Virus suppression

Abstract

The present invention relates to the use of at least a macrolide (such as ivermectin or moxidectin) to inhibit coronavirus in an individual to treat, prevent or reduce the risk of coronavirus infection.

Description

Technical Field

[0001] The present invention relates to the use of macrolides to inhibit coronaviruses. Specifically, the present invention relates to the use of macrolides (such as ivermectin and / or moxidectin) to inhibit coronaviruses, or to prevent or treat coronavirus infections in individuals in need thereof.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to Australian provisional patent application (application number 2020900778) filed on March 13, 2020 and Australian provisional patent application (application number 2020901549) filed on May 14, 2020. These applications are incorporated herein in their entirety by cross-reference. Background Art

[0004] Ivermectin is a U.S. Food and Drug Administration (FDA)-approved macrolide that is a broad-spectrum antiparasitic drug that has demonstrated potent antiviral activity against a wide range of viruses in recent years. Ivermectin was originally identified as an inhibitor of the interaction between the human immunodeficiency virus-1 (HIV-1) integrase protein (IN) and the importin (IMP) α / β1 heterodimer responsible for IN nuclear import, and has since been shown to inhibit IN nuclear import and HIV-1 replication. Other effects of ivermectin have been reported, but ivermectin has been shown to inhibit nuclear import of host and viral proteins, including the simian virus SV40 large tumor antigen (T-ag) and dengue virus nonstructural protein 5 (DENV NS5). Ivermectin has been shown to limit infection by viruses such as DENV, Venezuelan equine encephalitis virus (VEEV), and influenza, and the mechanism is thought to be due to the dependence of RNA viruses on IMP α / β1 during infection.

[0005] Moxidectin is another example of a macrolide. Moxidectin is an FDA-approved drug for the treatment of onchocerciasis in humans. It is also used in animals as an antiparasitic drug to prevent, treat, or control infections and infestations by worms, ectoparasites, insects, mites, and nematodes. It is particularly useful for controlling parasites such as lice and worms in cattle and sheep. Moxidectin can be administered to livestock and other animals in a variety of ways, including as a topical medication or "dip," subcutaneous injection, or oral pill or tablet. Moxidectin has been shown to limit infection by viruses such as H9 influenza.

[0006] The nucleocapsid protein of many coronaviruses is localized in the nucleus of the host cell, and IMPα / β1 is thought to play a role in the nuclear import of this protein. However, the nucleocapsid protein of SARS-CoV (essentially identical to that of SARS-CoV-2) is almost entirely localized in the cytoplasm. In addition, the ORF6 protein of SARS-CoV binds IMPα / β1 and sequesters it in the cytoplasm, preventing infected host cells from mounting an antiviral response through the STAT1 transcription factor, which requires IMPα / β1 to enter the nucleus. Therefore, IMPα / β1 inhibitors such as ivermectin are expected to release IMPα / β1 from ORF6, but this would also inhibit the STAT1-induced antiviral response, thereby exacerbating infection.

[0007] Contrary to expected effects, the inventors have determined that ivermectin and moxidectin inhibit SARS-CoV-2 and therefore these macrolides can be used to treat and prevent infection by SARS-CoV-2 and all members of the Coronaviridae (coronaviruses). Summary of the invention

[0008] In a first aspect, the present invention provides a formulation comprising at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof for use as an inhibitor of a coronavirus in an individual.

[0009] In a second aspect, the present invention provides a method for inhibiting a coronavirus in an individual, the method comprising administering to the individual a therapeutically effective amount of at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof.

[0010] In a third aspect, the present invention provides a method of treating an individual infected with a coronavirus, the method comprising administering to the individual a therapeutically effective amount of at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof.

[0011] In a fourth aspect, the present invention provides a method for preventing coronavirus infection in an individual, the method comprising administering to the individual a therapeutically effective amount of at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof.

[0012] In a fifth aspect, the present invention provides a method for reducing the risk of an individual being infected with a coronavirus, the method comprising administering to the individual at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof.

[0013] In a sixth aspect, the present invention provides a method for preventing antibody-dependent enhancement (ADE) coronavirus infection in an individual, comprising administering to the individual a therapeutically effective amount of at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof.

[0014] In one embodiment of the second to sixth aspects, the level of detectable coronavirus in the individual is reduced by at least 50%. For example, the level of detectable coronavirus in the individual can be reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9%.

[0015] The levels of detectable coronavirus can be reduced 1, 2, 3 days or longer after administration of the macrolide.

[0016] In one embodiment, the at least one macrolide is administered at a dosage of about 0.1 mg / kg to about 1.0 mg / kg of the subject's body weight.

[0017] In a seventh aspect, the present invention provides the use of at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof in the preparation of a medicament for preventing or treating coronavirus infection.

[0018] In an eighth aspect, the present invention provides the use of at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof in the preparation of a medicament for reducing the risk of contracting a coronavirus infection.

[0019] In a ninth aspect, provided is the use of at least one macrolide or a salt, hydrate, solvate, tautomer or stereoisomer thereof in the preparation of a medicament for preventing ADE coronavirus infection in an individual.

[0020] The at least one macrolide may be an avermectin or a milbemycin. For example, the avermectin may be selected from abamectin, dimadectin, doramectin, emamectin, eprinomectin, ivermectin, latidectin, lepimectin and selamectin.

[0021] In one embodiment, the avermectin is ivermectin.

[0022] The milbemycin may be selected from milbemectin, milbemycin D, milbemycin oxime, moxidectin and nemadectin.

[0023] In one embodiment, the milbemycin is moxidectin.

[0024] In another embodiment, the avermectin is ivermectin and the milbemycin is moxidectin.

[0025] In one embodiment, the individual is a human.

[0026] The coronavirus can be coronavirus 229E, coronavirus NL63, coronavirus OC43, SARS-CoV, MERS-CoV, SARS-CoV-2 or a SARS-CoV-2 variant. Preferably, the coronavirus is SARS-CoV-2 or a SARS-CoV-2 variant.

[0027] definition

[0028] In this specification, unless the context clearly requires otherwise, the word "comprise" or variations thereof (such as "includes", "comprising") are understood to mean the inclusion of a stated element, integer or step, or a group of elements, integers or steps, but not the exclusion of any other elements, integers, steps, or groups of elements, integers and steps.

[0029] In this specification, the term "consisting of" means consisting of only.

[0030] The term "consisting essentially of" means including the elements, integers or steps, but may also include other elements, integers or steps that do not substantially change or contribute to the work of the present invention. Therefore, in the context of the present invention, a composition "consisting essentially of" a specific macrolide means that the specific macrolide is an active agent, and the composition does not contain other (unspecific) active agents with therapeutic effects. However, it will be understood by those skilled in the art that trace amounts of other compounds (e.g., other macrolides) may exist, for example, as impurity 2. The composition may include inert ingredients, such as excipients, diluents, etc., even if these ingredients are not clearly identified.

[0031] Any discussion of documents, acts, materials, devices, papers or the like included in this specification is intended solely to provide a context for the present invention. It is not an admission that some or all of these matters form part of the prior art for this patent or that they were common general knowledge in the field relevant to the present invention in Australia before the priority date of each claim of this application.

[0032] Unless the context requires otherwise or there is a specific provision to the contrary, an integer, step, or element herein referred to as a singular integer, step, or element expressly includes both the singular and the plural form of the referenced integer, step, or element.

[0033] In the context of this specification, the terms "a" and "an" are used to refer to one or more (ie, at least one) grammatical objects of the article. For example, reference to "an element" refers to one element or more than one element.

[0034] In the context of this specification, the term "about" means that the reference to a number or value should not be regarded as an absolute number or value, but includes numbers or values ​​that are higher or lower than the boundaries of the range in accordance with the understanding of a person skilled in the art, including typical error ranges or instrument limitations. That is, the use of the term "about" is understood to refer to a range or approximate value that a person skilled in the art considers to be equivalent to the value in the case of achieving the same function or result.

[0035] The term "treatment" as used herein refers to therapeutic therapy, prophylactic therapy, palliative therapy and preventive therapy. Therefore, in the context of the present invention, the term "treatment" includes treating, improving or alleviating the severity of coronavirus infection or one or more of its related symptoms.

[0036] The term "effective amount" or "therapeutically effective amount" refers to the amount of a macrolide that is sufficient to produce the desired therapeutic or pharmacological effect in a treated individual. These terms are synonymous and are intended to define the amount of each macrolide to achieve the goal of improving the severity of the disease and / or the morbidity of each macrolide alone, while preferably avoiding or minimizing adverse side effects, including those typically associated with other treatments. Those skilled in the art can determine the effective dose using information known in the art and conventional methods.

[0037] More specifically, the term "effective amount" or "therapeutically effective level" and similar terms refer to the amount of macrolide that (i) treats a specific disease, condition or disorder, (ii) alleviates, improves or eliminates one or more symptoms of a specific disease, condition or disorder, (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition or disorder described herein, (iv) prevents or delays the progression of a specific disease, condition or disorder, or (v) reverses to some extent the damage caused before treatment. Reversal is not necessarily absolute. The amount administered may be higher than the amount required to achieve a therapeutic effect in vivo, but the absorption, distribution, metabolism and excretion (ADME) of the macrolide, the route of administration and the frequency of administration should be taken into account.

[0038] In alternative embodiments, the severity of symptoms associated with coronavirus infection can be reduced by administering a therapeutically effective amount of a macrolide to the individual. Reducing the severity of symptoms means that one or more symptoms are alleviated, improved or eliminated, or the occurrence of one or more symptoms is prevented or delayed.

[0039] "Pharmaceutical carriers, diluents or excipients" include, but are not limited to, any physiologically buffered (i.e., pH value of about 7.0 to 7.4) medium, which contains suitable water-soluble organic carriers, conventional solvents, dispersion media, fillers, solid carriers, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Suitable water-soluble organic carriers include, but are not limited to, saline, glucose, corn oil, dimethyl sulfoxide and gelatin capsules. Other conventional additives include lactose, mannitol, corn starch, potato starch, binders (e.g., microcrystalline cellulose), cellulose derivatives (e.g., hydroxypropyl methylcellulose), gum arabic, gelatin, pulverizers (e.g., sodium carboxymethyl cellulose) and lubricants (e.g., talc or magnesium stearate).

[0040] "Subject" includes any human or non-human mammal. Thus, in addition to being useful for human therapy, the compounds of the invention may also be used for veterinary therapy of mammals, including companion animals and farm animals, such as, but not limited to, dogs, cats, horses, cows, sheep, camels, chickens, and pigs. In a preferred embodiment, the subject is a human.

[0041] In the context of this specification, the term "administering" and variations thereof include contacting, applying, delivering or providing the compound or composition of the present invention to the individual by any appropriate means.

[0042] As used herein, the term "inhibition" in relation to coronavirus refers to a reduction in the binding of coronavirus-encoded proteins to cellular targets, a reduction in disease symptoms or severity, or a reduction in viral load.

[0043] Those skilled in the art will appreciate that the techniques described herein may be subject to variations and modifications other than those specifically described. It should be understood that the present invention includes all such variations and modifications. For the avoidance of doubt, the present invention also includes all steps, features and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations of any two or more of said steps, features or compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Relative amounts of viral RNA associated with (A) Vero / hSLAM cells infected with SARS-CoV-2 isolates or (B) in cell supernatants before and 1, 2, and 3 days after treatment with 5 μM ivermectin are shown. (C, D) Based on the detection of the E gene, a reduction of >5000 viral RNA was observed at 48 hours in both the supernatant and the cell pellets of samples treated with 5 μM ivermectin. (E, F) Almost identical results were obtained using primers specific for the SARS-CoV-2 RdRp gene.

[0045] Figure 2 Ivermectin and moxidectin strongly inhibit the replication of SARS-CoV-2 clinical isolate Australia / VIC01 / 2020. (A) Ivermectin and moxidectin reduce viral RNA in culture supernatants of Vero / hSLAM cells infected with SARS-CoV-2 clinical isolate Australia / VIC01 / 2020. (BD) Ivermectin and moxidectin reduce viral RNA in culture supernatants of Vero / hSLAM cells infected with SARS-CoV-2 clinical isolate Australia / VIC01 / 2020 (before addition of vehicle (DMSO), ivermectin (filled squares), or moxidectin (open squares)). Supernatants were analyzed for SARS-CoV-2 E gene at 24 hours (B), 48 hours (C), or 72 hours (D).

[0046] Figure 3 Indicated that ivermectin reduces the viral load in CALU-3 (human lung) cells infected with the SARS-CoV-2 clinical isolate Australia / VIC01 / 2020.

[0047] Figure 4 Indicates that multiple doses of ivermectin or moxidectin treatment enhance SARS-CoV-2 antiviral activity compared to single treatment. Vero / hSLAM (A / C) or CALU-3 (B / D) cells were infected with SARS-CoV-2 clinical isolate Australia / VIC01 / 2020 before addition of indicated concentrations of vehicle (DMSO), ivermectin (A / B), or moxidectin (C / D).

[0048] Figure 5 Indicates that ivermectin and moxidectin have preventive antiviral activity against SARS-CoV-2 in Vero / hSLAM cells. Vero / hSLAM cells were pretreated with the indicated concentrations of DMSO, ivermectin or moxidectin for 2 hours before infection with SARS-CoV-2 (MOI = 0.01) for 2 hours in the presence of DMSO or drugs.

[0049] Figure 6 Ivermectin and moxidectin are potent antiviral inhibitors of seasonal coronavirus 229E. Huh-7 cells were infected with 229E before addition of indicated concentrations of vehicle (DMSO), ivermectin (A), or moxidectin (B). Three-parameter dose-response curves were fitted using GraphPad prism to determine IC 50 (C) To assess the prophylactic potential, Huh-7 cells were pretreated with the indicated concentrations of DMSO and ivermectin before infection with 229E in the presence of DMSO or ivermectin. Samples were washed after adsorption and incubated in fresh medium containing DMSO or ivermectin for 48 h before quantification of viral load. DETAILED DESCRIPTION

[0050] The inventors have determined that the macrolides ivermectin and moxidectin can be used to inhibit SARS-CoV-2. In particular, ivermectin and moxidectin reduce the viral load of SARS-CoV-2 and can therefore be used to treat, reduce or prevent infection.

[0051] Therefore, the present invention provides at least one macrolide for inhibiting the use of coronavirus in an individual. The macrolide can be used to treat and / or prevent coronavirus infection, which requires one or more of the following conditions:

[0052] i) Reducing the viral load in individuals infected with coronaviruses, including but not limited to SARS, MERS, SARS-CoV-2, and in particular individuals infected with any isolate or strain of SARS-CoV-2.

[0053] ii) reducing or ameliorating an individual’s risk of infection with a coronavirus, including but not limited to SARS, MERS, SARS-CoV-2, and in particular any isolate or strain of SARS-CoV-2.

[0054] iii) Alleviate symptoms of coronavirus infection in individuals (infected with or suspected of being infected with a coronavirus, including but not limited to SARS, MERS, SARS-CoV-2, and in particular any isolate or strain of SARS-CoV-2).

[0055] iv) Reducing or ameliorating the risk of developing ADE coronavirus in individuals who have been infected or suspected of having been infected with coronavirus.

[0056] Alternatively, the patient may also receive other treatments so that a macrolide or its composition is administered in adjuvant therapy. Other therapies may be therapies for treating concomitant symptoms, including but not limited to pain relief, such as aspirin, acetaminophen, codeine and non-steroidal anti-inflammatory drugs (NSAIDS). In this regard, the invention provides a method for treating coronavirus infection, or reduces the method for the severity of symptoms of a patient suffering from or suspected of suffering from coronavirus infection, comprising administering at least one macrolide and another therapeutic drug to an individual in need thereof in an effective amount, wherein the macrolide administered reaches a therapeutically effective level in an individual.

[0057] The methods and uses described herein comprise administering at least one macrolide or a formulation comprising at least one macrolide to an individual exposed to, infected with, or suspected of being exposed to a coronavirus.

[0058] Administration of a macrolide to the individual can inhibit coronaviruses in the individual. This inhibition has many beneficial effects, such as reducing viral load, preventing or reducing the interaction of coronaviruses with cellular targets (such as IMPα / β1), and thereby preventing or reducing coronavirus infection of cells. These viral inhibition effects help prevent or treat (e.g., reduce the severity of) coronavirus infection.

[0059] As described above, the inhibition of coronavirus by macrolide can prevent or reduce the interaction between coronavirus and cellular targets (such as IMPα / β1), thereby reducing or preventing coronavirus infection of cells. Therefore, administering macrolide to the individual can reduce the risk of coronavirus infection.

[0060] Macrolides may be administered as a preventive measure, for example, to healthcare workers, caregivers, or people at risk of exposure to the coronavirus.

[0061] Although infection with one coronavirus serotype or strain may be protective against reinfection with the same serotype or strain, infection with other serotypes or strains may not protect an individual. Continuous infection with different serotypes is believed to lead to antibody-dependent enhancement (ADE) of infection. Therefore, in some embodiments of the present invention, the individual has been infected with a coronavirus, such as a different coronavirus serotype or strain. These individuals are at a higher risk of infection with an ADE coronavirus. The methods of the present invention are also suitable for treating, preventing or reducing the risk of infection with an ADE coronavirus.

[0062] ADE occurs when circulating antibodies from a previous infection with a different coronavirus serotype lead to an increase in viral load, caused by cross-reactive but non-neutralizing antibodies. ADE is known to occur with SARS-CoV and MERS-CoV. ADE also leads to increased expression of genes encoding the cytokines interleukin (IL) 8 and tumor necrosis factor (TNF) alpha, for example, which has been associated with the development of infection with more severe symptoms.

[0063] Therefore, in a further embodiment of the present invention, the present invention provides a method for preventing or reducing the severity of ADE coronavirus infection, which comprises administering an effective amount of a macrolide.

[0064] The present invention also provides the use of an effective amount of a macrolide in the preparation of a medicament for preventing ADE coronavirus infection in an individual in need thereof.

[0065] In some embodiments, at least one macrolide is administered to an individual infected with a coronavirus, and the amount of detectable coronavirus (cell-associated or free virus) is reduced by at least 50%. For example, the amount of the detectable coronavirus can be reduced by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9%.

[0066] Detectable coronavirus levels may decrease over 1, 2, 3 days, or longer.

[0067] IC of macrolides 50 is the response (reduction in detectable coronavirus) to a reduction in macrolide concentration by half. In some embodiments, the macrolide IC 50 Less than about 10 μM. For example, IC 50 It may be less than 10 μM, 9 μM, 8 μM, 7 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, or less than about 1 μM.

[0068] As a preparation containing a pharmaceutically effective amount of a macrolide, the macrolide can be co-administered with one or more pharmaceutically acceptable excipients, including carriers, vehicles and diluents. The term "excipient" herein refers to any substance used as a diluent, adjuvant or carrier, which is not a therapeutic agent itself, and is used to deliver a therapeutic agent to an individual, or is added to a pharmaceutical composition to improve its handling or storage properties, or to allow or promote the formation of a solid dosage form, such as a tablet, capsule, or a solution or suspension for oral, parenteral, intradermal, subcutaneous or external use. By way of example and not limitation, excipients may include diluents, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, glidants, stabilizers, and added substances, spices, pigments, aromatics, and added substances for masking or counteracting unpleasant tastes or odors, and substances for improving the appearance of the composition. Acceptable excipients include, but are not limited to, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, and cellulosic materials such as cellulose alkanoates and cellulose alkyl esters, low melting waxes, cocoa butter or cocoa powder, polymers such as polyvinyl pyrrolidone, polyvinyl alcohol, and polyethylene glycol, and other pharmaceutically acceptable materials. Examples of excipients and their use are described in Pharmaceutical Sciences, 20th Edition, Lippincott Williams & Wilkins, 2000. The choice of excipient will depend largely on various factors, such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0069] The macrolide and pharmaceutical composition of the present invention can be formulated for oral, injection, rectal, nasal, parenteral, subcutaneous, intravenous, external or intramuscular delivery. Non-limiting examples of specific formulation types include tablets, capsules, caplets, powders, granules, injections, ampoules, vials, ready-to-use solutions or suspensions, freeze-dried materials, creams, lotions, ointments, drops, suppositories and implants. Solid preparations such as tablets or capsules can contain any number of suitable pharmaceutically acceptable excipients or carriers described above. The compounds of the present invention can also be formulated for sustained delivery.

[0070] In one or more preferred embodiments, tablets and capsules for oral administration can be in unit dose presentation form and can contain conventional excipients, such as binders, such as gum arabic, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone; fillers, such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tableting lubricants, such as magnesium stearate, talc, polyethylene glycol or silicon dioxide; disintegrants, such as potato starch; or acceptable wetting agents, such as sodium lauryl sulfate. Tablets can be coated according to well-known methods in normal pharmaceutical practice.

[0071] Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be in the form of dry products to be reconstituted with water or other suitable vehicles before use. Such liquid preparations may contain conventional additives, such as suspending agents, such as sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, emulsifiers, such as lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), such as almond oil, oily esters, such as glycerol, propylene glycol or ethanol; preservatives, such as methyl or propyl parabens or sorbic acid; if desired, conventional flavoring or coloring agents may also be used.

[0072] For parenteral administration, including intravenous, intramuscular, subcutaneous or intraperitoneal administration, liquid unit dosage forms can be prepared by combining the compound and a sterile carrier, which is generally a sterile aqueous solution that is preferably isotonic with the blood of the recipient. Depending on the carrier and concentration used, the compound can be suspended or dissolved in a carrier or other suitable solvent. When preparing the solution, the compound can be dissolved in water for injection and filtered and sterilized before filling into a suitable vial or ampoule and sealing. Advantageously, agents such as local anesthetics, preservatives and buffers can be dissolved in the carrier. In order to improve stability, the composition can be frozen after being loaded into the vial and water can be removed under vacuum. Then, the dried lyophilized powder can be sealed in a vial, and a vial of water for injection or other suitable liquid can be provided to reconstitute the liquid before use. Parenteral suspensions are prepared in essentially the same manner, except that the compound is suspended in the carrier instead of being dissolved and cannot be sterilized by filtration. The compound can be sterilized by exposure to ethylene oxide before being suspended in a sterile carrier. A surfactant or wetting agent may be included in the composition to promote uniform distribution of the compound.

[0073] The lyophilized formulation is preferably reconstituted with a solution consisting essentially of water (e.g., USP WFI or water for injection) or bacteriostatic water (e.g., USP WF1 containing 0.9% benzyl alcohol). Alternatively, a solution comprising a buffer and / or excipients and / or one or more pharmaceutically acceptable carriers may be used. The liquid to be freeze-dried or lyophilized preferably contains all the ingredients required in the final reconstituted liquid formulation.

[0074] In some embodiments, the compositions of the invention are formulated as sustained-release formulations or depots. Exemplary sustained-release formulations or depots include microspheres; matrices; emulsions; lipid-based; polymer-based; nanoparticles; micelles; nanovesicles, such as liposomes, noisomes, transfersomes, disproportionate bodies, drugsomes, emulsomes or spanlastics, particularly liposomes; microparticles; nanoparticles, such as nanocapsules or nanospheres composed of, for example, lipids, proteins, natural or synthetic polymers (e.g., albumin, sodium alginate, chitosan, PLGA, PLA and / or polycaprolactone); or in situ gels, such as in situ hydrogel drug delivery systems.

[0075] The dosage regimen for administering a therapeutically effective amount of a macrolide and using the compounds and / or pharmaceutical compositions of the present invention for treating a disease depends on a variety of factors, including the age, weight, sex and medical condition of the individual, the severity of the disease, the route and frequency of administration, the specific compound used, and the pharmacokinetic properties (e.g., absorption, distribution, metabolism, excretion) of the individual being treated, and therefore may vary greatly. Such treatment may be performed as often as needed and for a period of time deemed necessary by the treating physician. Those skilled in the art will appreciate that it may be necessary to optimize the dosage regimen or therapeutically effective amount of the compound to be administered for each individual. For example, a pharmaceutical composition may contain an active ingredient in the range of about 0.1 mg to 2000 mg, for example, typically in the range of about 0.5 mg to 500 mg, about 1 mg to 200 mg, about 1 mg to 100 mg, and more typically between about 1 mg and 50 mg.

[0076] Depending on the route of administration and the frequency of administration, a dosage of about 0.01 mg / kg to 100 mg / kg body weight, or about 0.1 mg / kg to about 50 mg / kg body weight, or about 0.1 mg / kg to about 25 mg / kg body weight, or about 0.1 mg / kg to about 10 mg / kg body weight, or about 0.1 mg / kg to about 1 mg / kg body weight may be appropriate.

[0077] In one embodiment, the dosage of the macrolide is about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.8 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or about 1 mg / kg of body weight.

[0078] In some embodiments, the amount of macrolide administered is sufficient to achieve a plasma concentration of about 0.1 μM to about 100 μM, 0.1 μM to about 80 μM, 0.1 μM to about 60 μM, 0.1 μM to about 40 μM, 0.1 μM to about 20 μM, 0.1 μM to about 10 μM, or 0.1 μM to about 5 μM.

[0079] In some embodiments, the amount of macrolide administered is sufficient to achieve a plasma concentration of about 1 ng / ml to about 500 ng / ml, about 1 ng / ml to about 400 ng / ml, about 1 ng / ml to about 300 ng / ml, about 1 ng / ml to about 200 ng / ml, about 1 ng / ml to about 175 ng / ml, about 5 ng / ml to about 150 ng / ml, about 5 ng / ml to about 125 ng / ml, about 5 ng / ml to about 100 ng / ml, about 5 ng / ml to about 75 ng / ml, or about 5 ng / ml to about 50 ng / ml.

[0080] The compounds of the invention may be administered together with a pharmaceutical carrier, diluent or excipient as described above. Alternatively, or in addition, the compounds may be administered in combination with other drugs (e.g., other antiviral drugs and / or drugs suitable for relieving symptoms, such as analgesics or expectorants).

[0081] In defining the use of a compound of the invention and one or more other pharmaceutical agents, the term "combination therapy" or "adjunctive therapy" is intended to include administration of each agent in a sequential manner in a regimen that will provide the beneficial effects of the pharmaceutical combination, and is also intended to include co-administration of the agents in a substantially simultaneous manner, such as administration of the agents in a single formulation having a fixed ratio of the active agents or administration of the agents in multiple separate formulations of each agent.

[0082] According to various embodiments, one or more macrolides can be formulated or administered in combination with one or more other therapeutic agents. Therefore, according to various embodiments of the present invention, one or more macrolides can be included in a combined treatment regimen with other known treatments or therapeutic agents and / or adjuvants or prophylactic agents.

[0083] Many drugs are available for commercial use, clinical evaluation (such as amlodipine and losartan), and preclinical development that could be selected to treat coronavirus infection, including antiviral drugs such as protease inhibitors, helicase inhibitors, and entry inhibitors. Other antiviral drugs include oseltamivir Zanamivir Ribavirin, remdesivir, penciclovir, faviparvir, nafamostat, nitazoxanide, camostat mesylate, interferon alpha (such as interferon alpha B2), ritonavir, lopinavir, ASC09, azvudine, baloxavirmarboxil, daerunavir, cobistat, and chloroquine.

[0084] Other agents suitable for combination therapy are immunotherapies including convalescent plasma or monoclonal antibodies such as bamlanivimab, etesevimab, casirivimab, and imdevimab (anti-SARS-CoV-2 spike protein monoclonal antibody), or a combination thereof.

[0085] Those skilled in the art will recognize other suitable agents that can be used in combination therapy (e.g., nonsteroidal anti-inflammatory drugs, acetaminophen, codeine, corticosteroids (e.g., dexamethasone), interferons, etc.). For example, suitable drugs are listed in the Merck Index, An Encyclopaedia of Chemicals, Drugs and Biologicals, 12th Ed., 1996 and subsequent versions, the entire contents of which are incorporated herein by reference.

[0086] The combination regimen may involve, in each case, the administration of the active agents together, sequentially or at intervals, as the case may be. The combination of active agents including the compounds of the invention may be synergistic.

[0087] Co-administration of the macrolide may be effected by administering the same unit dose of the macrolide and the other active agent, or by administering the macrolide and one or more other active agents in separate and discrete unit doses simultaneously, or at similar times, or at different times, according to a dosing regimen or schedule. Sequential administration may be in any order desired, and a continued physiological effect of the first or initial compound may be desired while a second or subsequent compound is administered, particularly where a cumulative or synergistic effect is desired.

[0088] Co-administration of the macrolide may be effected by administering the compound and another active agent in the same unit dose, or by administering the compound and one or more other active agents in separate and discrete unit doses simultaneously, or at similar times, or at different times, according to a dosing regimen or schedule. Sequential administration may be in any order desired, and a continued physiological effect of the first or initial compound may be desired prior to administering the second or subsequent compound, particularly where a cumulative or synergistic effect is desired.

[0089] Coronavirus

[0090] As described herein, at least one macrolide can be used to inhibit coronavirus in an individual. In addition, the macrolide can be used to treat and / or prevent coronavirus infection.

[0091] The methods and uses are applicable to all members of the Coronaviridae (coronaviruses).

[0092] For example, the coronavirus can be an alphacoronavirus, such as coronavirus 229E, coronavirus NL63, porcine epidemic diarrhea coronavirus (PEDV), or porcine acute diarrhea syndrome coronavirus (SADS-CoV).

[0093] In one embodiment, the alphacoronavirus is coronavirus 229E or coronavirus NL63.

[0094] In some embodiments, the coronavirus can be a beta coronavirus, such as a mouse coronavirus. In other embodiments, the coronavirus is a beta coronavirus 1, such as coronavirus HKU1, mouse coronavirus, SARS (severe acute respiratory syndrome)-coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), MERS (Middle East respiratory syndrome) coronavirus, Middle East respiratory syndrome-related coronavirus, or coronavirus OC43.

[0095] In some embodiments, the coronavirus is coronavirus OC43, SARS-CoV, MERS-CoV, or SARS-CoV-2. In one embodiment, the coronavirus is SARS-CoV-2.

[0096] In some embodiments, reference to SARS-CoV-2 includes any reference to a SARS-CoV-2 isolate, SARS-CoV / 2 variant, SARS-CoV-2 variant of interest (VOI), or SARS-CoV-2 variant of concern (VOC).

[0097] Thus, in some embodiments, references to SARS-CoV-2 include references to known variants, such as the “UK variant” (called 20I / 501Y.V1, VOC 202012 / 01, or B.1.1.7), the “South African variant” (called 20H / 501Y.V2 or B.1.351), and the “Brazilian variant” (called P.1).

[0098] The "UK variant" is characterized by the N501Y mutation in the spike protein receptor binding domain (RBD) at position 501. The variant also has several other mutations, including P681H near the furin cleavage site and a 69 / 70 deletion that may lead to conformational changes in the spike protein.

[0099] The "South African variant" is characterized by multiple mutations in the spike protein, including K417N, E484K, and N501Y. The variant does not contain the 69 / 70 deletion.

[0100] The "Brazilian variant" is characterized by three mutations in the receptor binding domain of the spike protein: K417T, E484K and N501Y.

[0101] Other variants include those with a D614G mutation in the spike protein and the "cluster 5" variant, also known as the "ΔFVI-spike" variant discovered in Denmark, which is characterized by mutations including 69-70deltaHV (loss of histidine and valine residues at positions 69 and 70 of the spike protein), Y453F, I692V and S1147L.

[0102] The SARS-CoV-2 genomes of more than 150 isolates have been sequenced. One reference sequence is GenBank NC 045512 (Wang, C. et al. (2020) "The Establishment Of Reference Sequence for SARS-CoV-2 and variation analysis," J. Med. Virol. 2020 Jun; 92(6): 667-674 (published online on March 13, 2020). The sequences of multiple virus isolates (MN988668 and NC 045512, as well as MN938384.1, MN975262.1, MM985325.1, MN988713.1, MN994467.1, MN994468.1 and MN997409.1) were compared and found to have greater than 95% identity. Therefore, the methods described herein are applicable to SARS-CoV-2 variants having 95%, 96%, 97%, 98% or 99% sequence identity with SARS-CoV-2 isolates.

[0103] In other embodiments, the coronavirus is a gamma coronavirus such as infectious bronchitis virus, or a delta coronavirus such as spherical coronavirus HKU11 or porcine coronavirus.

[0104] Macrolide

[0105] Any macrolide or combination of macrolides is suitable for use in the methods and formulations described herein. Suitable macrolides are known and can be readily obtained commercially or by synthetic techniques known in the art.

[0106] Typically, the macrolide is an avermectin, a milbemycin, or a combination thereof. Avermectin and milbemycins contain a common macrolide (ML) ring. The main structural difference is that avermectin has a sugar group at C13 of the macrocycle, while the milbemycins are protonated at C13. Moxidectin (a milbemycin) has other differences, including a methyl oxime at C23.

[0107] Avermectin is a group of lactone compounds with five rings and 16 members. Abamectin is a 1a (>90%) and avermectin B 1b The compounds of the A-series are methoxylated at the 5-position, while the compounds of the B-series have an underivatized hydroxyl group at that position. The 1-subset compounds have an olefinic bond between C22 and C23; the 2-subset compounds have a hydroxyl group at the 23-position due to hydration of the double bond. They are believed to have very similar biological activities and toxicological properties. Among the other macrolides, the avermectins are generally characterized by the presence of a sugar substituent at the 13-position and a sec-butyl or isopropyl group at the 25-position.

[0108] Ivermectin is a chemically reduced 22,23-dihydro derivative of abamectin and is a derivative of 22,23-dihydroavermectin B. 1a (>90%) and 22,23-dihydroavermectin B 1b (<10%), which differs from abamectin in that it has a methylene component at position 26.

[0109] Eprinomectin is an aminoavermectin derived from avermectin B1 with a modified terminal oleandrose moiety, called 400-epiacetylamino-400-deoxy-avermectin B1. Doramectin is prepared by mutational biosynthesis and has a closer structural similarity to avermectin than to ivermectin. It has a different substituent at position 25 and no dihydro modifications at positions 22–23. It differs from ivermectin in that it has a cyclohexyl group at position C25 of the avermectin ring. Selamectin is a semisynthetic monosaccharide oxime derivative of doramectin. In terms of chemical structure and its monosaccharides, it is an intermediate between the disaccharides avermectins and milbemycins.

[0110] Milbemycin oxime is a natural compound isolated from Streptomyces hygroscopicus that contains the 16-membered macrolide structure of milbemycin. Nematicidin is naturally occurring and can be extracted from milbemycin. Moxidectin can be chemically derived from nematicidin by adding the oxime moiety at C-23.

[0111] Milbemycins are structurally similar to avermectins, but are not glycosylated and lack the bisoleandrosyl moiety at position 13. Milbemycins differ from avermectins not only by the lack of a glycosyl group at position 13, but they also differ from avermectin aglycones in that they are protonated at this position, whereas avermectin aglycones are hydroxylated at this position. They also have an ethyl or methyl group at position 25. Milbemycins can be distinguished by the substituents at positions 5 and 25. Moxidectin has a substituted olefin side chain at position 25 and a methyloxime group at position 23, both of which are unique to the drug and not found in other commercial milbemycins or avermectins.

[0112] Suitable avermectins are well known in the art, for example, avermectin-ivermectin and abamectin can be found in "Ivermectin and Abamectin", co-authored by MH Fischer and H. Mrozik, William C. Campbell, published by Springer Verlag in 1989, or Albers-Schonberg et al. (1981), "Avermectins Structure Determination", J. Am. Chem. Soc., 103, 4216-4221. For doramectin, "Veterinary Parasitology", vol. 49, No. 1, July 1993, 5-15 can be used as a reference. For milbemycins, please refer to Davies HG et al., 1986, “Avermectins and Milbemycins”, Nat. Prod. Rep., 3, 87-121, Mrozik H. et al., 1983, Synthesis of Milbemycins from Avermectins, Tetrahedron Lett., 24, 5333-5336.

[0113] Macrolides are natural products or semi-synthetic derivatives thereof, and are well known in the art. For example, in addition to "International Nonproprietary Names for Pharmaceutical Substances (INN)", WHO Drug Information, vol. 17, no. 4, pp. 263-286, (2003), suitable macrolides can also be found by references cited in "The Merck Index" 12. sup. th ed., S. Budavari, Ed., Merck & Co., Inc. Whitehouse Station, NJ (1996).

[0114] In some embodiments, the avermectin is abamectin, dimadectin, doramectin, emamectin, eprinomectin, ivermectin, latidectin, lepimectin, selamectin, or a combination thereof. In one embodiment, the avermectin is ivermectin.

[0115] In some embodiments, the milbemycin is milbemycin, milbemycin D, milbemycin oxime, moxidectin, or nemadectin. In one embodiment, the milbemycin is moxidectin.

[0116] In one embodiment, the avermectin is a combination of ivermectin and the milbemycin is moxidectin.

[0117] In one embodiment, at least one macrolide is a combination of at least one avermectin and at least one milbemycin. For example, the avermectin is selected from any one or more of abamectin, demadectin, doramectin, avermectin, eprinomectin, ivermectin, latidectin, lepimectin and selamectin; the milbemycin is selected from any one or more of milbemycin, milbemycin D, milbemycin oxime, moxidectin and nemadectin.

[0118] In one embodiment, the at least one macrolide is a combination of ivermectin and moxidectin.

[0119] In addition, if a macrolide is referred to by name, it is intended to encompass both solvated and unsolvated forms of the compound, where applicable. Thus, each macrolide (e.g., ivermectin or moxidectin) includes hydrated or solvated forms, as well as non-hydrated and non-solvated forms. The term "solvate" as used herein describes a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules (e.g., ethanol). When the solvent is water, the term "hydrate" is used.

[0120] The term "pharmaceutically acceptable salt" refers to a salt that is suitable for contact with human and animal tissues within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reaction, etc., and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. For a review of suitable salts, please refer to Stahl and Wermuth's Handbook of Pharmaceutical Salts: Properties, Selection (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art. Salts can be prepared in situ during the final separation and purification of the compounds of the present invention, or prepared separately by reacting a free base with a suitable organic acid. Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention can be prepared from inorganic acids or organic acids. Examples of such inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid and phosphoric acid. Suitable organic acids can be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic acids and sulfonic acids, exemplified by formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, fumaric, maleic, pyruvic, alkylsulfonic, arylsulfonic, aspartic, glutamic, benzoic, anthranilic, mesylic, methanesulfonic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic, pantothenic, sulfanilic, cyclamic, stearic, algenic, β-hydroxybutyric, galactaric and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include metal salts made from lithium, sodium, potassium, magnesium, calcium, aluminum and zinc, and organic salts made from organic bases such as choline, diethanolamine, morpholine, etc. Alternatively, suitable pharmaceutically acceptable base addition salts of the compounds of the invention include organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamines, meglumine (N-methylglucamine), procaine, ammonium salts, quaternary ammonium salts (e.g., tetramethylammonium salts), amino acid addition salts, such as salts containing glycine and arginine. In the case of solid compounds, those skilled in the art will appreciate that the compounds, agents and salts of the invention may exist in different crystalline or polymorphic forms, all of which are intended to fall within the scope of the invention and the specified macrolides.

[0121] The term "stereoisomer" as used herein refers to any two or more isomers having the same molecular structure and differing only in the three-dimensional arrangement of the atomic groups in space. Stereoisomers may be non-stereoisomers or enantiomers. It should be recognized that the compounds described herein may have asymmetric centers and therefore can exist in more than one stereoisomeric form. Therefore, the present invention also relates to compounds in substantially pure isomeric forms at one or more asymmetric centers, such as greater than about 90% ee (enantiomeric excess), such as about 95% or 97% ee or greater than 99% ee, and mixtures thereof, including racemic mixtures. Such isomers may be naturally occurring or prepared by asymmetric synthesis (e.g., using chiral intermediates) or chiral resolution.

[0122] In certain embodiments, the macrolide can be a tautomer. As used herein, the term "tautomer" is a kind of isomer, which includes two or more interconvertible compounds produced by at least one formal migration of hydrogen atoms and at least one valence change (e.g., single bond to double bond, triple bond to single bond, and vice versa). "Tautomerism" includes transfer or proton shift tautomerism, which is considered to be a subset of acid-base chemistry. "Proton shift tautomerism" or "proton shift tautomerization" involves proton migration and is accompanied by bond order changes. The exact ratio of tautomers depends on several factors, including temperature, solvent and pH. If tautomerism is possible (e.g., in solution), the chemical equilibrium of tautomers can be reached. Tautomerization (i.e., providing a reaction of tautomerism) can be catalyzed by acid or base, or it can occur in the absence of external reagents or presence.

[0123] It will be apparent to those skilled in the art that, unless otherwise specifically stated, reference generally to a "macrolide" or to a macrolide by name (e.g., ivermectin or moxidectin) refers to the compound and salts, hydrates, solvates, tautomers or stereoisomers thereof.

[0124] In order that the present invention may be more clearly understood, preferred embodiments are described with reference to the following examples and drawings.

[0125] Example

[0126] Example 1: Cell culture, viral infection and drug treatment

[0127] Vero / hSLAM cells were maintained in Earle's Minimum Essential Medium (EMEM) containing 7% fetal bovine serum (FBS) (Bovogen Biologicals, Keilor East, AUS), 2mM L-glutamine, 1mM sodium pyruvate, 1500mg / L sodium bicarbonate, 15mM HEPES, and 0.4mg / ml geneticin at 37°C, 5% CO2. Cells were seeded into 12-well tissue culture plates at an MOI of 0.1 (calculated per maintenance medium, but containing only 2% fetal bovine serum) for 2 hours 24 hours before infection with SARS-CoV-2 (Australia / VIC01 / 2020 isolate). The medium containing the inoculum was removed and replaced with 1mL of fresh medium (2% FBS) containing the indicated concentration of ivermectin or DMSO alone and incubated for 0-3 days as indicated. At appropriate time points, cell supernatants were collected and spun at 6000 g for 10 minutes to remove debris and the supernatant was transferred to a fresh collection tube. Cell monolayers were collected by scraping and resuspended in 1 mL of fresh medium (2% FBS). Toxicity controls were set up in parallel for each experiment performed on uninfected cells.

[0128] Example 2: Preparation of SARS-CoV-2 cDNA

[0129] RNA was extracted from 200 μL aliquots of supernatant or cell suspension using the QIAamp 96 Virus QIAcube HT kit (Qiagen, Hilden, Germany) and eluted with 60 μL. Reverse transcription was performed using the BioLine SensiFAST cDNA kit (Bioline, London, United Kingdom) with a total reaction mixture (20 μL) containing 10 μL RNA extract, 4 μL 5x TransAmp buffer, 1 μL reverse transcriptase, and 5 μL nuclease-free water. The reaction was incubated at 25°C for 10 min, 42°C for 15 min, and 85°C for 5 min.

[0130] Example 3: Detection of SARS-CoV-2 using TaqMan real-time RT-PCR assay.

[0131] The TaqMan RT-PCR assay used 2.5 μl of cDNA, 10 μl of Primer Design PrecisonPLUS qPCR Master Mix 1 μM forward (5′-AAA TTC TAT GGT TGG CAC AAC ATG TT-3′), 1 μM reverse (5′-TAG GCA TAG CTC TRT CAC AYT T-3′) primer targeting the BetaCoV RdRp (RNA-dependent RNA polymerase) gene or forward (5′-ACAGGT ACG TTA ATA GTT AAT AGC GT-3′) primer and 0.2 μM probe (5′-FAM-TGG GTT GGG ATTATC-MGBNFQ-3′), 1 μM reverse (5′-ATA TTG CAG CAG TAC GCA CAC A-3′) primer targeting the BetaCoV E gene and 0.2 μM probe (5′-FAM-ACA CTA GCC ATC CTT ACT GCG CTT CG-286NFQ-3'). Real-time RT-PCR assays were performed on an Applied Biosystems ABI 7500Fast Real-time PCR machine (Applied biosystem, FosterCity, CA, USA) with cycling conditions of 95°C for 2 min, 95°C for 5 s, and 60°C for 24 s. SARS-CoV-2 cDNA (Ct ~ 28) was used as a positive control. The calculated Ct values ​​were converted to the fold reduction of the treated samples compared with the control group using the ΔCt method (fold change of viral RNA = 2^ΔCt) and expressed as a percentage of the DMSO sample alone. IC was fitted using the 3-parameter dose-response curve in GraphPad Prism 50 value.

[0132] Example 3: Effects of Ivermectin and Moxidectin

[0133] To test the antiviral activity of ivermectin against SARS-CoV-2, Vero / hSLAM cells were infected with SARS-CoV-2 isolate Australia / VIC01 / 2020 at an MOI of 0.1 for 2 h, followed by the addition of 5 μM ivermectin. Supernatants and cell pellets were collected on days 0-3 and analyzed for SARS-CoV-2 RNA replication by RT-PCR ( Figure 1A / B). At 24 hours, there was a 93% reduction in viral RNA (indicating released viral particles) in the supernatant of samples treated with ivermectin compared to vehicle DMSO. Similarly, a 99.8% reduction in cell-associated viral RNA (indicating unreleased and unpackaged viral particles) was observed for ivermectin treatment. At 48 hours, this effect of ivermectin treatment increased to a 99.98 / 99.99% reduction in viral RNA compared to control samples, indicating that ivermectin treatment resulted in an effective loss of almost all viral material within 48 hours. Consistent with this idea, no further reduction in viral RNA was observed within 72 hours. No toxicity of ivermectin was observed in the sample wells or in the individual parallel drug samples at any time point tested.

[0134] To further determine the effectiveness of ivermectin, cells infected with SARS-CoV-2 were subjected to a series of ivermectin dilutions at 2 h post-infection, and supernatants and cell pellets were collected at 48 h for RT-PCR ( Figure 1 C / D). As described above, a reduction of >5000 in viral RNA was observed in the supernatants and cell pellets of samples treated with 5 μM ivermectin at 48 hours, equivalent to a 99.98% reduction in viral RNA in these samples. Again, no toxicity was observed at any ivermectin concentration tested. IC 50 Determined to be 2.8 μM ( Figure 1 C) and 2.4 μM ( Figure 1 D). Emphasize that the assay does specifically detect SARS-CoV-2 using the viral RdRp gene ( Figure 1 RT-PCR experiments were repeated using primers specific for the E / F gene instead of the E gene (described above), and almost identical results were observed for both released (supernatant) and cell-associated viruses. 50 Determined to be 2.5 μM ( Figure 1 E) and 2.2 μM ( Figure 1 F).

[0135] To test the antiviral activity of moxidectin against SARS-CoV-2 and compare it with the activity of ivermectin, Vero / hSLAM cells were infected with SARS-CoV 2 isolate Australia / VIC01 / 2020 at an MOI of 0.1 for 2 h, followed by the addition of 5 μM ivermectin or 5 μM moxidectin. Supernatants and cell pellets were harvested at 48 h (n=3, biological replicates), and viral loads were quantified by real-time PCR using a SARS-CoV-2E gene probe. Results represent mean ± SEM (n=3). p values ​​were determined by t-test with Welch correction ( Figure 2 A).

[0136] Infected Vero / hSLAM cells were infected with SARS-CoV-2 clinical isolate Australia / VIC01 / 2020 (MOI = 0.01) for 2 h, and then vehicle (DMSO), ivermectin (filled squares), or moxidectin (open squares) were added at the indicated concentrations. Figure 2 B) 48 hours Figure 2 C) or 72 hours ( Figure 2 D) Supernatants were subjected to SARS-CoV-2 E gene analysis. Results represent mean SEM (n=3). Figure 2 Ivermectin IC calculated in B 50 The value was 1.4 μM for 5-nitropropene and 1.3 μM for moxidectin. Figure 2 Ivermectin IC calculated in C 50 The value was 2.2 μM for 5-nitropropene and 1.3 μM for moxidectin. Figure 2 Ivermectin IC calculated in D 50 The value was 3.1 μM for ivermectin and 2.4 μM for moxidectin. That is, at 48 hours, the viral RNA (indicative of released viral particles) in the supernatant of samples treated with ivermectin was reduced by 99.5%, and the viral RNA in the supernatant of samples treated with moxidectin was reduced by 99.9% compared to the vehicle DMSO. No toxicity of moxidectin was observed at any time point tested, either in the sample wells or in the parallel single drug samples.

[0137] Taken together, these results confirm that ivermectin and moxidectin are effective antiviral agents that inhibit SARS-CoV-2 clinical isolates, and that both can control viral replication within 24 to 48 hours at a single dose, likely by affecting IMPα / β1-mediated nuclear transport of viral proteins. Ivermectin and moxidectin have established safety standards for human use and are FDA-approved for the treatment of human parasitic infections, making them attractive possibilities for use as broad-spectrum antiviral agents with other macrolides.

[0138] Example 4: Enhanced antiviral activity of ivermectin SARS-CoV-2 in human lung cells

[0139] CALU-3 (human lung) cells were infected with SARS-CoV-2 clinical isolate Australia / VIC01 / 2020 (MOI = 0.01) for 2 hours and then Figure 3 Vehicle (DMSO) or ivermectin was added at the indicated concentrations. Samples were collected 24 hours after infection, and viral load was quantified using real-time PCR of supernatants. Figure 3Results shown represent mean ± SD (n = 6) relative to DMSO alone. Three-parameter dose-response curves were fitted using GraphPad prism to determine IC 50 The value is 0.42μM.

[0140] Table 1 shows the average IC values ​​of CALU-3 cells compared with Vero / hSLAM cells tested under the same conditions. 50 values, indicating enhanced ivermectin SARS-CoV-2 antiviral activity in human lung cells.

[0141] Table 1: IC values ​​of CALU-3 cells compared to Vero / hSLAM cells 50 value.

[0142] Cell lines <![CDATA[IC 50 + / -SEM(n*)]]> Vero / hSLAM 2.37+ / -0.23(7) CALU-3 0.41+ / -0.15(4)

[0143] *n = number of independent experiments

[0144] Example 5: Multiple doses of ivermectin or moxidectin enhance the antiviral activity of SARS-CoV-2

[0145] Vero / hSLAM cells ( Figure 4 A / C) or CALU-3( Figure 4 B / D) Cells were infected with SARS-CoV-2 clinical isolate Australia / VIC01 / 2020 (MOI = 0.01) for 2 hours, and then added with concentrations of Figure 4 Vehicle (DMSO), ivermectin (A / B) or moxidectin (C / D) as indicated. Supernatants were collected at 8, 24, 32 and 48 hours and replaced with fresh medium containing the same concentration of drug or DMSO. Viral load of samples was quantified using real-time PCR. Results are presented as mean ± SD (n = 6) relative to DMSO alone under the same conditions. Three-parameter dose response curves were fitted using GraphPad prism. Figure 4 The data shown are a single representative plot from a series of two similar experiments.

[0146] Example 6: Ivermectin and moxidectin have prophylactic antiviral activity against SARS-CoV-2.

[0147] Vero / hSLAM cells were pretreated with the indicated concentrations of DMSO, ivermectin, or moxidectin for 2 h before infection with SARS-CoV-2 (MOI = 0.01) in the presence of DMSO or drugs for 2 h. Samples were washed after adsorption and incubated in fresh medium containing DMSO or drugs for 48 h, and viral load was quantified using real-time PCR of the supernatant. Figure 5Results shown represent mean ± SD (n = 6) relative to DMSO alone. Three-parameter dose-response curves were fitted using GraphPad prism to determine the IC of ivermectin. 50 The value is 1.6 μM for α-cadherin and 1.8 μM for moxidectin. The average IC values ​​of CALU-3 cells under the same conditions compared with the Vero / hSLAM cell assay are shown. 50 Values ​​(right table, n = independent experiments).

[0148] Table 2 IC of Vero / hSLAM cells treated prophylactically with ivermectin compared with moxidectin 50 value.

[0149] drug IC50+ / -SEM(n*) Ivermectin 1.73+ / -0.29(3) Moxidectin 1.66+ / -0.23(3)

[0150] *n = number of independent experiments

[0151] Example 7: Ivermectin and Moxidectin are effective antiviral inhibitors of seasonal coronavirus 229E

[0152] After adding the specified concentrations of vehicle (DMSO), ivermectin ( Figure 6 A) or moxidectin ( Figure 6 B) Before, Huh-7 cells were infected with 229E (MOI = 0.01) for 2 h. Samples were collected 48 h after infection and viral load was quantified using real-time PCR of supernatants. Results are presented as mean ± SD relative to DMSO alone (n = 6). Three-parameter dose-response curves were fitted using GraphPad prism to determine the IC of ivermectin 50 The value was 0.18 μM for 5-nitropropene and 0.24 μM for moxidectin.

[0153] To evaluate the preventive potential, Huh-7 cells were pretreated with the indicated concentrations of DMSO or ivermectin for 2 h before infection with 229E (MOI = 0.01) in the presence of DMSO or ivermectin. Figure 6 C). Samples were washed after adsorption (n=5) and incubated in fresh medium containing DMSO or ivermectin for 48 h, and viral load was quantified as described above. Three-parameter dose-response curves were fitted using GraphPad prism to determine the IC of ivermectin. 50 The value is 2.06μM.

[0154] Those skilled in the art will appreciate that, as shown in the specific embodiments, various changes and / or modifications may be made to the present invention without departing from the spirit or scope of the present invention as broadly described. Therefore, the present embodiments are to be considered in all aspects as illustrative and not restrictive.

Claims

1. Use of a macrolide or a salt thereof as an inhibitor of coronavirus in the preparation of a medicament for inhibiting coronavirus in an individual or treating an individual infected with coronavirus, wherein the macrolide is ivermectin and the coronavirus is coronavirus 229E or SARS-CoV-2.

2. Use of a macrolide or a salt thereof as an inhibitor of coronavirus in the preparation of a medicament for preventing individual coronavirus infection or reducing the risk of individual coronavirus infection, wherein the macrolide is ivermectin, and the coronavirus is coronavirus 229E or SARS-CoV-2.

3. Use of a macrolide or a salt thereof as an inhibitor of a coronavirus in the preparation of a medicament for preventing or treating a coronavirus infection in an individual, wherein the macrolide is ivermectin and the coronavirus is coronavirus 229E or SARS-CoV-2.

4. Use of a macrolide or a salt thereof as an inhibitor of coronavirus in the preparation of a medicament for reducing the risk of coronavirus infection in an individual, wherein the macrolide is ivermectin and the coronavirus is coronavirus 229E or SARS-CoV-2.

5. The use according to any one of claims 1 to 4, wherein The individual is a human.

6. The use according to any one of claims 1 to 4, wherein The coronavirus is SARS-CoV-2.

7. The use according to any one of claims 1 to 4, wherein The subject is a dog, cat, horse, cow, sheep, camel, chicken or pig.

8. The use according to any one of claims 1 to 4, wherein The level of detectable coronavirus in the individual is reduced by at least 50%.

9. The use according to claim 8, wherein The level of detectable coronavirus in the individual is reduced by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9%.

10. The use according to claim 8, wherein A reduction in the level of coronavirus can be detected 1 day, 2 days, 3 days or longer after administration of the drug.

11. The use according to any one of claims 1 to 4, wherein The macrolide or salt thereof is administered at a dosage of about 0.1 mg / kg to about 1.0 mg / kg of the subject's body weight.

Images