ANTIVIRAL USE OF CALIXARENES

Abstract

A calixarene compound in an antiviral effective amount for use as an antiviral substance in a pharmaceutical preparation for use in prophylactic or therapeutic treatment of a disease condition which is caused by or associated with an infection with a virus.

Claims

1. A method for prophylactically or therapeutically treating a disease condition which is caused by or associated with an infection of a subject with one or more viruses comprising at least one respiratory virus, comprising administering an antiviral effective amount of a calixarene compound to the subject.

2. The method of claim 1, wherein said one or more viruses are selected from: a) a Coronaviridae virus; b) an Adenoviridae virus; c) a Paramyxoviridae virus; or d) an Orthomyxoviridae virus.

3. The method of claim 1, wherein said one or more viruses comprise a coronavirus.

4. The method of claim 1, wherein said one or more viruses comprise any one or both of SARS-CoV-2 and influenza virus.

5. The method of claim 1, wherein the calixarene compound is formulated as a pharmaceutical preparation in admixture with a pharmaceutically acceptable carrier.

6. The method of claim 1, wherein the disease condition is common cold, infection of the nose, sinusitis, throat and larynx, bronchiolitis, diarrhea, rash on skin, or pneumonia, acute respiratory distress syndrome (ARDS).

7. The method of claim 1, wherein the antiviral effective amount is effective in preventing infection of susceptible cells by the virus, thereby treating the disease condition.

8. The method of claim 1, wherein the antiviral effective amount is administered locally or systemically.

9. The method of claim 1, wherein the calixarene compound is a calix[n]arene characterized by the structure (I): ##STR00004## wherein n=denotes 4, 5, 6, 7, or 8 and R is selected from the group consisting of —SO.sub.3R.sup.a, ORE, COOR.sup.a, hydrogen, or halogen, and each R.sup.a is hydrogen or methyl; or a pharmaceutically acceptable salt thereof.

10. The method of claim 9, wherein the calix[n]arene is a calix[4]arene with four intrannular hydroxyl groups on the lower rim and four sulfonate or carboxyilate groups on the upper rim.

11. The method of claim 5, wherein said pharmaceutical preparation is formulated for local administration to the upper and lower respiratory tract or by nasal, pulmonary, intraoral, ocular, or dermal delivery, or is formulated for systemic administration by intravenous, intramuscular, subcutaneous, intradermal, transdermal, or oral administration.

12. The method of claim 5, wherein said pharmaceutical preparation is administered to the subject as a spray, a powder, a gel, an ointment, a cream, a foam, or a liquid solution, a lotion, a gargle solution, an aerosolized powder, an aerosolized liquid formulation, granules, capsules, drops, tablet, syrup, lozenge, or a preparation for infusion or injection.

13. The method of claim 1, wherein the calixarene compound is applied into the subject's nose in an antiviral effective amount of 1-1000 μg per nostril.

14. The method of claim 1, wherein the calixarene compound is administered as the sole antiviral substance, or wherein treatment is combined with a further treatment with one or more active substances.

15. The method of claim 1, wherein the subject has been infected or is at risk of being infected with said virus.

16. A method for treating a biological surface to prevent virus infection and/or virus spread, comprising applying an antiviral effective amount of a calixarene compound to the biological surface preferably wherein said medicinal product is formulated for topical use, such as application to the upper and lower respiratory tract, nasal, pulmonary, intraoral, ocular, or dermal application.

17. The method of claim 2, wherein said one or more viruses are selected from: a) SARS-CoV-2, MERS-CoV, SARS-CoV-1, HCoV-OC43, HCoV-HKU1, HCoV-NL63, HCoV-229E, PEDV, or naturally-occurring variants or mutants of any of the foregoing; b) HAdVB, HAdVC, or HAdVD; c) RSV; or d) an influenza virus.

18. The method of claim 10, wherein the calix[n]arene is Sulfonatocalixarene or Sulfonatocalixarene sodium salt.

19. The method of claim 14, wherein the one or more active substances are selected from the group consisting of an antiviral substance, an anti-inflammatory substance, and an antibiotic substance.

20. The method of claim 16, wherein said calixarene compound is formulated for topical use for application to the upper and lower respiratory tract, or for nasal, pulmonary, intraoral, ocular, or dermal application.

Description

FIGURES

[0144] FIG. 1 shows microscopic pictures of Vero E6 cells in an in vitro model of SARS-CoV-2 infection when cells were infected in the presence of CX-1. A: Negative control: uninfected cells; B: Positive control: cells infected with SARS-CoV-2; C-E: Cells infected with SARS-CoV-2 in the presence of CX-1 used at 500, 100 or 20 μM concentrations, respectively. Cells were infected with MOI 0.01 SARS-Co-V2 virus, and microscopic pictures taken 48 hours post infection.

[0145] FIG. 2 shows microscopic pictures of Vero E6 cells in an in vitro model of SARS-CoV-2 infection when cells were infected first with virus then treated with CX-1. A: Negative control: uninfected cells; B: Positive control: cells infected with SARS-CoV-2; C-E: Cells infected with SARS-CoV-2 in the presence of CX-1 used at 500, 100 or 20 μM concentrations, respectively. Cells were infected with MOI 0.01 SARS-CoV-2 virus for 30 min, then culture medium was replaced to remove the virus and cell were cultured in the presence of CX-1, microscopic pictures taken 48 hours post infection.

[0146] FIG. 3 shows the antiviral effect of CX-1 in a prophylactic setting. Vero E6 cells were infected with SARS-CoV-2 in the presence of CX-1 at the indicated concentrations. After 30 min of incubation at 37° C., the virus-containing medium was replaced with fresh medium supplemented with the corresponding concentrations of CX-1. Following an additional 48 h incubation at 37° C., viral copy numbers were determined by qPCR.

[0147] FIG. 4 shows the influence of CX-1 as well as oseltamivir on the viral copy numbers obtained from apical washes of reconstituted nasal tissues at 24 h (panel A) or 48 h (panel B) after infection by influenza virus H1N1. Differences between two groups were tested by Student's t test using GraphPad Prism 6.0. Statistically significant differences are marked by asterisks. Graphs shows mean results ±standard error of mean of triplicates from one representative experiment. *P<0.05

[0148] FIG. 5 shows the effect of CX-1 as well as oseltamivir on the cilia beating frequency of primary human nasal epithelial cells at day 4 post-infection by influenza virus H1N1. Differences between two groups were tested by Student's t test using GraphPad Prism 6.0 Statistically significant differences are marked by asterisks.

[0149] Graphs shows mean±standard error of mean of triplicates from one representative experiment. ***P<0.001; ****P<0.0001.

[0150] FIG. 6: SEQ ID NO:1 (NCBI accession number QII57161.1, SARS-CoV-2, S-protein).

DETAILED DESCRIPTION OF THE INVENTION

[0151] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0152] The term “about” as used herein refers to the same value or a value differing by +/−10% or +/−5% of the given value.

[0153] A small molecule organic compound such as a calixarene compound as described herein may be used as a “physiologically acceptable salt”. The choice of salt is determined primarily by how acid or basic the chemical is (the pH), the safety of the ionized form, the intended use of the drug, how the drug is given (for example, by mouth, injection, or on the skin), and the type of dosage form (such as tablet, capsule, or liquid).

[0154] Exemplary salts which are physiologically acceptable are sodium salts. However, it is, also possible to employ, in place of the sodium salts, other physiologically acceptable salts, e.g., other alkali metal salts, alkaline earth metal salts, ammonium salts and substituted ammonium salts. Specific examples are the potassium, lithium, calcium, aluminum and iron salts. Preferred substituted ammonium salts are those derived, for example, from lower mono-, di-, or trialkylamines, or mono-, di- and trialkanolamines. The free amino acids per se can also be used. Specific examples are ethylamine, ethylenediamine, diethylamine, or triethylamine salts.

[0155] The term “pharmaceutically acceptable” also referred to as “pharmacologically acceptable” means compatible with the treatment of animals, in particular, humans. The term pharmacologically acceptable salt also includes both pharmacologically acceptable acid addition salts and pharmacologically acceptable basic addition salts.

[0156] The term “pharmacologically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compound of the disclosure, or any of its intermediates. Basic compounds of the disclosure that may form an acid addition salt include, for example, compounds that contain a basic nitrogen atom. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono-, di- or the triacid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of the compounds of the disclosure are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmacologically acceptable acid addition salts, e.g. oxalates, may be used, for example, in the isolation of the compounds of the disclosure, for laboratory use, or for subsequent conversion to a pharmacologically acceptable acid addition salt.

[0157] The term “pharmacologically acceptable basic salt” as used herein means any non-toxic organic or inorganic basic addition salt of any acid compound of the invention, or any of its intermediates, which are suitable for or compatible with the treatment of animals, in particular humans. Acidic compounds of the invention that may form a basic addition salt include, for example compounds that contain carboxylic acid, sulfonic acid, sulfinic acid, sulfonamide, N-unsubstituted tetrazole, phosphoric acid ester, or sulfuric acid ester. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art. Other non-pharmacologically acceptable basic addition salts, may be used, for example, in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmacologically acceptable basic addition salt. The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with a base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.

[0158] The term “antiviral” as used herein shall refer to any substance, drug or preparation, that effects the biology of a virus and attenuates or inhibits viral attachment, entry, replication, shedding, latency or a combination thereof, resulting in reduction of viral load or infectivity. The terms “attenuating,” “inhibiting,” “reducing,” or “prevention,” or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result, e.g., reduction in the risk of viral infection (pre-exposure), or reduction of post-exposure viral survival, load, or growth.

[0159] Exemplary antiviral preparations described herein are medicinal products, pharmaceutical preparations, or disinfectants, for in vivo, ex vivo or in vitro use.

[0160] The term “biological surface” as used herein shall refer to a surface comprising viable cells, such as mammalian (human or non-human animal) cells, including e.g., a biological tissue surface, such as a surface or epithelial or dermal tissue (e.g. skin), mucosal tissue, or membrane tissue.

[0161] The term “effective amount” with respect to an antiviral effect as used herein, shall refer to an amount (in particular a predetermined amount) that has a proven antiviral effect. The amount is typically a quantity or activity sufficient to, when applied to a surface or administered to a subject effect beneficial of desired results, including antiviral or clinical results, and, as such, an effective amount or synonym thereof depends upon the context in which it is being applied.

[0162] An effective amount of a pharmaceutical preparation or drug is intended to mean that amount of a compound that is sufficient to treat, prevent or inhibit a disease, disease condition or disorder. Such an effective dose specifically refers to that amount of the compound sufficient to result in healing, prevention or amelioration of conditions related to diseases or disorders described herein.

[0163] In the context of disease, effective amounts (in particular prophylactically or therapeutically effective amounts) of a calixarene compound as described herein are specifically used to treat, modulate, attenuate, reverse, or affect a disease or condition that benefits from its antiviral effect. The amount of the compound that will correspond to such an effective amount will vary depending on various factors, such as the given drug or compound, the formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, the assessment of the medical situations and other relevant factors, but can nevertheless be routinely determined by one skilled in the art.

[0164] A treatment or prevention regime of a subject with an effective amount of the calixarene compound described herein may consist of a single application or administration, or alternatively comprise a series of applications and administrations, respectively. For example, the calixarene compound may be used at least once a month, or at least once a week, or at least once a day. However, in certain cases of an acute phase, e.g. upon suspected or confirmed exposure to a virus, or after virus infection has been determined, the calixarene compound may be used more frequently e.g., 1-10 times a day.

[0165] Specifically, a combination therapy is provided which includes treatment with the preparation described herein and standard therapy of a coronavirus-caused disease.

[0166] Doses may be applied in combination with other active agents such as antiviral agents, anti-inflammatory drugs or antibiotics, e.g. upon the subject's risk of viral spread, so to prevent a pathogen associated reaction.

[0167] Treatment can be combined with an antiviral, anti-inflammatory or antibiotic treatment, preferably wherein a pharmaceutical preparation is administered before, during (e.g., by co-administration or in parallel), or after said antiviral, anti-inflammatory or antibiotic treatment.

[0168] Specifically, the calixarene compound described herein can be combined with an additional antiviral agent, which can be a calixarene compound, e.g. the same of a different compound. Specific embodiments refer to further antiviral agents selected from an ACE2 inhibitor, a viral protein M2 ion channel inhibitor, a neuraminidase inhibitor, an RNA replication and translation inhibitor and a polymerase inhibitor. The antiviral agent may be amantadine or rimantadine. Specifically, the antiviral agent may be oseltamivir, zanamivir, peramivir, ribavirin, lopinavir, or ritonavir. Specific further antiviral examples are those suitably used for biological surface treatment such as carrageenan, or those currently under investigation for treating SARS-Cov2 infections, such as hydroxychloroquine, or remdesivir.

[0169] Specifically, the calixarene compound is combined with an anti-inflammatory agent such as standard steroidal anti-inflammatory drugs, glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAID's). Suitable NSAID's include, but are not limited to ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid and celecoxib. Suitable steroidal anti-inflammatory agents include, but are not limited to, corticosteroids such as synthetic glucocorticoids. Specific examples are fluticasone, COX-2 inhibitors, ibuprofen, hydroxychloroquine, heparin, LMW heparin, hirudine, or immunosuppressants, such as azathioprine, cyclosporin A, or cyclophosphamide.

[0170] Specifically, the calixarene compound is combined with an antibiotic such as a beta lactam antibiotic, an aminoglycoside antibiotic, an ansamycin, a carbacephem, a carbapenem, a cephalosporin, a glycopeptide, a lincosamide, a lipopeptide, a macrolide, a monobactam, a nitrofuran, an oxazolidinone, a polypeptide, a sulfonamide, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Mupirocin, Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole, Trimethoprim, Teixobactin, Malacidins, Halicin, clindamycin, vancomycin, metronidazole, fusidic acid, thiopeptides, fidaxomicin, quinolons, tetracyclins, omadacycline, rifamycin, kibdelomycin, oxazolidinone, ketolides, thiazolides, amixicile, teicoplanin, ramoplanin, oritavancin, lantibiotics, capuramycin, surotomycin, thuricin, endolysin, avidocin CD, cadazolid, ramizol, defensins, ridinilazole, medium-chain fatty acids, phages, berberine, lactoferrin.

[0171] Specifically, treatment with the calixarene compound described herein can be combined with a treatment administering at least one other therapeutic agent selected from the group consisting of a corticosteroid, an anti-inflammatory signal transduction modulator, a 2-adrenoreceptor agonist bronchodilator, an anticholinergic, a mucolytic agent, hypertonic saline and other drugs for treating a Coronaviridae virus infections; or mixtures thereof. Specific pharmaceutical compositions may particularly include one or more anti-inflammatory agents, and/or analgesics, PPAR-γ agonists and immune response modulators.

[0172] The length of the treatment period depends on a variety of factors, such as the severity of the disease, either acute or chronic disease, the age of the patient, and the concentration of the calixarene compound. It will also be appreciated that the effective dosage used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.

[0173] According to a specific aspect, a medicinal product or pharmaceutical composition described herein contains an effective amount of the calixarene compound as defined herein. The preparation described herein may be provided for single or multiple dosage use.

[0174] Unit-dose or multi-dose containers may be used, for example, sealed ampoules and vials, or multi-use sprays, and may be stored comprising a liquid or dry phase, e.g., in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, or multiple doses, of the calixarene compound.

[0175] The term “single-dose” as used herein is understood in the following way. A single-dose or amount for single-use is the amount intended for administration that is meant for use in a single subject, such as a patient, either human or animal for a single case/procedure/administration. Packages comprising the single-dose are typically labelled as such by the manufacturer. The single-dose amount is specifically understood as a daily dose for an individual, like a child or adult, to provide an effective amount.

[0176] The medicinal product or pharmaceutical composition described herein is specifically provided as human or veterinary medicinal product or pharmaceutical composition. Medicinal products are understood as substances that are used to treat diseases, to relieve complaints, or to prevent such diseases or complaints in the first place. This definition applies regardless of whether the medicinal product is administered to humans or to animals. The substances can act both within or on the body.

[0177] The medicinal product or pharmaceutical composition described herein preferably contains one or more pharmaceutically acceptable auxiliaries and is in a pharmaceutical form which allows the active pharmaceutical compound to be administered with high bioavailability. Suitable auxiliaries may be, for example, based on cyclodextrins. Suitable formulations might for example incorporate synthetic polymeric nanoparticles formed of a polymer selected from the group consisting of acrylates, methacrylates, cyanoacrylates, acrylamides, polylactates, polyglycolates, polyanhydrates, polyorthoesters, gelatin, albumin, polystyrenes, polyvinyls, polyacrolein, polyglutaraldehyde and derivatives, copolymers and mixtures thereof.

[0178] Specific medicinal products or pharmaceutical compositions described herein comprise the calixarene compound and a pharmaceutically acceptable carrier or excipient. A “pharmaceutically acceptable carrier” refers to an ingredient in a formulation for medicinal or medical use, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative, and in particular saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like.

[0179] The calixarene compound as used herein can be formulated with conventional carriers and excipients, which will be selected in accord with ordinary practice.

[0180] Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antiviral, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible with an antiviral small molecule compound or related composition or combination preparation described herein.

[0181] According to a specific aspect, the calixarene compound can be combined with one or more carriers appropriate a desired route of administration. The calixarene compound may be e.g., admixed with any of lactose, sucrose, starch, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, polyvinyl alcohol, and optionally further tableted or encapsulated for conventional administration. Alternatively, the calixarene compound may be dispersed or dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cotton seed oil, sesame oil, tragacanth gum, and/or various buffers. Other carriers, adjuvants, and modes of administration are well known in the pharmaceutical arts. A carrier may include a controlled release material or time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well-known in the art.

[0182] Compounds as described herein may be provided in controlled release pharmaceutical (“controlled release formulations”) in which the release of the calixarene compound is controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

[0183] Pharmaceutical compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject agent is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action. Such dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, waxes, and shellac.

[0184] Additional pharmaceutically acceptable carriers are known in the art and described in, e.g., Remington: The Science and Practice of Pharmacy, 22.sup.nd revised edition (Allen Jr, L V, ed., Pharmaceutical Press, 2012). Liquid formulations can be solutions, emulsions or suspensions and can include excipients such as suspending agents, solubilizers, surfactants, preservatives, and chelating agents.

[0185] The preferred preparation is in a ready-to-use, storage stable form, with a shelf-life of at least one or two years.

[0186] The term “formulation” as used herein refers to a preparation ready-to-use in a specific way. Specifically, compositions described herein comprises the calixarene compound, and a pharmaceutically acceptable diluent, carrier or excipient.

[0187] According to a specific aspect, formulations are provided comprising pharmaceutically acceptable vehicles for nasal, intrapulmonary, oral, topical, mucosal or parenteral administration. Administration may also be intradermal or transdermal. Also, the present disclosure includes such compounds, which have been lyophilized and which may be reconstituted to form pharmaceutically acceptable formulations for administration.

[0188] Specific medicinal products or pharmaceutical compositions described herein are formulated for intranasal administration or by another topical route e.g., onto biological surfaces, including e.g., mucosa or skin. Pharmaceutical carriers suitable for facilitating such means of administration are well known in the art.

[0189] Specifically, a nasal spray may be used containing 0.1% or 0.15% (w/w) calixarene compound in an aqueous solution at pH 6.8±0.3, optionally further containing any one or more of citric acid monohydrate, disodium hydrogen phosphate dodecahydrate, edetate disodium, hypromellose, purified water, sodium chloride, and a preservative such as benzalkonium chloride.

[0190] To administer the calixarene compound by any route other than parenteral administration, it may be necessary to coat the active agent with, or co-administer the active agent with, a material to prevent its inactivation. For example, an appropriate carrier may be used, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.

[0191] The calixarene compound can be orally administered, for example, with an inert diluent or an assimilable or edible carrier. For example, a preparation may be enclosed in a hard- or soft-shell gelatin capsule, or compressed into tablets. For oral therapeutic administration, the calixarene compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the compound in the compositions and preparations may, of course, be varied. The amount of the calixarene compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.

[0192] Tablets will contain excipients, glidants, fillers, binders, disintegrants, lubricants, flavors and the like. Granules may be produced using isomaltose. It is furthermore preferred to provide for a preparation formulated to act at the site of the mucosa, e.g. at mucosal sites (such as nose, mouth, eyes, esophagus, throat, lung), e.g. locally without systemic action. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration generally will be isotonic.

[0193] The term “mucosal” with respect to administration or application or else mucosal use of a preparation for treating a subject or a respective formulation, refers to administration via the mucosal route, including systemic or local administration, wherein an active ingredient is taken up by contact with mucosal surfaces. This includes nasal, pulmonary, oral, or peroral administration and formulations, e.g. liquid, syrup, lozenge, tablet, spray, powder, instant powder, granules, capsules, cream, gel, drops, suspension, or emulsion.

[0194] Peroral formulations may include liquid solutions, emulsions, suspensions, and the like. The pharmaceutically acceptable vehicles suitable for preparation of such compositions are well known in the art. Typical components of carriers for syrups, elixirs, emulsions and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol and water. For a suspension, typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, tragacanth, and sodium alginate; typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate. Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.

[0195] Other compositions useful for attaining systemic delivery of the calixarene compound or respective preparations include sublingual, buccal and nasal dosage forms. Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose, or glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents.

[0196] The calixarene compound or respective preparations can also be administered topically to a subject, e.g., by the direct laying on or spreading of a composition containing same on the epidermal or epithelial tissue of the subject, or transdermally via a “patch”. Such compositions include, for example, lotions, creams, solutions, gels and solids. These topical compositions may comprise an effective amount, usually at least about 0.1 wt %, or even from about 1 wt % to about 5 wt %, of the calixarene compound. Suitable carriers for topical administration typically remain in place on the skin as a continuous film, and resist being removed by perspiration or immersion in water. Generally, the carrier is organic in nature and capable of having dispersed or dissolved therein the therapeutic agent. The carrier may include pharmaceutically acceptable emollients, emulsifiers, thickening agents, solvents and the like.

[0197] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (in particular where the compounds or pharmaceutically acceptable salts are water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In particular, the composition is specifically sterile and fluid to the extent that easy syringability exists; it is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0198] Suitable pharmaceutically acceptable vehicles include, without limitation, any non-immunogenic pharmaceutical adjuvants suitable for oral, parenteral, nasal, mucosal, transdermal, intravascular (IV), intraarterial (IA), intramuscular (IM), and subcutaneous (SC) administration routes, such as phosphate buffer saline (PBS).

[0199] The term “subject” as used herein shall refer to a warm-blooded mammalian, particularly a human being or a non-human animal, including e.g., dogs, cats, rabbits, horses, cattle, and pigs. In particular the treatment and medical use described herein applies to a subject in need of prophylaxis or therapy of a disease condition associated with a coronavirus infection. Specifically, the treatment may be by interfering with the pathogenesis of a disease condition where a coronavirus is a causal agent of the condition. The subject may be a patient at risk of such disease condition or suffering from disease.

[0200] The term “at risk of” a certain disease conditions, refers to a subject that potentially develops such a disease condition, e.g. by a certain predisposition, exposure to virus or virus-infected subjects, by exposure to a certain disease causing agent such as a virus, or that already suffers from such a disease condition at various stages, particularly associated with other causative disease conditions or else conditions or complications following as a consequence of viral infection. The risk determination is particularly important in a subject, where a disease has not yet been diagnosed. This risk determination therefore includes early diagnosis to enable prophylactic therapy. Specifically, the calixarene compound is used in subjects with a high risk, e.g. a high probability of developing disease.

[0201] The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. The term “patient” as used herein always includes healthy subjects. The term “treatment” is thus meant to include both prophylactic and therapeutic treatment.

[0202] Specifically, the term “prophylaxis” refers to preventive measures which is intended to encompass prevention of the onset of pathogenesis or prophylactic measures to reduce the risk of pathogenesis.

[0203] The term “therapy” as used herein with respect to treating subjects refers to medical management of a subject with the intent to cure, ameliorate, stabilize, reduce the incidence or prevent a disease, pathological condition, or disorder, which individually or together are understood as “disease condition”. The term includes active treatment, directed specifically toward the improvement of a disease condition, prophylaxis directed specifically toward the prevention of a disease condition, and also includes causal treatment directed toward removal of the cause of the associated disease condition. In addition, this term includes palliative treatment designed for the relief of symptoms rather than the curing of the disease condition, and further curing a disease condition directed to minimizing or partially or completely inhibiting the development of the associated disease condition, and supportive treatment employed to supplement another specific therapy directed toward the improvement of the associated disease condition.

[0204] The foregoing description will be more fully understood with reference to the following examples. Such examples are, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention.

EXAMPLES

Example 1: Preventing SARS-CoV-2 Infection of Cells by CX-1

[0205] To detect the effect of CX-1 (4-Sulfocalix[4]arene, Sigma Aldrich Cat #55523, Merck KGaA, Darmstadt, Germany) on SARS-CoV-2 infection, Vero E6 (monkey kidney) cells were infected with SARS-CoV-2 (Source: isolated by the Virology Laboratory, University of Pecs, Hungary The test was performed in two different settings. 1., virus infection was done in the presence of CX-1 or 2., cells were first infected with the virus, then treated with CX-1. The effectiveness of CX-1 to prevent infection was evaluated by microscopic examination of the cells and virus quantification was done by RT-PCR analysis or droplet PCR analysis of culture supernatants.

Experimental Procedure

[0206] Vero E6 cells (ATCC CRL-1586) were seeded on 96-well plates. After 2 days the cell cultures reached confluency and formed a homogenous monolayer. The cells were fed with fresh cell culture medium and infected with SARS-CoV-2 at MOI 0.01 (multiplicity of infection: 1 viral particle to 100 cells). In the “prophylactic setting” virus infection was done in the presence of CX-1 and after 30 min incubation, the culture medium was removed and replaced with fresh culture medium containing CX-1. In the “therapeutic setting” cells were first infected for 30 min, then culture medium was removed and replaced with culture medium containing CX-1. CX-1 was used at 500, 100 or 20 μM or at 300, 200, 100 and 50 μM concentrations in the prophylactic and therapeutic settings, respectively. The virus stock was prepared by propagation in Vero E6 cells and the infectious titre determined by using the plaque assay. 48 hours post infection the cells were evaluated by microscopic observation. Samples from the cell culture supernatant were taken at 24 and 48 hours for quantitative RT-PCR analysis. RNA was extracted with standard methods, and for viral copy number quantification droplet digital PCR technology was applied (Bio-Rad Laboratories Inc. QX200 Droplet Digital PCR System). The primers and probes used were specific for the SARS-CoV-2 RdRp gene (Reverse primer: CARATGTTAAASACACTATTAGCATA (SEQ ID NO:2), Forward primer: GTGARATGGTCATGTGTGGCGG (SEQ ID NO:3), Probe: FAM-CAGGTGGAACCTCATCAGGAGATGC-BBQ (SEQ ID NO:4)). The droplet PCR kit was used (BioRad ddPCR™, Bio-Rad Laboratories GmbH, Germany). The results of the RT-PCR reaction were quantified and calculated as viral particle/μl.

[0207] Results:

[0208] The infection and the cytopathogenic effect of the SARS-CoV-2 virus is detected by microscopic examination of the cells. The confluent, homogenous layer of cells is disrupted and “holes” appear indicating cell death due to the virus. In the presence of CX-1, the SARS-CoV-2 infected cells were significantly protected from dying providing evidence of direct anti-viral effect in both the prophylactic (FIG. 1) and therapeutic settings (FIG. 2). The protective effect was more prominent in the prophylactic setting and dependent on the concentration of CX-1 and was the most prominent at the highest concentration (500 μM) (FIG. 1). In the therapeutic setting, it was mainly the highest concentration of CX-1 that was effective (FIG. 2). These data suggest that CX-1 was efficient to prevent the infection of cell by SARS-CoV-2 both at the first encounter and after the virus already have propagated inside cells and started infecting other cells.

[0209] The protection from viral infection was further proven by quantitative PCR analysis of the reverse transcribed viral RNA. At 24 and 48 hours, post-infection in the prophylactic setting, an approximately 82% and 95% reduction in viral particles was observed in the presence of 500 μM CX-1 (Table 1). Upon testing lower dilutions of CX-1 in the preventive setting, we observed over 90% of reduction in viral genome copy number compared to infected, but non-treated control and even 50 μM of CX-1 was effective in reducing viral genome copy number by 30% (FIG. 3).

TABLE-US-00001 TABLE 1 Results of PCR quantification of viral particle numbers in cell culture supernatants of FIG. 1; B: virus infected and C: virus infected in the presence of CX-1 500 μM at 24 and 48 hours post-infection in triplicate measurements. Sample Sample Sample #1 #2 #3 average potency 24 h virus 2.34 3.73 1.94 2.67 virus + CX-1 0.00 0.00 1.48 0.49 82% 48 h virus 229.00 156.00 255.00 213.33 virus + CX-1 11.10 15.00 8.63 11.57 95% values represent viral particle number/μl cell culture supernatant

Example 2. Preventing Infection of MucilAir™ by Influenza a Virus H1N1

[0210] The effect of repeated dose CX-1 upon influenza H1N1 infection was tested on fully differentiated human nasal epithelial cells cultured at the air-liquid interface (MucilAir™ Pool, Epithelix Sarl-primary cells from a pool of 14 different normal nasal donors).

Experimental Procedure

[0211] Antiviral effect of CX-1 against influenza H1N1 was tested as described by Boda et al (Antiviral Research 156 (2018) 72-79). Briefly, following apical wash of MucilAir™ Pool with MucilAir™ culture media (200 μl for 10 min), 10 μl of CX-1 (4-Sulfocalix[4]arene, Sigma Aldrich Cat #55523, Merck KGaA, Darmstadt, Germany) at 500 μM concentration was applied to the apical side of MucilAir™ for 10 minutes. Immediately afterwards, 100 μl of influenza H1N1 (ATCC VR-95), from a stock of 10.sup.6 genome copy number per ml) was applied on the apical side and incubated for 3 h. The inoculum was removed by washing the apical side of the cells with 200 μl of MucilAir culture media (20 min incubation) thrice. After the 3.sup.rd wash, the same concentration of CX-1 was added to the apical side of the cells at 10 μl volume and incubated for 21 h. At 24 h, 48 and 72 h timepoints MucilAir™ cell washes were repeated after which CX-1 was added on the apical side as before. The basal culture medium was also removed and replaced with 500 μl of fresh culture medium, daily. From the basal wash LDH release was measured (to assess cell death at 96 h). All incubation steps were carried out at 34° C. at 5% CO.sub.2 and at 100% humidity. Viral copy number was determined from the apical washes: for this RNA was extracted with the QIAamp® Viral RNA kit (Qiagen) and the viral RNA was quantified by RT-PCR (QuantyTect Probe RT-PCR, Qiagen) with the qTOWER3 detection system. Ct data were reported normalized to the standard curve and expressed as genome copy number/ml.

[0212] Cilia beating frequency was measured on day 4 post-infection with a dedicated system. The system consists of three parts: a camera connected to a microscope, a PCI card and a specific package of software based on Fourier spectral analysis. The Cilia beating frequency is expressed as Hz. Two hundred and fifty-six images of a movie recorded from each well containing tissues were captured at high frequency rate (125 frames per second) and cilia beating frequency was calculated using a Cilia-X software (Epithelix San, France).

[0213] As negative control, uninfected cells were included. As positive control for infection, cells infected with influenza H1N1 but treated with buffer only were used). Antiviral effect of CX-1 was compared to the effect of oseltamivir carboxylate (oseltamivir from Carbosynth (Compton, UK) at 10 μM) added to infected cells in the basolateral compartment. Compounds were tested in triplicates.

[0214] Results:

[0215] Treatment of MucilAir™ cells with CX-1 resulted in a statistically significantly decrease in viral genome copy at 24 h post-infection, compared to buffer treated cells. The decrease in the viral copy was −0.9 log (88% reduction) at 24 h (FIG. 4 panel A) and ˜0.8 log (80.5% reduction) at 48 h post infection (FIG. 4 panel B).

[0216] Additionally, CX-1 treatment preserved the active cilia beating on the epithelial cells, the cilia beating frequency (Hz) on day 4 post infection was significantly higher on CX-1 treated cell, as on buffer treated cells (FIG. 5). CX-1 showed no cytotoxicity in non-infected cells.