibco (#15140); and 10 U/mL penicillin and 10 μg/mL streptomycin (only in assay media)). For the SARS-CoV-2 infection induced cytopathic effect (CPE) assay, cells were grown in MEM/10% HI FBS and harvested in MEM/1% PS/supplemented with 2% HI FBS. Cells were batch inoculated with SARS-CoV-2 USA_WA1/2020 (M.O.I.˜0.002), which resulted in 5%-10% cell viability 72 hours post-infection.

Compounds and Preparation of Stock Solutions

10 mM stocks solutions of the inhibitors in dimethyl sulfoxide (DMSO; D8418-Lot #SHBL5613) were purchased from Sigma Aldrich. Ambroxol Hydrochloride (AMB) (A9797—Lot #BCCB1637) and Bromhexine Hydrochloride (BHH) (17343—Lot #BCBJ8156V) were also purchased from Sigma Aldrich. Both compound samples were serially diluted 2-fold in DMSO nine times and screened in duplicates for the SARS-CoV-2 infection induced cytopathic effect (CPE) assay. The reference compounds used for the CPE and cytotoxicity assays were made available by SRI. Assay Ready Plates (ARPs; Corning 3764BC) pre-drugged with test compounds (90 nL sample in 100% DMSO per well dispensed using a Labcyte (ECHO 550)) were prepared in the Biosafety Level-2 (BSL-2) laboratory by adding 5 μL assay media to each well.

Specifically, Vero E6 cells selected for expression of the SARS-CoV-2 receptor (ACE2 or angiotensin-converting enzyme 2) were used for the CPE assay. Cells were grown in MEM/10% HI FBS supplemented and harvested in MEM/1% PS/supplemented with 2% HI FBS. Cells were batch inoculated with SARS-CoV-2 (M.O.I.˜ 0.002), which resulted in 5% cell viability 72 hours post-infection. Compound samples were serially diluted 2-fold in DMSO nine times and screened in duplicates. Assay Ready Plates (ARPs; Corning 3764 BC black-walled, clear bottom plates) pre-drugged with test compounds (90 nL sample in 100% DMSO per well dispensed using a Labcyte (ECHO 550)) were prepared in the BSL-2 lab by adding 5 μL assay media to each well. The plates were passed into the BSL-3 facility where a 25 μL aliquot of virus inoculated cells (4000 Vero E6 cells/well) was added to each well in Columns 3-22. The wells in Columns 23-24 contained virus infected cells only (no compound treatment). Prior to virus infection, a 25 μL aliquot of cells was added to Columns 1-2 of each plate for the cell only (no virus) controls. After incubating plates at 37° C./5% CO₂and 90% humidity for 72 hours, 30 μL of Cell Titer-Glo (Promega) was added to each well. Luminescence was read using a Perkin Elmer Envision or BMG CLARIOstar plate reader following incubation at room temperature for 10 minutes to measure cell viability. Raw data from each test well were normalized to the average (Avg.) signal of non-infected cells (Avg. Cells; 100% inhibition) and virus infected cells only (Avg. Virus; 0% inhibition) to calculate % inhibition of CPE using the following formula: % inhibition=100*(Test Cmpd−Avg. Virus)/(Avg. Cells−Avg. Virus). The SARS CPE assay was conducted in BSL-3 containment with plates being sealed with a clear cover and surface decontaminated prior to luminescence reading.

The cytotoxicity of AMB and BHH was assessed in a BSL-2 counter screen using the Cell Titer-Glo Luminescent Cell Viability Assay as previously described.⁸⁵Briefly, host cells in media were added in 25 μL aliquots (4000 cells/well) to each well of assay ready plates prepared with test compounds as above. Cells only (100% viability) and cells treated with hyamine at 100 μM final concentration (0% viability) serve as the high and low signal controls, respectively, for cytotoxic effect in the assay. DMSO was maintained at a constant concentration for all wells (0.3%) as dictated by the dilution factor of stock test compound concentrations. After incubating plates at 37° C./5% CO₂and 90% humidity for 72 hours, 30 μL CellTiter Glo (CTG) (G7573, Promega) was added to each well. To measure cell viability, luminescence was read using a BMG CLARIOstar plate reader following incubation at room temperature for 10 minutes.

The SARS-CoV-2 Spike—ACE2 binding assay kits (Cat #CoV-SACE2-1, Lot #062320 7066 and Lot #081120 7066) were purchased from RayBiotech (Norcross, GA). The manufacturer's protocol⁸⁰for the kits was adapted to determine the effect of AMB and BHH on the interaction between SARS-CoV-2 Spike (RBD) protein and recombinant human ACE2. The in vitro enzyme-linked immunoabsorbent assay (ELISA) was performed in a transparent flat-bottom 96-well plate. A 10 mM stock solutions of the compounds in Dimethyl sulfoxide (DMSO) with serially dilutions of the compounds in DMSO as follows: 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, and 0.1 μM for AMB and BHH. All experiments were performed in triplicate. Each plate contained positive controls (1% DMSO) and blank controls with no ACE2. Specifically, 1 μL of serially diluted compounds was incubated with recombinant SARS-CoV-2 Spike receptor binding domain (RBD) protein, pre-coated on the 96 well plates in 49 μL of 1× assay diluent buffer for 30 mins at room temperature (22° C.) with shaking at 180 rpm. Then, 50 μL of ACE2 protein in 1× assay diluent buffer was added into the 96 well plate and incubated for 2.5 hours at room temperature (22° C.) with shaking at 180 rpm. Thereafter, the solution was discarded and the plate was washed consecutively four times with 300 μL 1× wash buffer, followed by the addition of the detection antibody (anti-ACE2 goat antibody). The reaction was allowed to go on proceed for 1 hour at room temperature (22° C.) with shaking at 180 rpm. Then, the solution was discarded and the wash step was repeated as described above. Next, the HRP-conjugated anti-goat IgG was added to each well, and the reaction plate was further incubated for 1 hour at room temperature (22° C.) with shaking at 180 rpm. Again, the solution was discarded and the wash step was repeated as described above. Then, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) one-step substrate was added to each well, and reaction mixtures were incubated in the dark at room temperature (22° C.) with shaking at 180 rpm for 30 mins. The reaction was stopped by the addition of 50 μL stop solution. The absorbance was read at 405 nm using a Beckman Coulter DTX880 multimode plate reader. The background hydrolysis was subtracted and the data was fitted to a special bell-shaped dose-response curve equation using GraphPad prism software 8.4.3.

Results and Discussion
Cytotoxicity Effects of Amb and Bhh in Vero E6 Cells

Using a Cell Titer-Glo Luminescent Cell Viability Assay⁸⁵, the cytotoxicity of AMB and BHH was examined. The cytotoxic effects of the reference compounds in Vero E6 cells were also determined. The 50% cytotoxic concentration (CC₅₀) of AMB and BHH were greater than 30 μM.

When compared to the reference compounds tested, AMB and BHH displayed slightly higher percent maximum and minimum viability at the concentrations tested. Between the two compounds, a higher percent minimum viability for AMB (113.95%) was observed compared to BHH (103.87%) at 30 μM. These cytotoxicity results are consistent with the known clinical safety profiles of both compounds with AMB showing better pharmacokinetic and safety profiles compared to BHH.¹⁰²

Efficacy of AMB and BHH Against SARS-CoV-2 Infection Induced Cytopathic Effect (CPE) in Vero E6 Cells.

To identify inhibitors of SARS-CoV-2 infection for potential treatment of COVID-19, the in vitro antiviral activity of AMB and BHH was examined using a standard luminescent-based high-throughput screening (HTS) platform^{54, 85}for SARS-CoV-2 infection induced CPE in African Green Monkey Kidney Vero E6 cells. BHH inhibited SARS-CoV-2 infection induced CPE in vitro with 50% Inhibitory Concentration (IC₅₀) value at about 21.72 μM. AMB's IC₅₀was greater than 30 μM, which was the highest concentration tested FIGS. 5A-5B. At this maximum concentration, AMB displayed 14.25% inhibition of SARS-CoV-2 induced CPE and BHH exhibited the highest maximum inhibition (about 91.08% inhibition) at 30 μM.

The antiviral effects of AMB and BHH were compared to that of five other known inhibitors of SARS-CoV-2 in vitro: (i) Calpain Inhibitor IV, (ii) Chloroquine, (iii) Remdesivir, (iv) Hydroxychloroquine, and (v) Aloxistatin. The IC₅₀for most of the reference compounds (Calpain Inhibitor IV (0.29 μM), Chloroquine (3.56 μM), Hydroxychloroquine (5.16 μM), Remdesivir (8.54 μM)) were lower than the IC₅₀values for BHH and AMB. However, the IC₅₀of Aloxistatin (21.78 μM) was similar to that of BHH (21.72 μM). The IC₅₀values observed for the reference compounds are consistent with earlier reports.^{44-4713, 51, 74, 99}, While other cellular studies tested BHH and AMB at certain or single concentrations,^{9, 37, 93}these are the first data to demonstrate the IC₅₀determination for BHH and AMB against the novel SARS-CoV-2 infection induced CPE.

Effects of AMB and BHH on Spike (RBD) Protein Interaction

The effects of AMB and BHH on the binding affinity of rhACE2 and RBD of S glycoprotein at concentrations ranging from 100 μM to 100 nM were tested using an adapted in vitro enzyme-linked immunoabsorbent assay (ELISA).⁸⁰A unique dose-response curve for both compounds (using the special bell shape curve model) was identified. AMB displayed the highest inhibition of the Spike (RBD)-rhACE2 protein interaction at lower micromolar concentrations (ranging from 100 nM to 10 μM) than compared to higher concentrations of AMB from 50 μM (FIG. 5B). BHH inhibited the binding of the Spike (RBD) glycoprotein to the rhACE2 receptor at lower concentrations ranging from 100 nM to 10 μM, but enhanced the interaction at higher concentrations (i.e., from 50 μM) (FIG. 5A). Hence, the bell-shaped model generated two IC₅₀values (IC_{50_1}and IC_{50_2}) (FIG. 9). At the concentrations tested, AMB did not produce a stimulation or enhancement of binding of SARS-CoV-2's Spike (RBD) protein to rhACE2 receptor. Using the bell curve model, however, the GraphPad software generated a second IC₅₀at 232 μM for AMB, which was greater than the highest tested concentration (100 PM). The unconventional dose response curve observed in this protein interaction assay can be intrinsic to the mode of inhibition. Alternatively, it can be an indicator of one or more additional binding sites and/or targets (e.g., other sites on rhACE2 or the Spike (RBD) glycoprotein). This is the first report to show that AMB inhibited and interfered with the binding between rhACE2 receptor and SARS-CoV-2 S (RBD) glycoprotein in vitro and that BHH inhibited this interaction at lower concentrations and enhanced this interaction at higher concentrations.

C. Additional Examples Comprising AMB and BHH
Materials and Methods

BHH, AMB and Dimethyl sulfoxide (DMSO) (D8418-1Lot #SHBM4129) were purchased from Sigma Aldrich (St. Louis, MO). BHH and AMB were dissolved in 100% DMSO at a concentration of 50 mM and were further serially diluted in 100% DMSO.

Spike (RBD)—ACE2 binding assay kits (Cat #CoV-SACE2-1, Lot #062320 7066) were purchased from RayBiotech (RayBiotech, 2022). The enzyme-linked immunoabsorbent assay (ELISA) was performed in a transparent flat-bottom 96-well plate, following manufacturer instructions. Stock solutions of 10 mM of the compounds in DMSO were prepared and serially diluted as follows: 100, 50, 10, 5, 1, 0.5, and 0.1 μM for BHH and AMB. All experiments were done in triplicates and repeated thrice. Each plate contained a positive control (1% DMSO) and a blank control with no ACE2. 96 well plate pre-coated with recombinant SARS-CoV-2 Spike receptor-binding domain (RBD) protein were incubated 1 μL of serially diluted compounds in 49 L of 1× assay diluent buffer for a duration of 30 mins, at room temperature (22° C.) with shaking at 180 rpm. Thereafter, 50 μL of ACE2 protein in 1× assay diluent buffer was added into the 96 well plates, and the reaction was allowed to go on for 2.5 hr at room temperature (22° C.) with shaking at 180 rpm. The reaction mixture was discarded, and the plate was washed consecutively four times with 300 μL 1× wash buffer, followed by the addition of the detection antibody (anti-ACE2 goat antibody) further incubated for 1 hr at room temperature (22° C.) with shaking at 180 rpm. Thereafter, the solution was discarded, and the plate was washed as described above.

The HRP-conjugated anti-goat IgG was added to each well, and the reaction plates were further incubated for 1 hr at room temperature (22° C.) with shaking at 180 rpm. The solution again was discarded, and the final wash step was repeated as described above. in the final step, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) one-step substrate was added to the reaction mixtures and incubated in the dark at room temperature (22° C.) with shaking at 180 rpm for an additional 30 mins and then stopped by the addition of 50 μL stop solution. The absorbance was read immediately at 405 nm using a Beckman Coulter DTX880 multimode plate reader. The data were fitted after the background hydrolysis was adjusted using a special bell-shaped dose-response curve equation in GraphPad prism software 8.4.3.

ACE1 Activity Assay. ACE1 Inhibitor Screening Kit (Cat #MAK422) was purchased from Sigma-Aldrich (St. Louis, MO) and adapted to determine the effect of BHH and AMB on the enzymatic activity of ACE (Aldrich, 2022). The in vitro colorimetric assay was performed in a transparent flat-bottom 96-well plate. 50 mm and 10 mm solutions of Captropil, a known ACE1 inhibitor were used as a positive inhibitor control. Stock solutions of the compounds were prepared in 100% Dimethyl sulfoxide (DMSO) and further serially diluted in DMSO as follows: 500, 100, 50,10,5,1 and 0.5 μm for AMB and the positive control Captropil. All experiments were performed in quadruplicates. Each plate contained positive controls (1% DMSO) and blank controls with no ACE1 enzyme. Specifically, 2.5 μl of serially diluted compounds were pre-incubated with 2 μl of ACE1 protein diluted in 38 μl of 1× assay diluent buffer for about 15 mins, at room temperature (22° C.). 157.5 μl of 1×ACE1 assay buffer (197.5 μl for −ve control) was added into the 96 well plates. The solution was mixed well and incubated at 37° C. for 15-20 minutes, protected from light. After that, 40 μl of the substrate was added to each well and mixed well. The absorbance was measured immediately at 345 nm (A345) in kinetic mode for 60 minutes.

ACE2 Inhibitor Screening Assay. An ACE2 exopeptidase Inhibitor screening assay kit with the fluorogenic substrate (Catalogue #79923) was purchased from BPS Bioscience (San Diego, CA) and modified to measure the exopeptidase activity of ACE2 in the presence and absence of inhibitors (BPSBioscience, 2022). Following the manufacturer's directions, the fluorescence assay was carried out in a 96-well black flat-bottom plate with a final reaction volume of 50 μL. 50 mM stock solutions of BHH and AMB in 100% DMSO were prepared. Compounds were serially diluted in DMSO as follows: 500, 250, 100, 50, 10, 1, 0.1 μM. All experiments were done in triplicates and repeated twice. A positive control of enzyme treated with vehicle alone (2 percent DMSO) and a blank control with no enzyme were included on each plate.

Each reaction had 24 μL of purified recombinant human ACE2 protein (0.42 ng/L) in ACE2 buffer, 1 μL of the compound at different concentrations, and 25 μL ACE2 fluorogenic substrate. The final volume of the reaction was 50 μL. The reaction mixtures were incubated for 1 hr at room temperature (22° C.) and protected from light. After that, the fluorescence intensities (λExcitation=535 nm, λEmission=595 nm) were measured using a Spectramax iD3 multimode plate reader. The background hydrolysis was subtracted, and the data were fitted to a four-parameter logistic (variable slope) equation using GraphPad prism software 9.1.

Western blotting. A549 “Lung” cells (RRID:CVCL_COQ5) were harvested in a radioimmuno precipitation assay (RIPA) buffer (Tris 10 mM pH 7.4, containing 140 mM NaCl, 1 mM EDTA, 1 mM NaF, 0.10% SDS, 0.50% sodium deoxycholate 0.1% NP-40, 1% Triton X-100) in the presence of protease inhibitors (Thermo Fisher) at 24 hrs. and 48 hrs. Protein concentration was determined by was determined using bicinchoninic acid with bovine serum albumin as a standard (Thermo Fisher). Equal amounts of protein were loaded and separated by SDS-PAGE using 10% pre-casted gels (Biorad). Proteins were electroblotted onto nitrocellulose membranes (Bio-Rad). The blots were blocked using 5% (w/v) milk in Tris-buffered saline containing 0.5% (v/v) Tween-20. The membranes were then incubated with different antibodies. Antibodies directed against ACE1 (ab28311) and ACE2 (ab108209) were used and the GAPDH antibody was used as loading control for cell lysates. Primary and secondary antibodies were diluted in Tris-buffered saline containing (0.5% v/v) Tween-20 (TBS-T) supplemented with 5% milk. The bands were visualized by incubating the blots with enhanced chemiluminescence reagent (ECL, Biorad) and visualized using chemidoc. Band intensities were quantified by using imagelab software Biorad.

Pseud typed Lentivirus Assay. Cell growth conditions and Medium, ACE2 Human Kidney 293 recombinant Cells (ACE2 HEK-293-#79951), Thaw media (#60187-1), Growth media (#79801), Spike (SARS-CoV-2) Pseudotyped Lentivirus (Luciferase Reporter #79942), Spike S1 Neutralizing Antibody (#101024), and ONE-Step™ Luciferase Assay System (#60690-1) were purchased from BPS Biosciences (San Diego, CA). The pseudotyped lentivirus contained SARS-CoV-2 Spike protein (Genbank Accession #QHD43416.1) and the firefly luciferase gene under the control of the CMV promoter. Expression of luciferase can be detected after the addition of the substrate. When pseudovirions are incubated with a molecular inhibitor before infection, they will block the spike-mediated cell entry. The number of blocked viruses can be measured via luciferase reporter activity.

HEK293 cells were resuspended in thaw media (no puromycin) and cultured in a T25 flask at 37° C. in a CO2 incubator. After the first passage, cells were further cultured in growth media (contains puromycin). On the experiment day, ACE2-HEK293 were seeded at the density of 7500 cells per well into a white opaque 96-well cell culture microplate in 90 μl of growth medium. Cells were inoculated with 750 infectious particles (MOI=0.1) of SARS-CoV-2. Before infection, pseudotyped lentivirus (5 μl) was preincubated with AMB (5 μl) at concentrations ranging from 100, 50, 25, and 10 μM and with controls for 30 minutes at room temperature (RT); the reactions were then added to the cells. After 48 hours, 100 l Luciferase reagent (BPS Bioscience, catalog no. 60690) was added to measure luciferase activity. Luminescence was measured with Spectra max ID3 (molecular devices). The measured luminescence signal was directly proportional to the amount of Pseudo typed lentivirus successfully transduced into the cells. Data are presented as percent inhibition of entry of pseudo-type lentivirus into the HEK293-ACE2 cells (Bioscience, 2022). Infected cells and infected treatment cells (Spike neutralizing antibodies) were positive and negative controls. All experiments were done in duplicates and were repeated twice.

Antiviral assays using SARS-CoV-2 In vitro. Screening Strategy: a cell-based assay was employed for measuring the cytopathic effect (CPE) of the virus infecting Vero E6 host cells. The CPE reduction assay is a popular and widely used assay format to screen for antiviral drugs because of its ease of use in the high throughput screening (HTS). In this assay, host cells are infected within the assay, and the virus-infected host cells die as a result of the virus's hijacking of cellular mechanisms for replication processes. By monitoring the viability of host cells three days after virus inoculation, the CPE reduction assay indirectly measures the effect of antiviral drugs acting through various molecular mechanisms. Anti-viral compounds were identified as those that protect the host cells from the cytopathic effect of the virus, thereby increasing viability.

Infection-Induced Cytopathic Effect (CPE). Vero E6 cells selected for expression of the SARS CoV receptor (ACE2; angiotensin-converting enzyme 2) were used for the CPE assay (W et al., 2003). Cells were grown in MEM/10% HI FBS supplemented and harvested in MEM/1% PSG/supplemented 2% HI FBS. Cells were batch inoculated with either SARS CoV-2 (M.O.I.˜0.002), resulting in 5% cell viability 72 hours post-infection. Compound samples were serially diluted 2-fold in DMSO nine times. Assay Ready Plates (ARPs; Corning 3712BC) pre-drugged with test compounds (90 nL sample in 100% DMSO per well dispensed using a Labcyte ECHO 550) were prepared in the BSL-2 lab by adding 5 μL assay media to each well. The plates were passed into the BSL-3 facility, where a 25 μL aliquot of virus inoculated cells (4000 Vero E6 cells/well) is added to each well in columns 3-22. The wells in columns 23-24 contain virus-infected cells only (no compound treatment). Before virus infection, a 25 μL aliquot of cells is added to columns 1-2 of each plate for the cell only (no virus) controls. Plates were incubated at 37° C./5% C02 and 90% humidity for 72 hours, and then 30 μL of Cell Titer-Glo (Promega) is added to each well.

Luminescence reading employed a Perkin Elmer Envision or BMG CLARIOstar plate reader following incubation at RT for 10 minutes to measure cell viability. Raw data from each test well is normalized to the average signal of non-infected cells (Avg Cells; 100% inhibition) and virus-infected cells only (Avg Virus; 0% inhibition) to calculate % inhibition of CPE using the following formula: % inhibition=100*(Test Cmpd−Avg Virus)/(Avg Cells−Avg Virus). The SARS CPE assay is conducted in BSL-3 containment with plates being sealed with a clear cover and surface decontaminated prior to luminescence reading.

Cytotoxicity of BHH and AMB in CPE Assay. Compound cytotoxicity was assessed in a BSL-2 counter screen as follows: In each well of assay-ready plates prepared with test compounds as described above, host cells in media were added in 25 μl aliquots (4000 cells/well). Cells alone (100% viability) and cells treated with hyamine at a final concentration of 100M (0% viability) serve as the high and low signal controls for cytotoxic effect in the assay, respectively. The DMSO concentration is kept constant in all wells (0.3%), as determined by the dilution factor of stock test compound concentrations. After 72 hours of incubation at 37° C./5% CO2 and 90% humidity, 30 l Cell Titer-Glo (Promega) is added to each well. Following a 10-minute incubation at room temperature, luminescence was measured using a BMG PHERAstar plate reader to measure cell viability.

Nano luciferase Reporter Virus Assay. A549 cells expressing ACE2 (obtained from Ralph Barric at UNC) were grown in DMEM high glucose supplemented with 20% HI FBS, 1% NEAA, 100 μg/ml Blasticidin and split 1:6 every three days (Blasticidin was removed from the media one passage before using the cells in the assay). On the day of assay, the cells were harvested in DMEM supplemented with 2% HI FBS, 1% HEPES, 1% Pen/Strep. Assay-ready plates pre-drugged with test compounds were prepared in the BSL-2 lab by adding 5 μL assay media to each well. The plates and cells were then passed into the BSL-3 facility. A working stock of SARS CoV-2 nanoluciferase reporter virus (NLRV) passaged five times in A549 cells expressing ACE2 is diluted 6000-fold in media containing 160,000 cells per mL (MOI˜0.002) and stirred at 200 RMP for approximately 10 minutes. A 25 μL aliquot of virus inoculated cells (4000 cells) is added to each well in columns 3-24 of the assay plates. The wells in columns 23-24 do not contain test compounds, only virus-infected cells for the 0% inhibition controls. Prior to virus inoculation, a 25 L aliquot of cells is added to columns 1-2 (no test compounds) of each plate for the cell, only 100% inhibition controls. After incubating plates at 37° C./5% CO2 and 90% humidity for 72 hours, 30 μL of NanoGlo (Promega) was added to each well. Luminescence was read using a BMG CLARIOstar plate reader (bottom read) following incubation at room temperature for 10 minutes to measure luciferase activity as an index of virus titer. Plates were sealed with a clear cover and the surface is decontaminated prior to luminescence reading.

Cytotoxic effect in NLRV assay. Compound cytotoxicity was assessed in a BSL-2 counter screen as follows: In each well of assay-ready plates prepared with test compounds as described above, host cells in media were added in 25 μl aliquots (4000 cells/well). Cells alone (100% viability) and cells treated with hyamine at a final concentration of 100M (0% viability) serve as the high and low signal controls for cytotoxic effect in the assay, respectively. The DMSO concentration is kept constant in all wells (0.3%), as determined by the dilution factor of stock test compound concentrations. After 72 hours of incubation at 37° C./5% C02 and 90% humidity, 30 L Cell Titer-Glo (Promega) was added to each well. Following a 10-minute incubation at room temperature, Luminescence was measured using a BMG PHERAstar plate reader to measure cell viability.

Data analysis. The raw data from plate readers were imported into the activity base for all assays, where raw values were associated with compound IDs and test concentrations. For the antiviral NLRV assay, raw signal values were converted to % inhibition by the following formula: % inhibition=100×(test compound value−mean value infected cell controls)/(mean value uninfected cell controls−mean value infected cell controls). For the cell viability assay measuring compound cytotoxicity, % cell viability was calculated as follows: % viability=100*(test compound value−mean low signal control)/(mean high signal control−mean low signal control).

EC₅₀and CC₅₀values were calculated from a four-parameter logistic fit of data using the Xlfit module of Activity base with top and bottom constrained to 100 and 0%, respectively. Concentration-response graphs of compound combinations were produced in the Graphpad Prism software package. For synergy analysis, reduced data with associated concentrations and compound ids were imported, which applies models for the detection of synergistic interactions. The zero-interaction potency (ZIP) model was used.

Results and Discussion

BHH and AMB Disrupt rhACE2 and Spike (RBD) Protein Interaction

The interaction between the human ACE2 and receptor-binding domain of the SARS-CoV-2 spike protein is a crucial first step in the entry process required for viral infectivity. Using an adapted in vitro enzyme-linked immunosorbent assay (ELISA) (RayBiotech, 2022), the effect of AMB and BHH on the binding affinity of rhACE2 and RBD of S protein at concentrations ranging from 100 μM to 100 nM was evaluated. Prism graph pad version 8.4.3 was used to generate dose-response curves. A unique bell shape curve was observed for both compounds when fitted using a special bell-shaped curve model. AMB was more potent at disrupting the interaction between ACE2 and RBD than BHH.

The percent inhibition was greater at low concentrations of AMB than at high values. A bell-shaped curve with activation and inhibition at opposite ends was observed with BHH. BHH increased the binding of ACE2 and RBD at high concentrations of 100 and 50 μM but blocked the interaction at low concentrations. For both drugs, the bell-shaped model predicted two IC₅₀values. The dose-response curves for BHH and AMB are shown in FIGS. 10A and 10B. Table 1 displays the IC₅₀values for each drug obtained in this assay.

TABLE 1

AMB and BHH inhibits the interaction between rhACE2 and

SARS-COV-2 Spike (RBD) protein

Estimated Relative IC₅₀against rhACE2 and RBD Interaction Assay

Inhibitor ID
IC₅₀_1 (μM)
IC₅₀_2 (μM)

AMB
0.82
231.6

BHH
1.19
42.9

Without being bound to theory, these findings suggest that AMB's mechanism of action against SARS-CoV-2 comes from a direct antiviral effect rather than its predicted effects on TMPRSS2. Even though BHH and AMB are classed as TMPRSS2 inhibitors, current research has revealed that these two drugs do not directly block the TMPRSS2 activity. One plausible mechanism for antiviral action of AMB in SARS-CoV-2 could be inhibition of the interaction between ACE2 and the Spike protein.

BHH Inhibits the Exopeptidase Activity of Ace at High Concentration while AMB Showed No Inhibition

Over-activation of the renin-angiotensin has shown to lead to several disorders. ACE2 acts as the negative regulator of the pathway which converts Ang II to angiotensin 1-7. ACE1 on the other hand is responsible for the conversion Ang II to Ang 1. Thus, the ACE1/ACE2 balance is important to the natural function of the Renin-angiotensin pathway.

In order to determine if BHH and AMB interacts with ACE, the role of the two compounds on the exopeptidase activity of ACE were examined. Similar to the findings of the ACE2 assay, AMB did not inhibit the activity of ACE. Even in the presence of very high concentrations of AMB such as 500 μM, ACE was 100% active. BHH on the other hand showed activity against ACE1 at concentrations.

AMB has No Effect on Exopeptidase Activity of Ace while BHH Disrupts the Exopeptidase Activity of ACE2 at High Concentrations

To further understand the RBD-ACE2 interaction assay results, the role of both compounds on the exopeptidase activity of ACE2 was investigated. ACE2 exopeptidase activity is vital for normal physiology, and long-term inhibition of ACE2 could be detrimental to cardiovascular health. Therefore, to rule out the two compounds inhibiting ACE2 the effect of AMB and BHH on the exopeptidase activity of rhACE2 was determined using an adapted fluorometric assay (BPS Bioscience, 2022). The assay found that AMB did not inhibit the exopeptidase activity of rhACE2 even at a very high concentration. On the other hand, BHH completely inhibited the exopeptidase function of ACE2 at high concentrations from 50 μM to 500 μM with an IC₅₀value of 25.98 (FIG. 11). At a low concentration from 0.1 nm to 10 PM, BHH had no activity against ACE2. These results reveal that AMB does not inhibit the exopeptidase activity rhACE2, while BHH inhibits exopeptidase activity at high concentrations. Results for ACE are shown in FIG. 11A and ACE2 are shown in FIG. 11B (BHH (OJT009), AMB (OJTO10)).

The behavior of BHH in the two biochemical assays; ACE2 exopeptidase activity and rhACE2 and RBD interaction shed light on their mechanism of action. At a high concentration (above 50 μM), BHH inhibits the ACE2 activity but enhances the binding of ACE2 with RBD. While at a low concentration, it does not affect ACE2 activity but disrupts the interaction of ACE2 and RBD. According to these findings interactions of these compounds with ACE2 modulate the extent of binding between ACE2 and RBD.

BHH and AMB do not Impact ACE2 and Ace Expression Levels in A549 Cells

SARS-CoV-2 binds to target cells through ACE2 and this interaction with ACE2 receptor leads to its downregulation and this in turn decreases the degradation of angiotensin II (Ang-II) into angiotensin1-7 (Ang-(1-7). ACE2 converts Ang-II to Ang-(1-7) and prevents the effects of the ACE1/angiotensin II axis. Ang-II induces strong vasoconstriction, inflammatory effects, and profibrotic effects, while Ang-(1-7) exhibits antiproliferative, antiapoptotic, and mild vasodilation and protects against various cardiovascular diseases. Knowledge of how both of our compounds affect the expression of these proteins is key to avoid disrupting the balance in the RAAS pathway. BHH and AMB reduced SARS-CoV-2 and Delta Variant Pseudo typed Lentivirus Infection in ACE2 HEK-293 cells

Ability of BHH and AMB to block infection of cells with spike Pseudotyped Lentivirus particles was assessed. A preliminary screen was done with four concentrations ranging from 10 uM to 100 uM. BHH and AMB were found to effectively block the entry of pseudo spike protein into the cells. A dose-dependent neutralization was observed of viral particles with increasing concentrations for AMB, at 50 uM AMB showed a close to 50% reduction of the viral entry (Table 3). On the other hand, BHH showed a 50% reduction of viral entry at concentrations as low as 10 μM while viral entry at 100 μM was reduced up to 100%. This assay further validates the results from biochemical assays that the interaction of BHH and AMB with ACE2 and RBD is part of its mechanism of action for SARS-CoV-2 neutralization.

The efficacy of both compounds to neutralize the B.1.617.2 Delta Variant of SARS-CoV-2 was evaluated. A similar assay was performed with spike pseudo-type lentiviral particle-containing B.1.617.2 Delta Variant gene. Interestingly at the concentrations tested, AMB retained similar potency as of wild type SARS-CoV-2. AMB blocked the entry of viral particles by 50% at 50 uM and 100% at 100 uM. BHH blocked viral entry completely at 100 μM but showed a reduction in potency at 50 μM and 25 μM to around 10% at 10 μM. AMB was slightly more potent against the delta variant of SARS-CoV-2 than the wild-type virus (Table 2). Given that Pseudo typed assay only measures the effect of the compound on preventing the entry of spike protein into the cells, it further substantiates that BHH and AMB interacts with the entry receptors.

TABLE 2

BHH and AMB reduced SARS-COV-2 and Delta Variant

Pseudo typed Lentivirus Infection in ACE2 HEK-293 cells

Percent (%) Inhibition of Pseudo virus Entry

in HEK293 Kidney Cells

B.1.617.2

Percent Inhibition
SARS-COV-2
Delta Variant

Conc (μM)
BHH
AMB
BHH
AMB

100

89

95

50

46

48

25

12

19

10

2

6

BHH AND AMB REDUCED SARS-COV-2 INFECTION-INDUCED CYTOPATHIC EFFECT (CPE) IN VERO E6 CELLS

To determine whether the results obtained in biochemical, and Pseudo typed assay also apply to SARS-CoV-2 infection in vitro. The antiviral activity of AMB was evaluated using a standard luminescent-based high-throughput screening (HTS) platform for SARS-CoV-2 infection-induced CPE in African Green Monkey Kidney Vero E6 cells. As expected, the treatment with AMB inhibited SARS-CoV-2 infection-induced CPE in vitro with a 50% Inhibitory Concentration (IC₅₀) value at about ˜150 μM, while BHH had an IC₅₀of about 31.64 μM. The IC₅₀obtained in this assay for both compounds is almost two-fold higher than the IC₅₀in the Pseudotyped assay. One reason for the variability in IC₅₀values is the use of different cell lines. In the Pseudo virus assay, HEK293 kidney cells were used, while Vero E6 cells were used in the CPE assay. Moreover, Pseudotyped lentiviral assay only measures the entry of the spike particles into the cell. It doesn't quantify viral replication and other viral factors essential in the SARS-CoV-2 infection pathway. Even though Vero E6 cells are often used for SARS-CoV-2 infection and propagation, they were originally obtained from the African green monkey kidney and hence did not represent an ideal model for SARS-CoV-2 infection because it targets mainly the respiratory cells from the human lung. Moreover, the CPE assay is an indirect measure for viral replication by determining cell viability after the infection.

The antiviral activity of four known inhibitors SARS-CoV-2 were also evaluated as positive controls for the current assay: Calpain Inhibitor IV, Chloroquine, Remdesivir, and Aloxistatin. The IC₅₀values for most of the reference compounds (Calpain Inhibitor IV (3.73 μM), Chloroquine (8.85 μM), and Remdesivir (16 μM)) and Aloxistatin (4.58 μM) were significantly lower than the IC₅₀values of both BHH and AMB. The IC₅₀values for the reference substances obtained in our assay were like previously reported IC₅₀. FIG. 10 shows the dose-response curve for both BHH and AMB and reference compounds against SARS-CoV-2 infection-induced cytopathic effect. Table 3 shows the structure and effective concentration of the reference compounds.

TABLE 3

BHH, AMB and Reference Compounds show activity against

SARS-COV-2 Induced Cytopathic Effect in Vero E6 Cells

IC ₅₀
Maximum
Concentration at Maximum

Inhibitor ID
(μM)
Inhibition (%)
% Inhibition (μM)

AMB
>150
49.87
150

BHH
31.64
72.39
37.5

CalpainInhibitorIV
3.73
115.91
7.17

E64d (Aloxistatin)
4.59
130.81
15

Chloroquine
8.85
114.51
30

Remdesivir
16.07
120.89
30

BHH AND AMB ONLY AFFECTED CELL VIABILITY AT CONCENTRATIONS GREATER THAN 50 MM

The cytotoxicity of OJTOO9 and AMB and reference compounds were measured using a Cell Titer-Glo Luminescent Cell Viability Assay in Vero E6 cells and observed the 50% cytotoxic concentration (CC₅₀) of AMB was 183.71 μM and 58.33 μM for OT009. At the concentration of 300 μM, the percent viability was 0.55%, while maximum viability of 96% was seen at a low concentration of 0.59 μM The CC₅₀values were also obtained for the reference compounds. For reference compounds, the maximum tested concentration was 30 μM Table 4 represents the cytotoxicity and percent viability of cells for AMB and reference compounds.

TABLE 4

BHH and AMB only affected cell viability at concentrations greater than 50 μM

Min.
Conc. at Min.
Max.
Conc. at Max.

Cytotoxicity
Viability
% Viability
Viability
% Viability

Inhibitor ID
CC₅₀(μM)
(%)
(μM)
(%)
(μM)

AMB
183.71
0.55
300
96.64
0.59

BHH

CalpainInhibitor IV
>7.17
99.77
0.028
106.84
0.056

E64d (Aloxistatin)
>30.00
93.02
0.117
106.71
0.234

Chloroquine
>30.00
90.50
3.750
99.66
0.469

Remdesivir
>30.00
91.15
0.117
100.73
3.750

BHH AND AMB REDUCED VIRAL REPLICATION OF SARS-COV-2 NANOLUCIFERASE REPORTER VIRUS ASSAY 549 CELLS

After determining the IC₅₀in CPE, the potency of AMB in inhibiting the viral replication in A549 cells was measured using Nanoluciferase Reporter Virus Assay (NLRV). A549 cells are human epithelial lung cells that are optimized to express AEC2. Unlike CPE, Nanoluciferase assay is a direct measure of viral replication, and efficacies were evaluated by quantifying the viral level in the cells. Moreover, CPE assay is done Vero E6; in contrast, NLRV is done A549 lung cell line, representing a closer model for SARS-CoV-2 infection in vivo.

In the NLRV assay, the EC₅₀of AMB is 47 μM compared to 150 μM in the CPE assay. The ECso obtained by the NLRV assay is very close to what we observed in the Pseudotyped lentiviral assay. In addition to AMB, the antiviral activity of other inhibitors SARS-CoV-2 were evaluated as positive controls for the current assay: Calpain Inhibitor IV, Chloroquine, Remdesivir, Paxlovid, and Molnupiravir. Similar to AMB, reference compounds were also more potent in this assay compared to the CPE assay. The IC₅₀values for most of the reference compounds (Calpain Inhibitor IV (0.0005 μM), Chloroquine (3.3 μM), and Remdesivir (3.3 μM), Paxlovid (0.05 μM), and Molnupirvair (4.2 μM) were significantly lower than the IC₅₀values for AMB. Table 5 shows the IC₅₀value of AMB in comparison to reference compounds.

TABLE 5

BHH and AMB reduced viral replication of SARS-COV-2 Nanoluciferase

Reporter Virus Assay 549 cells

Maximum Inhibition
Concentration at Maximum %

Inhibitor ID
IC₅₀(μM)
(%)
Inhibition (μM)

AMB
47.33
99.73
300

CalpainInhibitorIV
0.0005
99.56
.0028

PF-07321332
0.057
99.67
0.5

Chloroquine
3.3
99.53
15

Remdesivir
0.336
99.58
1.5

Molnupiravir
4.2
91.12
10

BHH AND AMB SHOWED HIGH CYTOTOXICITY CONCENTRATIONS IN A549 LUNG CELLS USING NLRV ASSAY

The CC₅₀for AMB and reference compounds in A549 cells was determined. The cytotoxic concentration obtained in this assay was 137.18 μM a little less than the CPE assay. However, it was still very high compared to the reference compounds. At 300 μM, only 0.14% of cells were viable. Cytotoxicity for AMB and reference compounds are shown in Table 6.

TABLE 6

BHH and AMB only affected cell viability at concentrations greater than 50 μM

in A549 Cells infected with SARS-COV-2

Min.
Conc. at Min.

Conc. at Max.

Cytotoxicity
Viability
% Viability
Max. Viability
% Viability

Inhibitor ID
CC₅₀(μM)
(%)
(μM)
(%)
(μM)

AMB
137.44
0.14
300
100
2.34

CalpainInhibitorIV
>0.179
77.49
.0056
94.37
.090

Chloroquine
18.37
9.11
30
117.48
.023

Remdesivir
>3
84.65
0.094
95.46
.006

Paxlovid
>1
89.51
0.004
101.24
1

Molnupiravir
>10
88.88
0.014
98.56
.002

BHH AND AMB SHOWED HIGH EFFICACY AGAINST B.1.617.2 DELTA VARIANT INFECTION-INDUCED CYTOPATHIC EFFECT (CPE)

To validate the preliminary results of the Pseudotyped delta virus assay, the efficacy of both compounds in inhibiting delta viral replication in CPE assay was assessed. A luminescent-based high-throughput screening (HTS) platform was used to evaluate the antiviral activity of both BHH and AMB against delta variant of SARS-CoV-2 infection-induced CPE in African Green Monkey Kidney Vero E6 cells. AMB was found to inhibit B.1.617.2 Delta Variant infection-induced CPE in vitro with a 50% Inhibitory Concentration (IC₅₀) value at about 98 μM while BHH inhibited the delta variant infection with a IC₅₀value of 17.74 μM. The results were similar to what was observed earlier in the Pseudotyped assay. Both compounds are more potent against B.1.617.2 Delta Variant than wild typed SARS-CoV-2. In addition, to BHH and AMB, the antiviral activities of known SARS-CoV-2 inhibitors were tested as reference compounds: The IC₅₀values for the majority of the reference compounds (Calpain Inhibitor IV (0.124 μM), Chloroquine (8.3 μM), PF-07321332+EI (0.029 μM), Remdesivir+Pfz EI (0.17 μM), PF-07321332 (1.98 μM), E64d (Aloxistatin) (9.84 μM) and Remdesivir (8.24 μM) were lower than the IC₅₀values for both BHH and AMB. Table 7 shows the efficacy of both compounds in comparison to reference compounds.

TABLE 7

BHH and AMB showed high efficacy against B.1.617.2 Delta Variant

Infection-Induced Cytopathic Effect (CPE)

Inhibitor ID
IC ₅₀(μM)
Max. Inhibition (%)
Conc. at Max. % Inhibition (μM)

AMB
98.23
88.38
150

BHH
17.74
74.57
18.75

CalpainInhibitor IV
0.124
105.43
0.45

PF-07321332
1.98
96.28
10

Chloroquine
8.3
993.48
15

Remdesivir
8.24
104.26
15

PF-07321332 + EI
0.029
116.90
.08

Remdesivir + Pfz EI
0.170
88.29
.47

E64d (Aloxistatin)
9.82
85.17
15

The above data supports potent antiviral activity of BHH and AMB against SARS-CoV-2 in human lung epithelial and African green monkey kidney cells. The two compounds additionally inhibit SARS-CoV-2 and Delta variant and improve the viability of Vero E6 kidney cells. The two compounds forth inhibited viral replication in Nano luciferase reporter assay at micromolar concentrations. Additionally, the compounds prevented the entry of Pseudotyped spike particles into Vero E6 cells.

EC₅₀values of AMB were high against SARS-CoV-2. However, clinical concentrations approaching 100 μM are safe for long-term use in Parkinson's disease, Gaucher disease, and pregnant women. EC₅₀values of BHH were lower against SARS-CoV-2 around 30 μm but it has also been proven safe at high doses in clinical studies that investigated the influence of BHH on the metabolism of alveolar type II cells, which lead to an increase in the secretion of phospholipids into the alveolar space.

The ability of AMB and its progenitor drug BHH to modulate the interaction between the RBD of SARS-CoV-2 spike protein and human ACE2 was also investigate. The generated data support AMB and BHH inhibition of the interaction of SARS-CoV-2 spike protein receptor-binding domain (RBD) with human recombinant ACE2 (rhACE2) in nano to the micromolar range, thereby blocking its entry into human cells.

The above studies explored the possibility of AMB and BHH as potential effectors of the exopeptidase activity of ACE2. Using a fluorogenic assay, AMB was found to be without effect on the exopeptidase activity of ACE2, while BHH inhibits the exopeptidase activity of ACE2 at high concentrations. ACE2, a membrane-bound metalloprotease and an essential component in the Renin-Angiotensin system convert Angiotensin II (Ang II) to Angiotensin 1-7, a potent vasopressor. Although ACE2 facilitates viral entry, it protects against acute lung damage, implying that the ACE2/Ang 1-7 pathway must be carefully manipulated to reduce SARS-CoV-2-induced lung injuries. ACE2 counteracts ACE negative effects in the lungs by decreasing Ang II levels. Downregulation of ACE2 by SARS-CoV-2 creates an imbalance of ACE2 and ACE in the lungs. This further aggravates lung inflammation by increasing capillary permeability and leading to pulmonary edema. This further activates the release of inflammatory markers, which eventually lead to cytokine storm. Due to the physiological implications of an imbalanced RAAS pathway, the effects of the two compounds on the expression of ACE1 and ACE2 were investigated and indicated no changes to ACE1 and ACE2 at 50 μm at 24 hrs and 48 hrs after treatment.

The mechanism of action of BHH and AMB in SARS-CoV-2 inhibition was investigated using artificial intelligence to evaluate the binding parameters of AMB with RBD of Spike protein and ACE2. Using molecular docking techniques, we also assessed the effect of the compounds on the molecular interaction between RBD and ACE2. It was determined that the binding of the compounds at the RBD-ACE2 site does not alter the binding affinity and molecular interaction between RBD and ACE2. Whereas, the binding of AMB at the exopeptidase site of ACE2 considerably lowered the binding affinities between the proteins compared to the unbound, ACE2-RBD complex. The result further showed that AMB has a good affinity at the exopeptidase site of ACE2. Interestingly the binding energies of the Delta variant with ACE2 were greater than wild-type SARS-CoV-2 and, hence, more significant disruption of the complex was obtained. These findings validate the in vitro efficacies of AMB obtained in CPE, NLRV, and Pseudo virus assay.

The data provides multiple lines of evidence that AMB mediates inhibition of SARS-CoV-2 entry through its interactions with ACE2. Our findings suggest that AMB binding to rhACE2 may preserve its physiological function and will prevent non-target cardiac toxicities observed in other ACE2-modulating drugs.

Animal Study to Evaluate the Efficacy of AMB or BHH in Adult, Male Syrian Hamsters Challenged with Wild-Type SARS-CoV-2
Materials and Methods

AMB was formulated in solution (35 mg/mL) and administered QD by oral gavage. BHH was formulated in solution (12.5 mg/mL) and administered QD by oral gavage. Aqueous solution containing 2% (w/v) polysorbate 80 was employed as vehicle controls. Challenge Virus USA_WA1/2020 (SARS-CoV-2) originated from CDC and was provided by UTMB Galveston. The virus diluent was phosphate buffered saline (PBS). Animal test system was Syrian Hamster (Mesocricetus auratus), males aged 7-9 weeks and weighing 100-150 g on day 0 for study.

Prior to Day 0, eighteen male Syrian Hamsters were randomized into three groups as outlined in Group Assignment according to weight using Provantis Software. There were 2 cohorts with staggered dosing to accommodate the necropsy schedule. Three animals from each of the three groups were assigned to Cohort 1 and the remaining three animals in each group were assigned to Cohort 2. The animals were housed in the ABSL-3 facility. Treatment began on Day −1 and continued daily through Day 3. Animals in Group 1 were dosed with MDXC19T010AMB at 500 mg/kg/day (or 1.43 mL drug solution per 100 g hamster body weight), animals in Group 2 were dosed with MDXC19T009BHH at 200 mg/kg/day (or 1.6 mL drug solution per 100 g hamster body weight), and animals in Group 3 were treated with the vehicle control at 15 mL/kg (or 1.5 mL vehicle solution per 100 g hamster body weight). All hamsters were IN challenged with SARS CoV-2 on Day 0. Clinical course of disease, changes in body weight, viral load in rectal swabs, pharyngeal swabs and tissues collected at necropsy, as well as cytokine production were examined to assess therapeutic efficacy. Animals were euthanized and tissues were collected on Day 4.

Beginning on Day −1 and continuing daily at approximately 24-hour intervals through Day 3, animals were treated PO (oral gavage). The dose on Day 0 was administered prior to viral challenge. Animals in Group 1 was dosed with AMB at 500 mg/kg/day (or 1.43 mL drug solution per 100 g of hamster body weight), QD for 5 days. Animals in Group 2 were dosed with BHH at 200 mg/kg (or 1.6 mL drug solution per 100 g of hamster body weight) QD for 5 days. Animals in Group 3 were treated with the vehicle control at 15 mL/kg (or 1.5 mL vehicle solution per 100 g of hamster body weight) QD for 5 days. Cohorts were staggered to accommodate the necropsy schedule.

Animals were anesthetized with the solution of 1 mL of Ketamine HCl at 100 mg/mL plus 0.15 mL of Xylazine (100 mg/mL) administered IP as 0.1 mL per 100 g of body weight for the following procedures: Oral Gavage Treatment, IN Challenge, Blood collection, Pharyngeal and rectal swab collection, Preparation for euthanasia.

On Day 0, anesthetized hamsters in were inoculated with a total of 200 μL of SARS-CoV-2 challenge inoculum (IN) by instillation in the nostrils using a micropipette, 100 μL in each nostril. The animal was returned to its home cage and monitored for recovery from anesthesia.

Blood was collected from anesthetized animals into serum separator tubes (SST) at the time points. Blood was processed on the day of collection according to SR SOP and stored frozen at ≤−70° C. for cytokine analysis. Terminal blood collections can be performed through cardiac puncture.

Pharyngeal and rectal swabs were collected at study timepoints, Swab collections on Day 0 were performed prior to viral challenge. The collected material was transferred to a sterile tube containing viral transport media (1.0 mL), flash frozen, and stored at −70° C. until testing.

Necropsy and Macroscopic Observations. At the conclusion of the study, animals were euthanized and a limited gross necropsy was performed including evaluation of the organs and tissues in the thoracic, abdominal, and pelvic cavities with special attention directed to evidence of possible gavage trauma. The necropsy was also performed on the animals that were found dead. Tissue samples were collected according to the protocol and preserved frozen for a later analysis by qRT-PCR (left lung lobes, nasal turbinates and trachea), sgRT-qPCR (left lung lobes) and TCID₅₀(left lung lobes); lung, brain, heart, liver, kidney and spleen were collected and weighed as a whole organ, then approximately 75 mg of each tissue was homogenized with 3 mL of RNA shield. The right lung lobes were collected preserved in 10% formalin for histopathology.

Histology. The fixed lung tissues were trimmed, processed, and cut using a microtome (approximately 5 m sections). The tissue sections were mounted on glass slides, stained with hematoxylin and eosin, and examined microscopically.

Microscopic Observations. All slides were submitted to a veterinary pathologist for evaluation. Records of gross findings for a specimen from postmortem observations were available to the pathologist when examining that specimen. A four-step grading system were used to rank the severity of microscopic lesions for comparison among groups.

An aliquot of the challenge inoculum was back-titrated by TCID₅₀assay to confirm the actual delivered dose. Remaining challenge inoculum was retained and stored at −80±10° C.

Hamster sera collected at time points were tested for the cytokines IL-6, IL-10, and TNF-α using commercially available kits and following the manufacturers' instructions.

Viral loads in the tracheal lavage, nasal swab, and serum samples collected at time points were analyzed using a qualified SARS-CoV-2 RT-qPCR assay for viral genomes using Nucleocapsid (N) Gene RNA (N2 gRNA), and active replication using Envelope subgenomic RNA (E sgRNA). Subgenomic RNAs are only generated during productive infection and the sgRNA RT-qPCR assay might be more accurate for monitoring active SARS-CoV-2 virus replication. Samples recovered from test article and control inoculated animals were homogenized in 1×RNA Shield, extracted, and tested using primers and probe set (E sgRNA) as described by Corman et al. (2020) and W6lfel et al. (2020). QC controls were produced by infecting Vero E6 cells with SARS-CoV-2 isolate USA-WA1/2020 and freezing aliquots to use as controls for sgRNA extraction. Viral sgRNA were isolated from biological fluids using QIAamp 96 Virus QIAcube HT Kit with QIAcube robot, and stored at −65° C. or below. An RNA standard was produced by in vitro transcription and quantified using the Quant-iT Ribogreen RNA assay kit. Serially diluted, quantified RNA was used to generate a standard curve that allowed the quantitation of viral sgRNA in each biological sample. All RT-qPCR reactions were run on the QuantStudios 6.

50% Median Tissue Culture Infectious Dose. Lung samples were evaluated in the TCID₅₀assay to determine viral titers. Briefly, samples were initially diluted 1:10 and added in triplicate to 96-well plates previously seeded with Vero E6 cells. Each sample/replicate were 10-fold serially diluted seven times and allowed to incubate for 72±4 hours. Controls including virus (VC) and cell (CC) controls were included on each test plate. Cytopathic effect was quantified by Cell-Titer Glo (Promega). Plate cutoffs were calculated using the following formula:

[(CCaverage−VCaverage)/2)]+VCaverage

Wells above the cutoff were reported as negative for viral replication and wells below the cutoff were reported as positive for viral replication. TCID₅₀titers were calculated using the Reed and Muench method.

Results
Body Weights

All study groups exhibited similar pattern of body weight loss starting on Day 1 post challenge. The weight loss continued till the end of the study period. The peak weight loss was observed on Day 4 post challenge. The mean peak weight loss observed in treatment groups (Group 1 and 2) and Vehicle Group was −11% and 8%, respectively. No significant difference in weight loss between treatment and vehicle control groups was observed any of the days post challenge.

Back titration of the Cohort 1 and Cohort 2 challenge inocula resulted in 4.64×10⁶TCID₅₀/mL and 2.15×10⁷TCID₅₀/mL, respectively. The target challenge dose was 1×10⁴TCID₅₀per animal. The back-titration data suggest that the animals received 9.2×10⁵TCID₅₀virus and 4.3×10⁶TCID₅₀per dose for Cohort 1 and Cohort 2 indicating that animals received approximately 90-100 times more virus than the targeted dose.

Cytokine Analysis Via ELISA

Sera collected on Days-1, 2 and 4 were tested for the cytokines IL-6, IL-10, and TNF-α using commercially available kits and following the manufacturers' instructions. Significantly elevated (P<0.05) IL-6 levels were observed in the Vehicle control group on Day 4 following challenge when compared to Day-1 levels. Similarly Group 2 also had elevated (P<0.05) IL-6 levels on Day 4 compared to Day-1. The comparison of IL-6 levels between groups showed that the levels of IL-6 were significantly low (P<0.05) in Group 1 and Group 2 animals when compared to Group 3 on Day 4.

No significance difference IL-10 levels were observed between or within Groups 1, 2 and 3 on any of the days tested. TNF-α levels were not altered in any of the groups on Days-1, 2 or 4 following challenge. However, significantly low expressions of TNF-α were observed in Group 1 and Group 2 compared to Group 3 animals on Days −1 (P<0.05), 2 (P<0.05), and 4 (P<0.001).

Pharyngeal Swabs

N2 gRNA: SARS-CoV-2 genomes were detected on Day 0 in all groups. Viral load reached its peak on Day 1 in Group 3 (1.41E+08 copies/swab) treated with the vehicle control and Groups 2 (4.93E+07 copies/swab) dosed with BHH and on Day 2 in Group 1 (9.01E+06 copies/swab) dosed with AMB. Peak viral load was 16-fold lower in Group 1 and 3-fold lower in Group 2 compared to Group 3. Overall Gmean for Days 1-4 shows 4-fold lower viral load in Group 1 (4.93E+07 copies/swab) and 2-fold lower viral load in Group 2 (4.93E+07 copies/swab) as compared to Group 3 (4.93E+07 copies/swab).

E sgRNA: No SARS-CoV-2 active replication was detected on Day 0 in any group. Viral replication load reached its peak on Day 1 in Groups 3 (9.59E+05 copies/swab) treated with the vehicle control and Groups 2 (2.65E+05 copies/swab) dosed with BHH and on Day 2 in Group 1 (2.79E+04 copies/swab) dosed with AMB. Peak viral replication load was 34-fold lower in Group 1 and 4-fold lower in Group 2 compared to Group 3. Overall Gmean for Days 1-4 shows 5-fold lower viral load in Group 1 (1.89E+04 copies/swab), and 2-fold lower viral load in Group 2 (4.82E+04 copies/swab) as compared to Group 3 (9.69E+04 copies/swab).

Nasal Turbinate

N2 gRNA: SARS-CoV-2 genome load was 2-fold lower in Group 1 (3.57E+10 copies/gram) and in Group 2 (3.57E+10 copies/gram) as compared to Group 3 (5.59E+10 copies/gram).

E sgRNA: SARS-CoV-2 active replication load was 2-fold lower in Group 1 (1.09E+08 copies/gram) and similar in Group 2 (1.67E+08 copies/gram) as compared to Group 3 (2.23E+08 copies/gram).

Trachea

N2 gRNA: SARS-CoV-2 genome loads in Group 1 (6.24E+08 copies/gram) and Group 3 (6.57E+08 copies/gram) were similar while Group 2 (1.21E+09 copies/gram) showed 2-fold higher viral load as compared to Group 3.

E sgRNA: SARS-CoV-2 active replication loads in Group 1 (1.11E+06 copies/gram) and Group 3 (1.23E+06 copies/gram) were similar while Group 2 (2.58E+06 copies/gram) showed 2-fold higher replication load as compared to Group 3.

Lungs

N2 gRNA: SARS-CoV-2 genome load was 2-fold higher in Group 1 (4.03E+09 copies/gram) and 3-fold higher in Group 2 (6.20E+09 copies/gram) as compared to Group 3 (2.09E+09 copies/gram).

E sgRNA: SARS-CoV-2 active replication load was 2-fold higher in Group 1 (2.78E+07 copies/gram) and 3-fold higher in Group 2 (4.41E+07 copies/gram) as compared to Group 3 (1.71E+07 copies/gram).

Lung samples collected on Day 4 were evaluated in the TCID₅₀assay to determine viral titers. No difference in lung viral load was observed between treatment (Group 1 and Group 2) and vehicle control group (Group 3).

Rectal Swabs

N2 gRNA: SARS-CoV-2 genomes were detected on Day 0 in all groups. Viral load reached its peak on Day 1 in Group 3 (1.22E+05 copies/swab) treated with the vehicle control and on Day 2 in Group 1 (9.75E+04 copies/swab) and Group 2 (1.54E+05 copies/swab) dosed with AMB and BHH, respectively. Peak viral load was similar in Groups 1, 2 and 3. Overall Gmean for Days 1-4 showed 2-fold higher viral load in Group 2 (1.00E+05 copies/swab) as compared to Group 3 (5.82E+04 copies/swab).

E sgRNA: No SARS-CoV-2 active replication was detected on Day 0 in any group. Only 5 animals were positive on other days. Viral replication load reached its peak on Day 3 in Group 3 (3.38 copies/swab) treated with the vehicle control, on Day 1 in Group 1 (20.15 copies/swab) and on Day 2 in Group 2 (4.29 copies/swab) dosed with AMB and BHH, respectively. Peak viral replication load was 6-fold higher in Group 1 compared to Group 3. Overall Gmean for Days 1-4 showed 3-fold higher viral load in Group 1 (4.21 copies/swab) as compared to Group 3 (1.36 copies/swab).

Discussion

In this study, the efficacy of AMB or BHH were evaluated in adult, male Syrian hamsters challenged with wild-type SARS-CoV-2. Eighteen male Syrian hamsters, six per group, were assigned to the following three groups: Group 1 (500 mg/kg/day AMB), Group 2 (200 mg/kg/day BHH), and Group 3 (vehicle, 15 mL/kg aqueous solution containing 2% (w/v) polysorbate-80). There were two cohorts with staggered dosing. The animals were treated with test articles orally starting on Day −1 and continued daily through Day 3. All hamsters were challenged intranasally with SARS-CoV-2 approximately 1×10⁴TCIDs_oper animal on Day 0. All animals were euthanized on Day 4.

All study groups exhibited similar pattern of body weight loss starting on Day 1 post-challenge. The weight loss was continued till the end of study period. No significant difference in weight loss between treatment and vehicle control groups were observed on any of the day post-challenge.

Pathologically, the incidence of gross lesions was highest in the vehicle control group. The incidence and severity of microscopic lesions in the AMB, BHH and vehicle control groups were similar, and therefore, prophylactic efficacy of AMB and BHH was not discernible based upon the pathology evaluation.

Peak viral load determined by N2 gRNA RT-qPCR in pharyngeal swabs was 16-fold lower in Group 1 and 3-fold lower in Group 2 compared to vehicle control group. Overall Gmean for Days 1-4 showed 4-fold lower viral load in Group 1 and 2-fold lower viral load in Group 2 as compared to Group 3. Similarly replicating viral load determined by E sgRNA RT-qPCR showed that peak viral replication load was 34-fold lower in Group 1 and 4-fold lower in Group 2 compared to Group 3. Overall replicating viral load Gmean for Days 1-4 showed 5-fold lower viral load in Group 1, and 2-fold lower viral load in Group 2 as compared to Group 3. RT-qPCR of nasal turbinates showed that total viral load and active replication viral load were two fold lower in Group 1 and Group 2 compared to Group 3 animals. The data indicates treatment with AMB (Group1) and BHH (Group 2) reduced viral load in pharyngeal swabs and nasal turbinates and the reduction in viral load was more in AMB treated group. However, the total viral load and active replication viral load in lungs trachea and rectal swabs were similar or higher in treatment groups compared to vehicle control group.

The back-titration data suggested that the animals received 9.2×10⁵TCID₅₀virus and 4.3×10⁶TCID₅₀per dose for Cohort 1 and Cohort 2 indicating that animals received approximately 90-100 times more virus than the targeted dose.

No significance difference in IL-10 levels were observed between or within Groups 1, 2, and 3 on any of the days tested. Increased serum IL-6 levels were observed in Group 2 and Vehicle control animals following SARS CoV-2 infection on Day 4. Previous studies have shown that increasing levels of IL-6 correlate with disease severity. However, analysis of IL-6 levels between groups showed that the levels of IL-6 were significantly lower (P<0.05) in Group 1 and Group 2 animals when compared to Group 3 on Day 4. Similarly, significantly low expressions of TNF-α were observed in Group 1 and Group 2 compared to Group 3 animals on Day −1 (P<0.05), Day 2 (P<0.05) and Day 4 (P<0.001). The data suggest that treatment with AMB and BHH reduced IL-6 and TNF-α levels in animals challenged with SARS CoV-2 virus.

No difference in lung viral load was observed between treatment (Group 1 and Group 2) and vehicle control group (Group 3).

Overall, the animals treated with AMB and BHH tolerated the administered doses well up to 5 days post-administration. The efficacy data suggest that the oral administration of AMB and BHH reduced gross lesions in the lung and IL-6 and TNF-α levels in serum of the animals challenged with SARS CoV-2 virus. Treatment with AMB and BHH also reduced viral load in pharyngeal swabs and nasal turbinates and the reduction in viral load was more in AMB treated group. However, no difference in the lung, trachea and rectal swab viral loads as well as the incidence and severity of microscopic lesions in the AMB, BHH and vehicle control groups were observed in the current study. Optimization of treatment doses and a reduced challenge virus titer may be used in future studies to evaluate these test articles in hamster model.

Summary of Experiments Comprising AMB and BHH

The crystal structure of full length human ACE2 revealed that the RBD on SARS-CoV-2 S1 binds directly to the metallopeptidase domain (MPD) of ACE2 receptor.^{98, 100, 105, 107}Here, AMB and BHH were examined as potential effectors of the interaction between SARS-CoV-2's Spike glycoprotein receptor binding domain and recombinant human ACE2 receptor, which is an interaction that is important in the pathways required for viral entry into host cells¹⁰⁵and initiation of pathogenesis. Using a sensitive ELISA⁸⁰, AMB and BHH modulated the interaction of rhACE2 and S (RBD) with AMB being the most potent effector. Significant inhibition of the interaction between the Spike (RBD) of SARS-CoV-2 and rhACE2 by AMB at low micromolar concentrations provided strong evidence that this pharmacophore is a novel SARS-CoV-2 entry inhibitor and a potential COVID-19 therapeutic. The data also demonstrated that BHH enhanced the interaction between Spike (RBD) protein and rhACE2 at higher concentrations and inhibited it at lower concentrations. This is the first report describing these two compounds as potent effectors of the binding of the Spike (RBD) protein to rhACE2.

The unconventional dose-response curve observed in the interaction studies indicates that there can be more than one binding site on rhACE2 and/or the Spike (RBD) glycoprotein for BHH and AMB, which can elicit potent inhibition of interaction at lower micromolar concentrations. This can also explain the enhancement of interaction by BHH at higher concentrations. To this end, a molecular dynamic study revealed that two different regions within the RBD of the Spike glycoproteins of SARS-CoV-2 interacted differently with ACE2 in the presence of high salt concentrations (E1 was more hydrophobic while E2 favored more polar interactions).⁵²

The effect of AMB and BHH on SARS-CoV-2 infection induced CPE in vitro was also examined using a simple and rapid cellular high throughput screening assay.^{54, 85}While AMB was more potent than BHH against the rhACE2-RBD interaction, AMB had a higher IC₅₀than BHH in the CPE assay for antiviral activity. The IC₅₀values of the compounds in the cellular CPE assay were much higher than the IC_50sin the rhACE2-Spike (RBD) protein interaction assay, although the special bell curve produced two IC_50sdue to the mode of inhibition at lower concentrations versus higher concentrations. Together, the data provided herein demonstrate that AMB targeted a novel protein-protein interaction.

A comparative analysis of the dose-response curves of antiviral activity and cytotoxicity of AMB and BHH with five other known inhibitors (“reference compounds”) of SARS-CoV-2 in vitro was performed. The IC₅₀range for BHH and AMB were similar to that of Aloxistatin, but higher than the other reference compounds (i.e., Chloroquine, Hydroxychloroquine, Remdesivir, and Calpain Inhibitor IV). The cytotoxicity of AMB and BHH in Vero E6 cells, however, displayed higher percent maximum and minimum viability at the concentrations tested when compared to the reference compounds. Moreover, AMB had a slightly higher percent minimum viability when compared to BHH, which is consistent with other reported safety studies for both compounds that demonstrate that AMB has superior safety profile than BHH.¹⁰²Thus, AMB and its progenitor BHH can be used as chemical probes to study the biology of host-pathogen interaction in the context of SARS-CoV-2 infections, particularly in the pre-clinical development of novel entry inhibitors.

Both AMB and BHH have been used for clinically for treatment of respiratory conditions because of their multiple pharmacologic effects and safety profile.^{28, 60, 73, 76, 113}In addition to their impact on lung physiology and function with regards to mucociliary clearance, mucokinetic properties, and stimulation of surfactant production, they have also elicited anti-inflammatory, antioxidative and anesthetic effects.^{28, 60, 73, 76, 113}Both compounds induced cellular autophagic-lysosome pathway.^{16, 23, 58}AMB has also reportedly been involved in the modulation of the homeostasis of ions such as hydrogen, calcium, and sodium.^{27, 102}Moreover, previous studies have shown that both AMB and BHH could enhance the lung levels of certain antibiotics when used in combination.^{16, 23}Additionally, AMB has gained attention clinically as a potential drug for treatment of neurodegenerative diseases.^{57, 58,}

Previously, AMB has been shown to inhibit certain viruses in vitro and in vivo. One proposed mechanism included preventing the release of RNA into the cytoplasm by increasing the endosomal pH.^{106, 109}BHH demonstrated inhibitory activity against TMPRSS2 at low micromolar concentrations.⁵³However, unlike AMB and BHH, Camostat, another known TMPRSS2 inhibitor reversed TMPRSS2-mediated enhancement of SARS-CoV-2 infection.³⁷This indicates that AMB and BHH can have additional modes of action.³⁷The data provided herein demonstrate that AMB and BHH are effectors of the RBD-rhACE2 interaction further the understanding of SARS-CoV-2 inhibition.

BHH targets the interaction between RBD and rhACE2 by binding to the exopeptidase site of rhACE2, while AMB did not impact the exopeptidase activity. These findings reveal that the binding of BHH to rhACE2 inhibits its exopeptidase activity at concentrations starting around 50 μM. Investigations through western blot analysis and semi quantitative RT-PCR indicated that BBH does not affect rhACE2 protein and gene expression at 50 μM. Our findings also demonstrate that BHH effectively inhibits the cellular entry and subsequent replication of both SARS-CoV-2 and the B.1.617.2 Delta Variant, as evidenced by three distinct assays: the Infection Induced Cytopathic Effect (CPE), Nanoluciferase reporter assay (NLRV), and Pseudotyped Lentiviral assay. Also, through molecular dynamic simulation and the analysis of best-fit docking complexes, we have elucidated the binding sites of BHH and examined its molecular interactions with both host and viral receptors. These data support BHH and AMB as pharmacophores for drug development against SARS-CoV-2 infection.

The animal study evaluated the efficacy of AMB or BHH in adult, male Syrian hamsters challenged with wild-type SARS-CoV-2. Eighteen male Syrian hamsters, six per group, were assigned to the following three groups: Group 1 (500 mg/kg/day AMB), Group 2 (200 mg/kg/day BHH), and Group 3 (vehicle, 15 mL/kg aqueous solution containing 2% (w/v) polysorbate-80). There were two cohorts with staggered dosing. The animals were treated with test articles orally starting on Day-1 and continued daily through Day 3. All hamsters were challenged intranasally with SARS-CoV-2 approximately 1×10⁴TCID₅₀per animal on Day 0. All animals were euthanized on Day 4. Overall, the animals treated with AMB and BHH tolerated the administered doses well up to 5 days post-administration. The efficacy data suggest that the oral administration of AMB and BHH reduced gross lesions in the lung and IL-6 and TNF-α levels in serum of the animals challenged with SARS CoV-2 virus. Treatment with AMB and BHH also reduced viral load in pharyngeal swabs and nasal turbinates and the reduction in viral load was more in AMB treated group.

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The following references cited herein are hereby incorporated by reference.

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	Number	Date	Country
	63065401	Aug 2020	US
	63076936	Sep 2020	US

	Number	Date	Country
Parent	17402419	Aug 2021	US
Child	18639661		US

COMPOSITIONS FOR AND METHODS OF INHIBITING SARS-COV-2 INFECTION

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