CA3106787A1 - Use of amphotericin b in the treatment of coronavirus diseases - Google Patents

Abstract

A use of amphotericin B for the treatment or prevention of SARS-CoV-2 infection in a mammal. Also disclosed are oral formulations comprising amphotericin B for such use.

Description

USE OF AMPHOTERICIN B IN THE TREATMENT OF CORONAVIRUS DISEASES
FIELD OF THE INVENTION
The present invention is directed to the treatment of coronavirus diseases, including the use of Amphotericin B for the treatment of COVID-19 infections in humans.
BACKGROUND OF THE INVENTION
The appearance of COVID-19 on the world stage has affected every population in the world.
Causing millions of infected individuals, a number which is continuously increasing and is showing no signs of slowing down.
The advent of an effective, safe and proven vaccine while very recent will not have any impact on the millions of individuals already infected with the virus. Nor will the vaccine be of any help to treat, minimize the after effects of the infection in recovered patients. Many of the recovered patients have lingering symptoms ranging in severity from mild to debilitating. To date there have been over ly million individuals infected with COVID-19 in the United States several millions more across the globe and until the vaccination programs in all countries have run their course and have provided vaccines to all of those who want to be vaccinated, it is expected, given current infection trends, that several million more individual will have become infected with COVID-19 and for those who will have survived this infection, many will live with post-infection symptoms.
In light of this, it is of paramount importance to develop some treatment compositions which are based on widely available compounds, which have received regulatory approval in many countries and which can either help in the prevention of COVID-19 infection and/or treat individuals which have been infected with COVID-19.
To that end, amphotericin B is an antifugal antibiotic used for serious fungal infections and leishmaniasis. The fungal infections it is used to treat include aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, and cryptococcosis.
Common side effects include a reaction with fever, chills, and headaches soon after the medication is given, as well as kidney problems. Allergic symptoms including anaphylaxis may occur. Other serious side effects include low blood potassium and inflammation of the heart. It appears to be relatively safe in Date Recue/Date Received 2021-01-22

2 pregnancy. There is a lipid formulation that has a lower risk of side effects.
It is said that it mechanism of action by interfering with the cell membrane of the fungus.
Amphotericin B was first isolated from Streptomyces nodosus in 1955 and came into medical use in 1958. It is on the WHO List of Essential Medicines, which is a compilation of the safest and most effective medicines needed in the world. It is available under a number of various formulations and is available as a generic medicine.
One of the main uses of amphotericin B is treating a wide range of systemic fungal infections. Due to its extensive side effects, it is often reserved for severe infections in critically ill, or immunocompromised patients. It is considered first line therapy for invasive mucormycosis infections, cryptococcal meningitis, and certain aspergillus and candidal infections. In use since 1958, one of the reasons for its enduring appeal is that there have been very few pathogens which have adapted to it and developed resistance to treatment.
Amphotericin B is also used in the treatment of individuals infected with for life-threatening protozoan infections such as visceral leishmaniasis and primary amoebic meningoencephalitis.
Amphotericin B can also be used to treat the following common and dangerous fungi: Aspergillus fumigatus; Candida albicans; Candida krusei; Candida glabrata; Cryptococcus neoformans; and Fusarium oxysporum.
Amphotericin B is available in several different types of formulations including: intravenous; and liposomal and oral. As amphotericin B by itself does not dissolve in a standardsaline solution at a p11 of 7, it was originally developed in a formulation which uses sodium deoxycholate to improve its solubility.
Amphotericin B deoxycholate (ABD) is administered intravenously.
Other formulations containing amphotericin B haeen developed in order to improve the tolerability of amphotericin and reduce toxicity, several lipid formulations. It was found that liposomal formulations have less renal toxicity than the form of amphotericin B deoxycholate. It was also found that the liposomal formulations had fewer infusion-related reactions. Consequently, the liposomal formulation of amphotericin B are more expensive than the original amphotericin B
deoxycholate formulations.
An oral preparation of amphotericin B is known to exist but is not widely available. The amphipathic nature of amphotericin along with its low solubility and permeability has posed major hurdles Date Recue/Date Received 2021-01-22

3 for oral administration given its low bioavailability. In the past it had been used for fungal infections of the surface of the GI tract such as thrush, but has been replaced by other antifungals such as nystatin and fluconazole. Recent advances in drug delivery has led to the development of a novel nanoparticulate drug delivery systems consisting of a lipid-based drug delivery systems including cochleates, self-emulsifying drug delivery systems, solid lipid nanoparticles and polymeric nanoparticles -such as Amphotericin B in pegylated polylactide coglycolide copolymer nanoparticles which have demonstrated potential for oral formulation of amphotericin B.
In light of the current state of the art, there exists a need for therapeutic compounds capable of impacting COVID-19 in such a manner that it slows down its physiological impact on an infected individual. Given the haste and the magnitude of the pandemic, it is highly advantageous to be able to use an already approved drug. The present disclosure meets this need by providing compositions and methods for the treatment of coronavirus infections, including SARS-CoV-2 infection, and related diseases and disorders.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a therapy for the prevention/stabilization/reduction of risks and/or symptoms associated with a coronavirus infection in a mammal.
According to a preferred embodiment of the present invention, there is provided a use of Amphotericin B for the treatment of a coronavirus infection and/or the prevention/stabilization/reduction of risks associated with a coronavirus infection in a mammal.
BRIEF DESCRIPTION OF THE FIGURES
The invention may be more completely understood in consideration of the following description of various embodiments of the invention in connection with the accompanying figure, in which:
Figure 1 is a graphical representation of the concentration-response curves for Nystatin from the first testing series;
Figure 2 is a graphical representation of the concentration-response curves for Amphotericin B
from first the testing series;
Date Recue/Date Received 2021-01-22

4 Figure 3 is a graphical representation of the concentration-response curves for Remdesevir from the second testing series; and Figure 4 is a graphical representation of the concentration-response curves for Amphotericin B
from the second testing series.
DESCRIPTION OF THE INVENTION
The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles of the present invention.
These examples are provided for the purposes of explanation, and not limitation, of those principles and of the invention.
According to an aspect of the present invention, there is provided the use of Amphotericin B for the treatment of a coronavirus infection in a mammal. In certain embodiments, the coronavirus infection is an infection by alpha coronavirus 229E, alpha coronavirus NL63, beta coronavirus 0C43, beta coronavirus HKU1, or SARS-CoV-2. In particular embodiments, the mammal is infected with SARS-CoV-2 and/or has been disagnosed with COVID-19 and/or has symptoms thereof.
In certain embodiments, the disclosure includes a method of treating or preventing a coronavirus infection in a mammal in need thereof, comprising providing to the mammal an effective amount of AmpB.
In particular embodiments, the coronavirus is SARS-CoV-2. In particular embodiments, the method inhibits viral replication. In certain embodiments, the mammal in need thereof is infected with or at risk of infection by SARS-CoV-2, or has been diagnosed with or suspected to have COVID-19. In certain embodiments, the mammal is a human. In certain embodiments, the AmpB is provided orally or is formulated for oral administration. In particular embodiments, the mammal is provided an oral formulation of AmpB, e.g., any of those disclosed herein, including but not limited to those described in any of Tables 1-7.
In certain embodiments, the disclosure includes a method of inhibiting replication of a coronavirus in a mammal in need thereof, comprising providing to the mammal an effective amount of AmpB. In particular embodiments, the coronavirus is SARS-CoV-2. In certain embodiments, the mammal in need thereof is infected with SARS-CoV-2, or has been diagnosed with COVID-19. In certain embodiments, the Date Recue/Date Received 2021-01-22

5 mammal is a human. In certain embodiments, the AmpB is provided orally or is formulated for oral administration. In particular embodiments, the mammal is provided an oral formulation of AmpB, e.g., any of those disclosed herein, including but not limited to those described in any of Tables 1-7.
In another embodiment, the disclosure provides a method of treating, inhibiting, reducing the likelihood of, or preventing a fungal infection secondary to infection with a coronavirus, such as, e.g., a SARS-CoV-2 infection, comprising providing to the mammal an effective amount of AmpB. In particular embodiments, the fungal infection is a yeast infection. In certain embodiments, the yeast is Candida, such as, e.g., Candida albicans. In certain embodiments, the mammal in need thereof is infected with or at risk of infection by SARS-CoV-2, or has been diagnosed with or suspected to have COVID-19. In certain embodiments, the mammal is a human. In certain embodiments, the AmpB is provided orally or is formulated for oral administration. In particular embodiments, the mammal is provided an oral formulation of AmpB, e.g., any of those disclosed herein, including but not limited to those described in any of Tables 1-7.
In particular embodiments of any of the methods disclosed herein, the mammal is provided with a pharmaceutical composition or formulation comprising the AmpB. In certain embodiments, the pharmaceutical composition or formulation is disclosed in any of U.S. Patent No. 8,592,382, U.S. Patent No. 8,673,866, PCT Patent Application Publication No. W02016/112339, U.S.
Patent Application No.
15/541,236 (published as U520180000854), U.S. Patent Application No.
16/487,356 (published as U520200155583), PCT Application Publication No. W02018/156,585, or PCT
Application Publication No. W02020/028508, each of which is herein incorporated by reference in its entirety.
In certain embodiments, the AmpB is provided to the mammal in an amphotericin B formulation comprising, (a) amphotericin B;
(b) one or more fatty acid glycerol esters; and (c) one or more polyethylene oxide-containing phospholipids or one or more polyethylene oxide-containing fatty acid esters.
In particular embodiments, the amphotericin B formulation is provide orally.
In one embodiment, the amphotericin B is present in the formulation in an amount from about 0.5 to about 10 mg/mL of the formulation. In one embodiment, the amphotericin B is present in the formulation Date Recue/Date Received 2021-01-22

6 in about 5 mg/mL. In another embodiment, the amphotericin B is present in the formulation in about 7 mg/mL. In particular embodiments, the formulation comprises: (a) amphotericin B; (b) one or more fatty acid glycerol esters; (c) one or more polyethylene oxide-containing fatty acid esters; and, optionally, (d) a tocopherol polyethylene glycol succinate.
In one embodiment, the fatty acid glycerol esters comprise from about 32 to about 52% by weight fatty acid monoglycerides. In one embodiment, the fatty acid glycerol esters comprise from about 30 to about 50% by weight fatty acid diglycerides. In one embodiment, the fatty acid glycerol esters comprise from about 5 to about 20% by weight fatty acid triglycerides. In one embodiment, the fatty acid glycerol esters comprise greater than about 60% by weight oleic acid mono-, di-, and triglycerides.
In one embodiment, the polyethylene oxide-containing phospholipids comprise a C8-C22 saturated fatty acid ester of a phosphatidyl ethanolamine polyethylene glycol salt. In one embodiment, the polyethylene oxide-containing phospholipids comprise a distearoylphosphatidyl ethanolamine polyethylene glycol salt. In one embodiment, the distearoylphosphatidyl ethanolamine polyethylene glycol salt is selected from the group consisting of a distearoylphosphatidyl ethanolamine polyethylene glycol 350 salt, a distearoylphosphatidyl ethanolamine polyethylene glycol 550 salt, a distearoylphosphatidyl ethanolamine polyethylene glycol 750 salt, a distearoylphosphatidyl ethanolamine polyethylene glycol 1000 salt, a distearoylphosphatidyl ethanolamine polyethylene glycol 2000 salt, and mixtures thereof. In one embodiment, the distearoylphosphatidyl ethanolamine polyethylene glycol salt is present in the formulation in an amount from 1 mM to about 30 mM based on the volume of the formulation. In one embodiment, the distearoylphosphatidyl ethanolamine polyethylene glycol salt is an ammonium salt or a sodium salt.
In one embodiment, the polyethylene oxide-containing fatty acid esters comprise a polyethylene oxide ester of a C8-C22 saturated fatty acid. In one embodiment, the polyethylene oxide-containing fatty acid esters comprise a polyethylene oxide ester of a C12-C18 saturated fatty acid. In one embodiment, the polyethylene oxide-containing fatty acid esters is selected from the group consisting of Laurie acid esters, palmitic acid esters, stearic acid esters, and mixtures thereof. In one embodiment, the polyethylene oxide-containing fatty acid esters comprise a polyethylene oxide having an average molecular weight of from about 750 to about 2000. In one embodiment, the ratio of the fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is from about 20:80 to about 80:20 v/v. In one embodiment, the ratio of the fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 60:40 v/v. In one Date Recue/Date Received 2021-01-22

7 embodiment, the formulation further comprises glycerol in an amount less than about 10% by weight. In one embodiment, the formulation is a self-emulsifying drug delivery system.
In certain embodiments, the formulation further comprises a tocopherol polyethylene glycol succinate. In particular embodiments, the tocopherol polyethylene glycol succinate is present in the formulation from about 0.1 to about 10 percent by volume based on the total volume of the formulation.
Structurally, tocopherol polyethylene glycol succinates have a polyethylene glycol (PEG) covalently coupled to tocopherol (e.g., .alpha.-tocopherol or vitamin E) through a succinate linker. Because PEG is a polymer, a variety of polymer molecular weights can be used to prepare the TPGS. In one embodiment, the TPGS is tocopherol polyethylene glycol succinate 1000, in which the average molecular weight of the PEG
is 1000. One suitable tocopherol polyethylene glycol succinate is vitamin E
TPGS commercially available from Eastman. In one embodiment, the tocopherol polyethylene glycol succinate is present in the formulation in about 5 percent by volume based on the total volume of the formulation. In one embodiment, the formulation further comprises glycerol in an amount less than about 10% by weight. In one embodiment, the formulation is a self-emulsifying drug delivery system.
Certain amphotericin B formulations disclosed herein include one or more fatty acid glycerol esters, and typically, a mixture of fatty acid glycerol esters. The fatty acid glycerol esters useful in the formulations can be provided by commercially available sources. A representative source for the fatty acid glycerol esters is a mixture of mono-, di-, and triesters commercially available as PECEOL® (Gattefosse, Saint Priest Cedex, France), commonly referred to as "glyceryl oleate" or "glyceryl monooleate." When PECEOLTM is used as the source of fatty acid glycerol esters in the formulations, the fatty acid glycerol esters comprise from about 32 to about 52% by weight fatty acid monoglycerides, from about 30 to about 50% by weight fatty acid diglycerides, and from about 5 to about 20% by weight fatty acid triglycerides. The fatty acid glycerol esters comprise greater than about 60% by weight oleic acid (C18:1) mono-, di-, and triglycerides.
Other fatty acid glycerol esters include esters of palmitic acid (C16) (less than about 12%), stearic acid (C18) (less than about 6%), linoleic acid (C18:2) (less than about 35%), linolenic aid (C18:3) (less than about 2%), arachidic acid (C20) (less than about 2%), and eicosenoic acid (C20:1) (less than about 2%).
PECEOLTM can also include free glycerol (typically about 1%). In one embodiment, the fatty acid glycerol esters comprise about 44% by weight fatty acid monoglycerides, about 45% by weight fatty acid diglycerides, and about 9% by weight fatty acid triglycerides, and the fatty acid glycerol esters comprise about 78% by weight oleic acid (C18:1) mono-, di-, and triglycerides. Other fatty acid glycerol esters include esters of palmitic acid (C16) (about 4%), stearic acid (C18) (about 2%), linoleic acid (C18:2) (about Date Recue/Date Received 2021-01-22

8 12%), linolenic aid (C18:3) (less than 1%), arachidic acid (C20) (less than 1%), and eicosenoic acid (C20:1) (less than 1%).
As used herein, the term "polyethylene oxide-containing fatty acid ester"
refers to a fatty acid ester that includes a polyethylene oxide group (i.e., polyethylene glycol group) covalently coupled to the fatty acid through an ester bond. Polyethylene oxide-containing fatty acid esters include mono- and di-fatty acid esters of polyethylene glycol. Suitable polyethylene oxide-containing fatty acid esters are derived from fatty acids including saturated and unsaturated fatty acids having from eight (8) to twenty-two (22) carbons atoms (i.e., a polyethylene oxide ester of a C8-C22 fatty acid). In certain embodiments, suitable polyethylene oxide-containing fatty acid esters are derived from fatty acids including saturated and unsaturated fatty acids having from twelve (12) to eighteen (18) carbons atoms (i.e., a polyethylene oxide ester of a C12-C18 fatty acid). Representative polyethylene oxide-containing fatty acid esters include saturated C8-C22 fatty acid esters. In certain embodiments, suitable polyethylene oxide-containing fatty acid esters include saturated C12-C18 fatty acids. The molecular weight of the polyethylene oxide group of the polyethylene oxide-containing fatty acid ester can be varied to optimize the solubility of the therapeutic agent (e.g., amphotericin B) in the formulation. Representative average molecular weights for the polyethylene oxide groups can be from about 350 to about 2000. In one embodiment, the average molecular weight for the polyethylene oxide group is about 1500. In this embodiment, the amphotericin B
formulations include one or more polyethylene oxide-containing fatty acid esters, and typically, a mixture of polyethylene oxide-containing fatty acid esters (mono- and di-fatty acid esters of polyethylene glycol). The polyethylene oxide-containing fatty acid esters useful in the formulations can be provided by commercially available sources.
Representative polyethylene oxide-containing fatty acid esters (mixtures of mono- and diesters) are commercially available under the designation GELUCIRE (Gattefosse, Saint Priest Cedex, France).
Suitable polyethylene oxide-containing fatty acid esters can be provided by GELUCIRE 44/14, GELUCIRE 50/13, and GELUCIRE 53/10. The numerals in these designations refer to the melting point and hydrophilic/lipophilic balance (HLB) of these materials, respectively.
GELUCIRE 44/14, GELUCIRE 50/13, and GELUCIRE 53/10 are mixtures of (a) mono-, di-, and triesters of glycerol (glycerides) and (b) mono- and diesters of polyethylene glycol (macrogols).
The GELUCIRE can also include free polyethylene glycol (e.g., PEG 1500). Laurie acid (C12) is the predominant fatty acid component of the glycerides and polyethylene glycol esters in GELUCIRE 44/14.
GELUCIRE 44/14 is referred to as a mixture of glyceryl dilaurate (Laurie acid diester with glycerol) and PEG dilaurate (Laurie acid diester with polyethylene glycol), and is commonly known as PEG-32 glyceryl laurate (Gattefosse) Date Recue/Date Received 2021-01-22

9 lauroyl macrogo1-32 glycerides EP, or lauroyl polyoxylglycerides USP/NF.
GELUCIRE® 44/14 is produced by the reaction of hydrogenated palm kernel oil with polyethylene glycol (average molecular weight 1500). GELUCIRE 44/14 includes about 20% mono-, di- and, triglycerides, about 72% mono- and di-fatty acid esters of polyethylene glycol 1500, and about 8% polyethylene glycol 1500. GELUCIRE
44/14 includes Laurie acid (C12) esters (30 to 50%), myristic acid (C14) esters (5 to 25%), palmitic acid (C16) esters (4 to 25%), stearic acid (C18) esters (5 to 35%), caprylic acid (C8) esters (less than 15%), and capric acid (C10) esters (less than 12%). GELUCIRE 44/14 may also include free glycerol (typically less than about 1%). In a representative formulation, GELUCIRE 44/14 includes lauric acid (C12) esters (about 47%), myristic acid (C14) esters (about 18%), palmitic acid (C16) esters (about 10%), stearic acid (C18) esters (about 11%), caprylic acid (C8) esters (about 8%), and capric acid (C10) esters (about 12%).
Palmitic acid (C16) (40-50%) and stearic acid (C18) (48-58%) are the predominant fatty acid components of the glycerides and polyethylene glycol esters in GELUCIRE
50/13. GELUCIRE 50/13 is known as PEG-32 glyceryl palmitostearate (Gattefosse), stearoyl macrogolglycerides EP, or stearoyl polyoxylglycerides USP/NF). GELUCIRE 50/13 includes palmitic acid (C16) esters (40 to 50%), stearic acid (C18) esters (48 to 58%) (stearic and palmitic acid esters greater than about 90%), Laurie acid (C12) esters (less than 5%), myristic acid (C14) esters (less than 5%), caprylic acid (C8) esters (less than 3%), and capric acid (C10) esters (less than 3%). GELUCIRE 50/13 may also include free glycerol (typically less than about 1%). In a representative formulation, GELUCIRE 50/13 includes palmitic acid (C16) esters (about 43%), stearic acid (C18) esters (about 54%) (stearic and palmitic acid esters about 97%), lauric acid (C12) esters (less than 1%), myristic acid (C14) esters (about 1%), caprylic acid (C8) esters (less than 1%), and capric acid (C10) esters (less than 1%) Stearic acid (C18) is the predominant fatty acid component of the glycerides and polyethylene glycol esters in GELUCIRE 53/10. GELUCIRE
53/10 is known as PEG-32 glyceryl stearate (Gattefosse). In one embodiment, the polyethylene oxide-containing fatty acid ester is a lauric acid ester, a palmitic acid ester, or a stearic acid ester (i.e., mono- and di-lauric acid esters of polyethylene glycol, mono- and di-palmitic acid esters of polyethylene glycol, mono- and di-stearic acid esters of polyethylene glycol). Mixtures of these esters can also be used.
For embodiments that include polyethylene oxide-containing fatty acid esters, the ratio of the fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is from about 20:80 to about 80:20 v/v. In one embodiment, the ratio of the fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 30:70 v/v. In one embodiment, the ratio of the fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 40:60 v/v. In one embodiment, the ratio of the fatty acid glycerol Date Recue/Date Received 2021-01-22

10 esters to polyethylene oxide-containing fatty acid esters is about 50:50 v/v.
In one embodiment, the ratio of the fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 60:40 v/v. In one embodiment, the ratio of the fatty acid glycerol esters to polyethylene oxide-containing fatty acid esters is about 70:30 v/v.
As used herein, the term "polyethylene oxide-containing phospholipid" refers to a phospholipid that includes a polyethylene oxide group (i.e., polyethylene glycol group) covalently coupled to the phospholipid, typically through a carbamate or an ester bond. Phospholipids are derived from glycerol and can include a phosphate ester group and two fatty acid ester groups. Suitable fatty acids include saturated and unsaturated fatty acids having from eight (8) to twenty-two (22) carbons atoms (i.e., C8-C22 fatty acids). In certain embodiments, suitable fatty acids include saturated C12-C18 fatty acids. Representative polyethylene oxide-containing phospholipids include C8-C22 saturated fatty acid esters of a phosphatidyl ethanolamine polyethylene glycol salt. In certain embodiments, suitable fatty acids include saturated C12-C18 fatty acids. The molecular weight of the polyethylene oxide group of the polyethylene oxide-containing phospholipid can be varied to optimize the solubility of the therapeutic agent (e.g., amphotericin B) in the formulation. Representative average molecular weights for the polyethylene oxide groups can be from about 200 to about 5000 (e.g., PEG 200 to PEG 5000).
In one embodiment, the polyethylene oxide-containing phospholipids are distearoyl phosphatidyl ethanolamine polyethylene glycol salts. Representative distearoylphosphatidyl ethanolamine polyethylene glycol salts include distearoylphosphatidyl ethanolamine polyethylene glycol 350 (DSPE-PEG-350) salts, distearoylphosphatidyl ethanolamine polyethylene glycol .. 550 .. (D SPE -PEG-550) .. salts, distearoylphosphatidyl ethanolamine polyethylene glycol .. 750 .. (D SPE -PEG-750) .. salts, distearoylphosphatidyl ethanolamine polyethylene glycol 1000 (DSPE-PEG-1000) salts, distearoylphosphatidyl ethanolamine polyethylene glycol 1500 (DSPE-PEG-1500) salts, and distearoylphosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG-2000) salts. Mixtures can also be used. For the distearoylphosphatidyl ethanolamine polyethylene glycol salts above, the number (e.g., 350, 550, 750, 1000, and 2000) designates the average molecular weight of the polyethylene oxide group.
The abbreviations for these salts used herein is provided in parentheses above.Suitable distearoylphosphatidyl ethanolamine polyethylene glycol salts include ammonium and sodium salts.
The chemical structure of distearoylphosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG-2000) ammonium salt is illustrated in FIG. 1B. Referring to FIG. 1B, the polyethylene oxide-Date Recue/Date Received 2021-01-22

11 containing phospholipid includes a phosphate ester group and two fatty acid ester (stearate) groups, and a polyethylene oxide group covalently coupled to the amino group of the phosphatidyl ethanolamine through a carbamate bond.
The polyethylene oxide-containing phospholipid affects the ability of the formulation to solubilize a therapeutic agent. In general, the greater the amount of polyethylene oxide-containing phospholipid, the greater the solubilizing capacity of the formulation for difficulty soluble therapeutic agents. The polyethylene oxide-containing phospholipid can be present in the formulation in an amount from about 1 mM to about 30 mM based on the volume of the formulation. In certain embodiments, the distearoylphosphatidyl ethanolamine polyethylene glycol salt is present in the formulation in an amount from 1 mM to about 30 mM based on the volume of the formulation. In one embodiment, the distearoylphosphatidyl ethanolamine polyethylene glycol salt is present in the formulation in about 15 mM
based on the volume of the formulation.
In certain embodiments, the AmpB is provided to the mammal in a solid dosage form (e.g., solid or semi-solid dosage forms) comprising amphotericin B. In particular embodiments, the solid dosage form comprises an oral formulation disclosed herein. In some embodiments, the solid dosage form comprises amphotericin B and at least one lipophilic component which are coated on a solid carrier. In other embodiments, the % w/w of amphotericin B in the solid dosage form is greater than a % w/w of the at least one lipophilic component. In further embodiments, the % w/w of amphotericin B
is in the range of about 20% to about 30% of the total weight of the solid dosage form. In some embodiments, amphotericin B is present in the solid dosage form in an amount in the range of from about 50 mg to about 200 mg. In other embodiments, amphotericin B is present in amount of about 100 mg. In still other embodiments, wherein the amphotericin B is present in amount of about 150 mg. In particular embodiments, the formulation is present in a hard shell capsule. In particular embodiments, the amphotericin B
formulation is provide orally.
In certain embodiments, the solid dosage form in any of those shown in Tables 1-7.
In some embodiments, the at least one lipophilic component is selected from the group consisting of a polyethylene oxide-containing fatty acid ester, fatty acid glycerol ester, and a combination thereof. In some embodiments, the solid dose formulation comprises AmpB, a polyethylene oxide-containing fatty acid ester, and fatty acid glycerol ester.
Date Recue/Date Received 2021-01-22

12 The solid dosage forms of the present disclosure can be prepared by any suitable method, including granulation of the therapeutic agent (e.g. amphotericin B) with excipients (e.g. fillers, glidants, lubricants, etc. known in the art and described herein), extrusion of the therapeutic agent with excipients, direct compression of the therapeutic agent with excipients to form tablets, etc. In particular embodiments, the solid dosage forms the present disclosure can be prepared by coating the active agent, e.g. amphotericin B
on a solid carrier. The solid carrier can be any material upon which a drug-containing composition can be coated and which is suitable for human consumption. Any conventional coating process can be used. For example, the therapeutic agent, e.g. amphotericin B can be dissolved or suspended in a suitable solvent (e.g., ethanol), together with an optional binder, or alternatively one or more of the lipophilic components described herein, and deposited on the solid carrier by methods known in the art, e.g. fluidized bed coating or pan coating methods. The solvent can be removed e.g. by drying, or in situ during the coating process (e.g., during fluidized bed coating), and/or in a subsequent drying step.
In some embodiments, the solid carrier may be an inert bead or an inert particle. In other embodiments, the solid carrier a non-pareil seed, an acidic buffer crystal, an alkaline buffer crystal, or an encapsulated buffer crystal. In some embodiments, the solid carrier may be a sugar sphere, cellulose sphere, lactose sphere, lactose-microcrystalline cellulose (MCC) sphere, mannitol-MCC
sphere, or silicon dioxide sphere. In other embodiments, the solid carrier may be a saccharide, a sugar alcohol, or combinations thereof. Suitable saccharides include lactose, sucrose, maltose, and combinations thereof. Suitable sugar alcohols include mannitol, sorbitol, xylitol, maltitol, arabitol, ribitol, dulcitol, iditol, isomalt, lactitol, erythritol and combinations thereof. In embodiments, the solid carrier may be formed by combining any of the above with a filler. Examples of suitable fillers which may be used to form a solid carrier include lactose, microcrystalline cellulose, silicified microcrystalline cellulose, mannitol-microcrystalline cellulose and silicon dioxide. In other embodiments, the dosage form disclosed herein does not include a solid carrier. In other embodiments, the disclosure provides for a capsule comprising a solid dosage form described herein.
In certain embodiments, the solid dose comprises a formulation disclosed in any of Tables 1-5.
Table 1: Formulation 1 Item Ingredient % w/w mg/unit g/batch a Amphotericin B 23.0 100 4.60 b Mannitol 160C 34.5 150 6.90 c Tabulo se 101 34.3 149 6.85 d Colloidal silicon dioxide 2.3 10 0.46 e TPGS 0.2 1 0.05 Date Recue/Date Received 2021-01-22

13 f Peceol 2.3 10 0.46 g Gelucire 44/14 2.3 10 0.46 h Ethanol 100% (evaporated during the process) (30.0) -(6.00) i Magnesium stearate 1.1 5 0.23 Total: 100 435 20 Items a-h are internal phase components, and item i is the external phase component.
Table 2: Formulation 2 Item Ingredient `)/0 w/w mg/unit g/batch a Amphotericin B 23.0 100 4.60 b Prosolv 1-1D90 66.0 287 13.21 c Croscarmellose sodium 5.0 22 1.00 d TPGS 0.2 1 0.05 e Peceol 2.3 10 0.46 f Gelucire 44/14 2.3 10 0.46 g Ethanol 100% (evaporated during the process) (30.0) -(6.00) h Magnesium stearate 1.1 5 0.23 Total: 100 435 20 Items a-g are internal phase components, and item h is the external phase component.
Table 3: Formulation 3 Item Ingredient % w/w mg/unit g/batch a Amphotericin B 23.0 100 4.60 b Tabulose 101 66.0 287 13.21 c Plasdone K-29/32 5.0 22 1.00 d TPGS 0.2 1 0.05 e Peceol 2.3 10 0.46 f Gelucire 44/14 2.3 10 0.46 g Ethanol 100% (evaporated during the process) (30.0) -(6.00) h Magnesium stearate 1.1 5 0.23 Total: 100 435 20 Items a-g are internal phase components, and item h is the external phase component.
Table 4: Formulation lA
Item Ingredient % w/w mg/unit g/batch a Amphotericin B 23.0 100 23.0 b Mannitol 160C 34.5 150 34.5 c Tabulose 101 34.3 149 34.3 d Colloidal silicon dioxide 2.3 10 2.3 e TPGS 0.2 1 0.2 f Peceol 2.3 10 2.3 g Gelucire 44/14 2.3 10 2.3 h Ethanol 100% (evaporated during the process) (30.0) - (30.0) i Magnesium stearate 1.1 5 1.1 Total: 100 435 100 Items a-h are internal phase components, and item i is the external phase component.
Date Recue/Date Received 2021-01-22

14 Table 5: Formulation 2A
Item Ingredient "4 w/w mg/unit g/batch a Amphotericin B 23.0 100 23.0 b Prosolv 1-1D90 66.0 287 66.0 c Croscarmellose sodium 5.0 22 5.0 d TPGS 0.2 1 0.2 e Peceol 2.3 10 2.3 f Gelucire 44/14 2.3 10 2.3 g Ethanol 100% (evaporated during the process) (30.0) -(30.0) h Magnesium stearate 1.1 5 1.1 Total: 100 435 100 Items a-g are internal phase components, and item h is the external phase component.
In certain embodiments, the solid dose comprises a formulation disclosed in any of Tables 6-7.
Table 6: iCo /Wasan Liquid Formulation, Formulation 4 and Formulation 5 iCo /Wasan Formulation Formulation 4 Formulation 5 Ingredient 07/dose % w/w mg/dose % w/w mg/dose % w/w Amphotericin B 150 mg 0.5 4 0.5 100 11.1 TPGS 1.5 mL 5 40 5 40 4.4 Peceol 14.25 mL 47.3 380 47.3 380 42.2 Gelucire 44/14 14.25 mL 47.3 380 47.3 380 42.2 Total: 30 mL 100 804* 100 900* 100 *=0.95 mL
Table 7: 100 mg Amphotericin B Lipid Formulation 5A
Ingredient mg/dose % w/w g/batch Amphotericin B 100 11.1 40.0 TPGS 40 4.4 16.0 Peceol 380 42.2 152.0 Gelucire 44/14 380 42.2 152.0 Total: 900 100 360 Date Recue/Date Received 2021-01-22

15 According to a one embodiment of the present invention, Amphotericin B, which is an antifungal antibiotic, would be useful in treating COVID-19 because of its actions as at interacts with S-protein on virus and blocks access to ACE-2 receptor. Amphotericin B is known to target Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1 is an enzyme that in humans is encoded by the CTDSP1 gene (1170 nM) and HTT (3320 nM).
It is known that amphotericin B is conventionally indicated for the treatment of patients with serious systemic fungal infections and certain protozoal infections. The intravenous (W) doses of amphotericin B as a colloidal complex with sodium deoxycholate are 17.5 to 21 mg/day, up to a maximum of 105 mg/day in a 70 kg adult. The IV doses for the drug encapsulated in liposomes or in complex with phospholipids (liposomal) are 210 to 350 mg/day. Therapy continues for several months depending on the infection. Injections into the spinal canal use doses between 0.1 and 1.5 mg, administered at intervals ranging from daily to weekly. Amphotericin B is also used orally with doses of 10-400 mg/day, dermally for localized infections (doses not specified) and by aerosol inhalation (INH) with a daily dose of 8 to 40 mg.
Amphotericin B is an antifungal antibiotic which acts by binding to sterols (lipid building blocks of the fungal cell wall) in the cell membrane of sensitive fungal species.
This leads to alterations in cell permeability, leakage of cellular constituents and cell death. While amphotericin B has a higher affinity for the sterol components of fungal cell membranes, it can also cause toxicity in mammalian cells through a similar mechanism.
Its mechanism related to interfering with Covid-19 is thought to be through an interaction with 5-protein on virus and blocks access to ACE-2 receptor Amphotericin B was shown to interact with the S-protein of the SARS-CoV-2 virus thus blocking its interaction with ACE-2 receptor with IC50 < 1 M. Amphotericin B, once it is bound to the S-protein, the interaction of this complex with ACE-2 was no longer energetically favored interaction - i.e., this ligand acted as desired inhibitor that can efficiently block the interaction of the SARS-CoV S-protein with ACE-2. Among a number of drugs tested, amphotericin B is one of the most promising, since it demonstrated the highest docking energy to the SARS-CoV-2 S-protein.
Date Recue/Date Received 2021-01-22

16 It is important to know that the pharmacokinetic profile of amphotericin B
depends significantly on the formulation used. Following W administration of the conventional, non-liposomal form, amphotericin B is widely distributed. Amphotericin B is excreted very slowly by the kidneys, with <10%
excreted as a biologically active form. While the metabolic pathway of amphotericin B is unknown, the systemic elimination half-life is 24 hours but increases to 15 days with long-term use. Systemic absorption following aerosol inhalation dosing was also low and in many cases serum levels were below the limit of detection.
In a 91-day rat oral study a slight nasal discharge was observed at doses >
250 mg/kg/day. A no-observed-adverse-effect level (NOAEL) was not identified in this study.
Intraperitoneal administration in mice for 71 days showed no toxicity with doses up to 100 mg/kg/day (NOAEL).
Oral administration to dogs for 187 days resulted in slight weight gain and occasional emesis at >125 mg/kg/day and kidney and bladder toxicity at 500 mg/kg/day. A NOAEL was not identified. In a 2-month study, W administration to dogs resulted in kidney toxicity (no further details provided) at dose of 16.5 mg/kg/day.
A literature review Amphotericin B was found to be neither mutagenic nor clastogenic in a battery of in vitro and in vivo assays.
In Vitro Cell testing A number of different compounds were tested for efficacy in in vitro testing using the viral strain: SARS-CoV-2/Canada/ONNIDO-01/2020Nero'76/p.2. The results are tabulated in Table El below. Figures 1 and 2 are concentration response curves generated during this testing series for each one of the compounds tested.
Table El- Summary of Preliminary Results from VIDO-InterVac in Vero-76 Kidney Cells (Viral Strain used: (SARS-CoV-2/Canada/ONNID0-01/2020/Vero'76/p.2)*
Drug EC50 SI Al Score Amphotericin B 1149 nM"" (range) 3.0 0.2 Nystatin >3600 nM 1.41 0 "D614G variant that's been circulating across the continent since early 2020 and there have been no appreciable differences in transmission or virulence across cities in North America that would suggest any mutational changes in the virus resulting in a new variant.
**Activity based on concentrations used to treat tapeworms (3.10-p to 3.10-s M) and systemic fungal infections (1000-2000 nM).
Date Recue/Date Received 2021-01-22

17 Only Amphotericin showed a significant reduction in the TCID50 titer, with the 50% effective concentration (EC50) of 2.8 to 1149 nM. Nystatin, a compound similar to Amphotericin B was substantially less active and would not be considered a suitable choice for further testing for use a COVID-19 treatment compound. Figures 1 and 2 highlight the difference in concentration curve response for both Nystatin and Amphotericin B. The selectivity index (SI) was calculated as the ratio of CC50 to EC50. The SI for both compounds was > 1, which indicates that the compounds are more effective than they are toxic to the cells.
Second testing series Experimental Design:
Efficacy was tested in parallel in African green monkey kidney (Vero E6) cells. Each test compound was tested individually. Technicians were blinded to the identification of the drug being tested.
Each of the concentrations was evaluated in triplicate for efficacy. Vero E6 cells were cultured in 96 well plates prior to the day of the assay. Cells were greater than 90% confluency at the start of the study. Each of the test article concentrations was evaluated in triplicate.
Test article concentrations was tested in two different conditions: 1) Pre-treatment for 24 4 hours prior to virus inoculation followed by treatment immediately after removal of virus inoculum or 2) treatment only with test article added immediately following removal of virus inoculum.
Remdesivir was added immediately following removal of virus inoculum. For pre-treatment and treatment, wells were overlaid with 0.2 mL DMEM2 (Dulbecco's Modified Eagle Media (DMEM) with 2% Fetal Bovine Serum (FBS) with test articles at concentrations as delineated in Section 9.8). Following the 24 4 hour pre-treatment, cells were inoculated at a MOI of 0.001 TCID50/cell with SARS-CoV-2 and incubated for 60-90 minutes.
Immediately following the 60-90 minute incubation, virus inoculum was removed, cells washed and appropriate wells overlaid with 0.2 mL DMEM2 (DMEM with 2% FBS with test or control articles) and incubated in a humidified chamber at 37 C 2 C in 5 2% CO2. At 48 6 hours post inoculation, cells were fixed and evaluated for the presence of virus by immunostaining assay Justification: The immunostaining assay utilized modified the incubation time to 48 hours. A 24 4 hour pre-treatment of the cells was included for selected test articles.
Table E2-Efficacy in African green monkey kidney (Vero E6) cells infected with Virus (Viral Strain used:2019 Novel Coronavirus, Isolate USA-WA1/2020 (SARS-CoV-2)Code Date Recue/Date Received 2021-01-22

18 Drug EC50 ECioo comment Amphotericin B 31.8 nM"" (range) Achieved Remdesevir 1.15um Achieved Positive control **Activity based on concentrations used to treat tapeworms(3.10-910 3.10-5M) and systemic fungal infections(1000-2000 nM).
The above results of Table E2 and accompanying Figures 3 and 4 confirm the results obtained in the first testing series and confirm the versatility of Amphotericin B as a replication blocker of COVID-19 as the tests were carried out on different strains of COVID-19. The above results also indicate that in this test, Amphotericin B was clearly superior to Remdesevir in terms of EC50 as also evidenced in Figures 3 and 4.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by those skilled in the relevant arts, once they have been made familiar with this disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.
Date Recue/Date Received 2021-01-22

Claims (6)

19

1. A use of amphotericin B for the prevention of SARS-CoV-2 infection in a mammal.

2. A use of amphotericin B for the treatment of COVID-19 in a mammal.

3. A use of amphotericin B for the prevention of the replication of the COVID-19 virus in mammalian cells.

4. A use of an oral formulation comprising amphotericin B for the prevention of SARS-CoV-2 infection in a mammal.

5. A use of an oral formulation comprising amphotericin B for the treatment of COVID-19 in a mammal.

6. A use of an oral formulation comprising amphotericin B for the prevention of the replication of the COVID-19 virus in mammalian cells.
Date Recue/Date Received 2021-01-22