INFECTIONS

FIELD

[0001] This disclosure relates to pharmaceutical compositions, pharmaceutical combinations, and methods of treatment of viral infections.

BACKGROUND

[0002] Recurrent infections caused by RNA viruses such as influenza and coronaviruses including SAR- CoV-2 threaten economies and millions of lives world wide. For mitigating detrimental impact on societies caused by these pathogens safe and effective drugs and drug combinations that can be used for treating viral infections in out of hospital settings are particularly useful. Antiviral drugs have traditionally been developed by directly targeting essential viral components. Flowever, this strategy frequently fails if viruses rapidly mutate and generate either drug- or vaccine resistant strains. Identifying cellular host cellular factors that are critical for virus replication, but are dispensable for the host, offers a new strategy for antiviral drug development that overcomes the limitation of virus direct approaches because it is less likely that viruses will mutate to replace missing cellular functions which means that targeting host mechanisms for antiviral therapy reduces the chance of generating drug resistant mutants.

SUMMARY

[0003] In one embodiment, a pharmaceutical composition is provided. The pharmaceutical composition includes atovaquone; at least one substance selected from a second group of substances consisting of ademetionine, albendazole, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib; and a pharmaceutically acceptable carrier.

[0004] In another embodiment, a pharmaceutical is provided. The pharmaceutical composition includes cepharantine; at least one substance selected from a second group of substances consisting of ademetionine, albendazole, atovaquone, celastrol, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, niclosamide, raloxifene, sorafenib, or tipifarnib; and a pharmaceutically acceptable carrier.

[0005] In another embodiment, a pharmaceutical composition is provided. The pharmaceutical composition includes one substance selected from a first group of substances consisting of ademetionine, albendazole, atovaquone, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib; at least one substance selected from a second group of substances selected from the group consisting of ademetionine, albendazole, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib; and a pharmaceutically acceptable carrier, wherein the not more than one substance selected from a first group of substances and the at least one substance selected from a second group of substances are not the same.

[0006] In another embodiment, a method for treating viral infections in a mammal is provided. The method includes administering to said mammal in need of such treatment an effective amount of one of the above pharmaceutical compositions.

DETAILED DESCRIPTION

[0007] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s).

[0008] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0009] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by embodiments of the present disclosure. As used herein, “about” may be understood by persons of ordinary skill in the art and can vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” may mean up to plus or minus 10% of the particular term.

[0010] The terms "treating" and “effective amount”, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment", as used herein, unless otherwise indicated, refers to the act of treating as "treating" is defined immediately above. The term “treating” also includes adjuvant and neo-adjuvant treatment of a subject.

[0011] Aspects of the present disclosure include novel combinations, pharmaceutical compositions and methods of for treating and/or preventing viral infections in a mammal where the combinations and pharmaceutical compositions comprise atovaquone in combination with at least one substance selected from a second group of substances consisting of ademetionine, albendazole, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib or comprise cepharantine with at least one substance selected from a second group of substances consisting of ademetionine, albendazole, atovaquone, celastrol, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, niclosamide, raloxifene, sorafenib, or tipifarnib or comprising not more than one substance selected from a first group of substances consisting of ademetionine, albendazole, atovaquone, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib in combination with at least one substance selected from a second group of substances selected from the group consisting of ademetionine, albendazole, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib, wherein the not more than one substance selected from a first group of substances and the at least one substance selected from a second group of substances are not the same.

[0012] In addition, the drug formulations, pharmaceutical compositions, combinations and method of treatment embodiments of the present disclosure can be used not only for humans but also for animals.

[0013] Examples of animals include mammals, birds, reptiles, amphibians, and fish.

[0014] It has been found that SARS-CoV-2 Is not detectable in the vaginal fluid of women with severe covid-19 infection ( See, Qiu Let al. SARS-CoV-2 Is Not Detectable in the Vaginal Fluid of Women with Severe COVID-19 Infection. Clin Infect Dis. 2020 Jul 28;71 (15):813-817.) For identifying defense mechanisms protecting vaginal tissues in patients with severe SARS CoV-2 infections a topological data analysis approach referred to in United States Patent No. 11120346, the disclosure of which is incorporated herein by reference in its entirety . For identifying molecular processes which render Vaginal tissues resistant against viral infections in contrast to mucosal tissues of the Gl tract and the lung (See, Kumamoto Y, Iwasaki A. Unique features of antiviral immune system of the vaginal mucosa. Curr Opin Immunol. 2012;24(4):411 - 416. doi:10.1016/j.coi.2012.05.006). Vaginal tissue proteins were identified that are capable of interacting with SARS CoV-2 interactome proteins referred to in Ostaszewski M., Mazein A., Gillespie M.E. et al. in “ (2020) COVID-19 Disease Map. Tissue proteins used in this analysis were derived from the protein atlas (See, Uhlen M., Fagerberg L, Hallstrom B.M. et al. (2015) Science, 347, 1260419) using a technology referred to in United States Patent No. 11120346. This comparative analysis identified a set of vaginal tissue proteins directly interacting with the SARS CoV-2 interactome shown in Table 1.

[0015]

[0016] Network protein identified in Table 1 were then analyzed using the String platform. ( See, Szklarczyk D, et al., STRING v11 : protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res. 2019 Jan; 47:D607-613 ) which identified that host proteins identified in Table 1 regulate molecular processes associated with cellular stress responses and antigen processing as shown in Table 2.

[0017]

[0018] Next, using again the methodology referred to in United States Patent No.

11120346, drugs were identified that are capable of interacting with host proteins identified in Table 1 that interact with SARS CoV-2 interactome proteins and, using data published by the National Center for Advancing Translational Sciences ( See, Brimacombe, Kyle R et al. An OpenData portal to share COVID-19 drug repurposing data in real time. bioRxiv 2020.06.04.135046), we selected drugs capable of inhibiting SARS Cov-2 replication in Vero 6 cells. These associations are shown on the right in Table 1. For examining effects of drug combinations targeting host proteins involved in the regulation of molecular processes involved in the regulation of stress responses a protein network fragment shown in Table 3 was selected regulating cellular stress responses containing proteins that can be targeted by imatinib, atovaquone and cepharantine.

[0019]

[0020] For examining effects of drug combinations on the target circuit regulating cellular stress responses shown in Table 2 by targeting either HOST- HOST protein interaction or HOST-VIRUS protein interactions we determined effects of (1) Atovaquone(H) and Niclosamide (V) and (2) Atovaquone (H) and Cephrantin (H) combinations on the viability of SARS CoV-2 infected Vero 6 cells using checkerboard titration virus neutralization assays.

[0021] A pharmaceutical composition and embodiments thereof of the present disclosure can be directly using a variety of drug formulations. Such drug formulation contains combinations of active ingredients. Further, the drug formulation can be produced by an arbitrary method that has been well known in the art of drug formulation by mixing the active ingredient with at least one type of pharmacologically acceptable carrier or vehicle. It is desirable that the most effective administration route of drug formulation would be selected for treatment. Examples thereof include oral administration, topical administration and parenteral administration such as intravenous, intraperitoneal, or subcutaneous administration. However, oral administration is preferable. Examples of dosage forms that can be used for administration include: oral agents such as tablets, powders, granules, pills, suspensions, emulsions, infusions and decoctions, capsules, syrups, liquid, elixirs, extracts, tinctures, and fluid extracts; and parenteral agents such as parenteral injections, intravenous fluids, creams, and Suppositories. The composition in the form of an oral composition is preferably used.

[0022] In the case involving the use of liquid preparations such as syrup appropriate for oral administration, the preparations can be formulated by addition of water, Sugars such as Sucrose, Sorbitol, and fructose; glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, and soybean oil; antiseptics such as p- hydroxy benzoate esters; parahydroxy benzoate derivatives such as methyl parahydroxy benzoate; preservatives such as Sodium benzoate; and flavors such as Strawberry flavor and peppermint flavor.

[0023] In addition, in the case involving the use of tablets, powders, and granules that are appropriate for oral administration the preparations can be formulated by addition of sugar such as lactose, glucose, sucrose, mannitol, and sorbitol; starch from potatoes, wheat, and corn; an inorganic substance such as calcium carbonate, calcium sulfate, sodium bicarbonate, and sodium chloride; an excipient of a plant-derived powder such as crystalline cellulose, a sweetroot powder and gentian powder, a disintegrator Such as starch, agar, gelatin powder, crystalline cellulose, carmellose sodium, carmellose calcium, calcium carbonate, sodium bicarbonate, and sodium alginate; a lubricant such as magnesium stearate, talc, hydrogenated plant oil, macrogol, and silicone oil; a binder such as polyvinyl alcohol, hydroxypropyl cellulose, methyl cellulose, ethylcellulose, carmellose, gelatin, and starch paste liquid; a surfactant such as fatty acid ester, and a plasticizer such as glycerine.

[0024] It is also possible to add an additive generally used for foods and beverages to the drug formulation appropriate for oral administration. Examples of additives include sweeteners, colorants, preservatives, thickening stabilizers, antioxidants, coloring agents, bleaches, antifungal agents, gumbases, bittering agents, enzymes, gloss agents, acidulants, seasonings, emulsifiers, fortifiers, production agents, aroma chemicals, and spice extracts. [0025] The pharmaceutical composition formulation embodiments of the present disclosure appropriate for oral administration may be directly used in the form of, for example, a powder food product, a sheet-type food product, a bottled food product, a canned food product, a retort food product, a capsule food product, a tablet food product, a liquid food product, or a drink. In addition, the drug formulation may be used in the form of food or beverage such as health food, functional food, nutritional supplement, or food for specified health use. For example, a parenteral injection appropriate for parenteral administration comprises preferably a sterilized aqueous agent which contains a composition of the present disclosure and which is isotonic to the blood of a recipient. For example, for a parenteral injection, an injectable solution is prepared with the use of a pharmaceutically acceptable carrier or vehicle comprising a salt solution, a glucose solution, or a mixture of a salt Solution and a glucose solution.

[0026] In addition, it is also possible to add at least one supplemental component to a parenteral agent, wherein Such components can be selected from the group consisting of diluents, antiseptics, flavors, excipients, disintegrators, lubricants, binders, surfactants, and plasticizers, which are described above for an oral agent.

[0027] An embodiment of the present disclosure includes a combination comprising atovaquone in combination with at least one substance selected from a second group of substances consisting of ademetionine, albendazole, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib.

[0028] An embodiment of the present disclosure includes a pharmaceutical composition comprising atovaquone in combination with at least one substance selected from a second group of substances consisting of ademetionine, albendazole, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib, and a pharmaceutically acceptable carrier. [0029] An embodiment of the present disclosure includes a combination comprising cepharantine with at least one substance selected from a second group of substances consisting of ademetionine, albendazole, atovaquone, celastrol, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, niclosamide, raloxifene, sorafenib, or tipifarnib.

[0030] An embodiment of the present disclosure includes a pharmaceutical composition comprising cepharantine with at least one substance selected from a second group of substances consisting of ademetionine, albendazole, atovaquone, celastrol, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, niclosamide, raloxifene, sorafenib, or tipifarnib, and a pharmaceutically acceptable carrier.

[0031] An embodiment of the present disclosure includes a combination comprising one substance selected from a first group of substances consisting of ademetionine, albendazole, atovaquone, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib in combination with at least one substance selected from a second group of substances selected from the group consisting of ademetionine, albendazole, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib.

[0032] An embodiment of the present disclosure includes a pharmaceutical composition comprising one substance selected from a first group of substances consisting of ademetionine, albendazole, atovaquone, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib in combination with at least one substance selected from a second group of substances selected from the group consisting of ademetionine, albendazole, celastrol, cepharantine, chloroquine, cycloheximide, diethylstilbestrol, gemcitabine, homoharringtonine, hydroxy progesterone, imatinib, imipramine, indomethacin, ivermectin, raloxifene, sorafenib, or tipifarnib, and a pharmaceutically acceptable carrier.

[0033] An embodiment of the present disclosure includes method of using of a pharmaceutical composition or combination embodiment of the present disclosure for the treatment of viral infections in a mammal.

[0034] Experimental:

[0035] Experiment 1 - A virus neutralization assay was performed using Vero E6 cells which are susceptible to SARS-CoV-2 infection. On the day of assay, the monolayer of Vero E6 cells is exposed to a series of dilutions of each test agent, or dilutions of both agents as a combination, in a checkerboard assay format (triplicate wells per dose). Standardized SARS-CoV-2 virus stock calculated to yield approximately 1x 10 ² TCID5o/ml is added after 1 hour at 37°C and 5% CO2, pre-treatment; the plates are incubated for 3 days, and the wells will be scored for the presence or absence of SARS-CoV-2 cytopathic effects (CPE) in the cells. Controls present on each assay plate include Remedesivir as direct acting antiviral compound control (at highest assay concentration) that lacks virus to ensure that the compound itself does not cause CPE. There are also negative control wells (without compound or virus) to verify that the serum-free media does not cause CPE. A back-titer of the virus is performed that serves to verify that the titer of the standardized virus is within an acceptable range. The titer is determined as the inverse of the last dilution of serum that inhibited the viral infection (cells that do not display CPE). Only results are considered when all these controls meet acceptance criteria,

Substances meeting these criteria are subjected to checkerboard titration for determining concentration dependency of drug combinations on viral neutralization.

[0036] Data from the drug combination are compared to the activity of single agents and to the positive controls used in both in vitro assays and animal studies. The potency of both agents as single drugs and the combination to inhibit SARS-CoV-2 entry and replication in Vero E6 cells (checkerboard titration) is analyzed for each compound’s individual minimal inhibitory activity (MIC) and the Fractional Inhibitory Concentration (FIC) index value will be calculated. The FIC index value considers the combination of antivirals that produces the greatest change from the individual antiviral's MIC. To quantify the interactions between the antivirals being tested (calculation of the FIC index is used. The FIC Index value will be used to categorize the interaction of the two antivirals tested, where synergy is defined as a FIC <0.5, additive/indifference as FIC = 0.5 - 4.0, and antagonism as FIC >4.0. The dose-effect curves and ECso values are also compared. This information will provide a range of dose combinations that are synergistic.

[0037]

Results

[0038] Combination experiment 1 shows that Niclosamide is non cytotoxic (CYT) and non-efficacious at concentrations ranging from 0.12 to 1 ,1 micro mol and that atovaquone is non cytotoxic at concentrations ranging from 0.12 to 3,3 micro molar and inhibits virus induced cytopathic effects (CPE) to 50% at a concentration of 3.3 micro mol. The addition of atovaquone to niclosamide dose dependency increases the cytotoxic effects of niclosamide in that the addition of 1.1 micromole atovaquone renders non cytotoxic concentration of niclosamide ( 0.12-3.37 micromole ) cytotoxic. Atovaquone at a lower concentration of 0.37 micromole marginally increases the efficacy of low doses of niclosamide ( 0.12 to 0.37 micromolar) which are inactive without the addition of atovaquone. This result suggests that targeting host viral protein interactions can increase antiviral efficacy but within a very narrow margin of safety suggesting that targeting the circuit in table 2 is important for both virus and host.

[0039]

[0040] Experiment 2 - A similar virus neutralization assay to that conducted in Experiment 1 was performed using neither cepharantine nor atovaquone. Experiment 2 shows that neither cepharantine nor atovaquone are cytotoxic at the highest concentration tested. The addition of cepharantine to atovaquone at all doses tested does not affect the cytotoxicity of either drug. Combining atovaquone and cepharantine unexpectedly results in a dose dependent substantially increase in antiviral efficacy to an extent that renders inactive concentration of cepharantine ( 0.04-0.12 micromole) in combinations with inactive concentration of Atovaquone (0.12-0.37) producing superior antiviral efficacy that approaches the maximal inhibition of atovaquone at the highest doses. Since cepharantine suffers from poor bioavailability this result of experiment 2 indicates that the combination of atovaquone with cepharantine has the capacity to provide oral active antiviral activity with a toleration profile that may allow use of this combination in out of hospital setting. In addition, the results produced in drug interaction experiment 1 and 2 indicates that targeting Host -Host protein interaction in cellular stress response regulating circuits identified in Table and 2 provides q novel strategy for identifying host directed drug combinations for treatment of viral infections.

[0041] In vivo efficacy evaluation

[0042] In vivo efficacy evaluation of Atovaquone and Cepharanthine alone and in combination against SARS-CoV-2 in Syrian Golden Hamster Model [Study Protocol Reference:! Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure development. Masaki Imai et al., PNAS 2020, 117(28)16587-16595]

[0043]

[0044] Results

[0045] Data Analysis One way ANOVA- Dunnett’s multiple comparison test [0046] 1. Statistically significant reduction in lung viral loads was seen with atovaquone once-a-day treatment at 200mg/kg in comparison with infection control.

[0047] 2. Cepharanthine once-a-day treatment at 8mg/kg exhibited a marginal but statistically not significant reduction in lung viral loads in comparison with infection control

[0048] 3. Combination treatment of atovaquone and cepharanthine also demonstrated significant reduction in lung viral loads at 4th day post infection.

[0049] 4. The efficacy outcome for the combination treatment correlated with improved body weights and histopathology of lungs.

[0050] 5. Overall, the study demonstrated efficacy of atovaquone and cepharanthine combination in hamsters against COVID-19 infection. Results suggest atovaquone and cepharanthine combination as a potential candidate for the treatment of COVID-19.

[0051] All publications, including but not limited to, issued patents, patent applications, and journal articles, cited in this application are each herein incorporated by reference in their entirety.

[0052] Thus, while there have been shown, described and pointed out, fundamental novel features of the present disclosure as applied to the exemplary embodiments thereof, it will be understood that various omissions and substitutions and changes in the form and details of devices and methods illustrated, and in their operation, may be made by those skilled in the art without departing from the spirit or scope of the present disclosure. Moreover, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, are within the scope of the present disclosure. Moreover, it should be recognized that structures and/or elements and/or method steps shown and/or described in connection with any disclosed form or embodiment of the present disclosure may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto. [0053] This written description uses examples as part of the disclosure, including the best mode, and also to enable any person skilled in the art to practice the disclosed implementations, including making and using any devices or systems and performing any incorporated methods. The patentable scope is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0054] While there have been shown, described and pointed out, fundamental features of the present disclosure as applied to the exemplary embodiments thereof, it will be understood that various omissions and substitutions and changes in the form and details of compositions, devices and methods illustrated, and in their operation, may be made by those skilled in the art without departing from the spirit or scope of the present disclosure. Moreover, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, are within the scope of the present disclosure. Moreover, it should be recognized that structures and/or elements and/or method steps shown and/or described in connection with any disclosed form or embodiment of the present disclosure may be incorporated in any other disclosed or described or suggested form or embodiment as a general matter of design choice. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.