ANTIVIRAL USE OF LIRAGLUTIDE AND GEFITINIB

Abstract

The present invention refers to a pharmaceutical preparation comprising liraglutide or gefitinib or a salt, solvate or combination thereof, in an effective amount for use in prophylactic or therapeutic treatment of a disease condition which is caused by or associated with an infection by a coronavirus.

Claims

1. A method for prophylactic or therapeutic treatment of a disease condition which is caused by or associated with an infection by a coronavirus, comprising the step of administering a pharmaceutical preparation comprising an effective amount of liraglutide, gefitinib, or a salt or solvate thereof to a subject.

2. The method of claim 1, wherein the coronavirus is a β-coronavirus.

3. The method of claim 1, wherein the pharmaceutical preparation is a medicinal product or a drug product comprising liraglutide or gefitinib and a pharmaceutically acceptable carrier.

4. The method of claim 1, wherein the disease condition is common cold, infection of the nose, throat and larynx, sinusitis, bronchiolitis, diarrhea, rash on skin, pneumonia, acute respiratory distress syndrome (ARDS), symptoms of the central nervous system, hepatic steatosis, portal fibrosis, occurrence of lymphocytic infiltrates and ductular proliferation, lobular cholestasis, acute liver cell necrosis, central vein thrombosis, renal proximal tubular injury, focal pancreatitis, adrenocortical hyperplasia, lymphocyte depletion of spleen and lymph nodes, alveolar damage specifically characterized by edema, hyaline membranes, proliferation of pneumocytes and fibroblasts, or endothelial damage.

5. 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.

6. The method of claim 1, wherein liraglutide comprises the sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(1)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly (SEQ ID NO: 1).

7. The method of claim 6, wherein the pharmaceutical preparation comprises about 50 ng to 1 g of liraglutide.

8. The method of claim 1 wherein gefitinib comprises the following structure: ##STR00010##

9. The method of claim 8, wherein the pharmaceutical preparation comprises about 50 ng to 1 g of gefitinib.

10. The method of claim 1, wherein said pharmaceutical preparation is formulated for local administration or for systemic administration.

11. The method of claim 1, wherein said pharmaceutical preparation is administered to the subject as a spray, a powder, a gel, an ointment, a cream, a foam, a liquid solution, a lotion, a patch, a gargle solution, an aerosolized powder, an aerosolized liquid formulation, granules, or capsules.

12. The method of claim 1, wherein the pharmaceutical preparation is administered in combination with another active agent selected from the group consisting of an antiviral agent, an anti-inflammatory agent, and an antibiotic.

13. The method of claim 1, wherein the pharmaceutical preparation is administered in combination with an active agent selected from the group consisting of suramin, raloxifene, maraviroc, miglustat, quinacrine, glatiramer acetate, auranofin, and dexamethasone.

14. The method of claim 1, wherein the subject has been infected or is at risk of being infected with coronavirus.

15. A pharmaceutical preparation comprising liraglutide, gefitinib, or a salt or solvate thereof in an effective amount for prophylactic or therapeutic treatment of a disease condition which is caused by or associated with an infection by a coronavirus, further comprising an active agent selected from the group consisting of suramin, raloxifene, maraviroc, miglustat, quinacrine, glatiramer acetate, auranofin, and dexamethasone.

16. The method of claim 2, wherein the β-coronavirus is selected from the group consisting of SARS-CoV-2, MERS-CoV, SARS-CoV-1, HCoV-OC43, and HCoV-HKU1, or mutants thereof.

17. The method of claim 10, wherein said pharmaceutical preparation is formulated for nasal, pulmonary, intraoral, ocular, or dermal use or for application to the upper and lower respiratory tract.

18. The method of claim 1, wherein said pharmaceutical preparation is administered to the subject by parenteral administration.

19. The method of claim 12, wherein the pharmaceutical preparation comprises liraglutide, gefitinib, or a salt or solvate thereof as the sole active agent.

Description

FIGURES

[0069] FIG. 1: Cytokine release assay of interleukin 6 (IL-6) in response to nucleocapsid (N) stimulation or Mock stimulation as control. Immune modulation by gefitinib (GEF) alone was significantly stronger than by dexamethasone (DEX) alone. Numbers at the bottom of the boxplot represent replicate numbers of the experiments. The strongest immunomodulatory effect was observed in combination of GEF and DEX.

[0070] FIG. 2: Cytokine release assay of interleukin 17 (IL-17), tumor necrosis factor beta (TNF-beta) and IL-21 in response to nucleocapsid (N) stimulation or Mock stimulation as control. Immune modulation by gefitinib (GEF, 5 μM), liraglutide (LIR, 2 μg/ml), and dexamethasone (DEX, 5 μM) alone as well as in combination is shown relative to stimulation with the positive control resiquimod (R848, 200 ng/ml).

[0071] FIG. 3: Cytokine release assay of interleukin 6 (IL-6) in response to spike (S), spike trimer (St) or Mock stimulation as control. Immune modulation by gefitinib (GEF, 0.5 μM and 5 μM) alone is shown in comparison to pomalidomide (POM, 100 ng/ml) which does not show immune modulation. Numbers at the bottom of the bar chart represent replicate numbers of the experiments.

[0072] FIG. 4: Genes from the COVID-19 model are displayed that were up- or down-regulated 2-fold in the comparison of SARS-CoV2 nucleocapsid (i.e. upon stimulation with nucleocapsid (N) protein) vs Mock are “normalized” in their expression by both gefitinib (GEF) and liraglutide (LIR).

DETAILED DESCRIPTION

[0073] 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.

[0074] The term “about” as used herein refers to the same value or a value differing by +/−10% or +/−5% of the given value.

[0075] Compounds such as gefitinib and liraglutide 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).

[0076] 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 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.

[0077] 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.

[0078] 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. Specifically, gefitinib may be present as gefitinib hydrochloride salt.

[0079] Liraglutide may also be present in the composition in the form of base or in the form of its salts or mixtures thereof. Representative example of salts includes salts with suitable inorganic acids such as hydrochloric, hydrobromic, and the like. Representative examples of salts also include salts with organic acids such as formic acid, acetic acid, propionic acid, lactic acid, tartaric acid, ascorbic acid and the like. Representative examples of salts also include salt with base such as triethanolamine, diethylamine, meglumine, arginine, alanine, leucine, diethylethanolamine, olamine, triethylamine, tromethamine, choline, trimethylamine, taurine, benzamine, methylamine, dimethylamine, trimethylamine, methylethanolamine, propylamine, isopropylamine, adenine, guanine, cytosine, thymine, uracil, thymine, xanthine, hypoxanthine and like. Liraglutide may also be present as liraglutide acetate. In another embodiment, liraglutide is present as a tromethamine salt.

[0080] Liraglutide may also be present as functional variant or conjugate.

[0081] The term “solvate” refers to a compound in the solid state, where molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent for therapeutic administration is physiologically tolerable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water, but may also be isopropanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid and amino ethanol. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate can be dried or azeotroped under ambient conditions.

[0082] The term “effective amount” with respect to an antiviral, anti-inflammatory or anti-coagulant effect as used herein, shall refer to an amount (in particular a predetermined amount) that has a proven antiviral, anti-inflammatory or anti-coagulant effect. The amount is typically a quantity or activity sufficient to, when 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.

[0083] 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.

[0084] In the context of disease, effective amounts (in particular prophylactically or therapeutically effective amounts) of liraglutide, or gefitinib as described herein are specifically used to treat, modulate, attenuate, reverse, or affect a disease or condition that benefits from its antiviral, anti-inflammatory or anti-coagulation 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.

[0085] 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 “preventing”, 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.

[0086] The term “anti-coagulant” as used herein shall refer to any substance, drug or preparation, that effects coagulation of blood. The terms “attenuating”, “inhibiting”, “reducing”, or “preventing”, 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., prolonging clotting time.

[0087] The term “anti-inflammatory” refers to any substance, drug or preparation, that reduces inflammation or swelling.

[0088] A treatment or prevention regime of a subject with an effective amount of liraglutide, gefitinib or combination thereof described herein may consist of a single application or administration, or alternatively comprise a series of applications and administrations, respectively. For example, gefitinib, or liraglutide 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, gefitinib, or liraglutide may be used more frequently, e.g. 1-10 times a day.

[0089] Specifically, a combination therapy is provided which includes treatment with the preparation described herein and standard therapy of a coronavirus-caused disease.

[0090] 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.

[0091] 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. The agents can be in separate containers or mixed in a single container.

[0092] 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 gefitinib, liraglutide or combination thereof. 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.

[0093] Specifically, the preparation is administered in combination with one or more of suramin, raloxifene, maraviroc, miglustat, quinacrine, glatiramer acetate and auranofin.

[0094] Specifically, the preparation comprises one or more of suramin, raloxifene, maraviroc, miglustat, quinacrine, glatiramer acetate, auranofin and dexamethasone.

[0095] Suramin is an anti-parasitic gent. Suramin has the molecular formula C.sub.51H.sub.40N.sub.6O.sub.23S.sub.6 and is of following structure:

##STR00002##

[0096] Raloxifene (Evista™) is an estrogen receptor modulator and has the structure:

##STR00003##

[0097] Maraviroc, a 4,4-Difluoro-N—R1S)-3-{(1R,3s,5S)-3-[3-methyl-5-(propan-2-yl)-4H-1,2,4-triazol-4-yl]-8-azabicyclo[3.2.1]octan-8-yl}-1-phenylpropyl] cyclohexanecarboxamide is an anti-infective agent and CCR5 co-receptor antagonist. It has the structure

##STR00004##

[0098] Miglustat (N-butyl-deoxynojirimycin, N-butylmoranoline), is a small molecule effective as anti-infective agent having the following structure

##STR00005##

[0099] Quinacrine (Mepacrine) is a medication with several uses. It is related to chloroquine and mefloquine, and has the structure

##STR00006##

[0100] Glatiramer acetate (also known as Copolymer 1, Cop-1, or Copaxone) is an immunomodulator medication currently used to treat multiple sclerosis. It has the structure

##STR00007##

[0101] Auranofin (brand name Ridaura™) is an anti-inflammatory agent and has the structure

##STR00008##

[0102] Dexamethasone is a glucocorticoid medication, used in the treatment of inflammatory and autoimmune diseases and is sold under the brand names Dextenza®, Ozurdex®, Neofordex®. It has the structure

##STR00009##

[0103] Dexamethasone may be administered in the form of a tablet, specifically containing the compound in an amount of about 0.1 to 10 mg/tablet, specifically about 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg.

[0104] Dexamethasone may be administered as solution, specifically in a range of 0.1 to 5 mg/dose, specifically 0.1, 0.2, 03, 0.4, 0.5, 0.6, 0.7, 0.7, 0.9, 1, 2, 3, 5, 5 mg/dose.

[0105] The preparation described herein may be provided for single or multiple dosage use.

[0106] 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, comprising liraglutide or gefitinib or a salt, solvate or combination thereof.

[0107] 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.

[0108] The pharmaceutical preparation or medicinal product described herein is specifically provided as human or veterinary pharmaceutical composition or medicinal product. 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.

[0109] The pharmaceutical preparation 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.

[0110] Specific medicinal products or pharmaceutical compositions described herein comprise liraglutide or gefitinib or combination thereof and a pharmaceutically acceptable carrier or excipient.

[0111] 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 the like.

[0112] Liraglutide or gefitinib as used herein can be formulated with conventional carriers and excipients, which will be selected according ordinary practice.

[0113] Commercially available liraglutide and gefitinib formulations may also be used for the prophylactic or therapeutic treatment of a disease condition which is caused by or associated with an infection by a coronavirus described herein.

[0114] 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.

[0115] According to a specific aspect, liraglutide, gefitinib or a salt, solvate or combination thereof can be combined with one or more carriers appropriate a desired route of administration. Liraglutide, gefitinib or combination thereof 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, polyvinylpyrrolidone, polyvinyl alcohol, and optionally further tableted or encapsulated for conventional administration. Alternatively, liraglutide and gefitinib 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.

[0116] The compounds as described herein may be provided in controlled release pharmaceutical (“controlled release formulations”) in which the release of liraglutide or gefitinib is controlled and regulated to allow less frequency dosing or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

[0117] 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.

[0118] 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, LV, 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.

[0119] The preferred preparation is in a ready-to-use, storage stable form, with a shelf-life of at least one or two years.

[0120] The term “formulation” as used herein refers to a preparation ready-to-use in a specific way. Specifically, compositions described herein comprises liraglutide or gefitinib or a salt, solvate or combination thereof, and a pharmaceutically acceptable diluent, carrier or excipient.

[0121] Specifically, gefitinib 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, gefitinib 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 gefitinib in therapeutically useful compositions is such that a suitable dosage will be obtained.

[0122] 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.

[0123] Pharmaceutical compositions suitable for injectable use, specifically for administering liraglutide, 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.

[0124] Suitable pharmaceutically acceptable vehicles include, without limitation, any non-immunogenic pharmaceutical adjuvants suitable for oral, parenteral, nasal, mucosal, transdermal, intravascular, intraarterial, intramuscular, and subcutaneous administration routes, such as phosphate buffer saline.

[0125] Liraglutide and gefitinib may be administered simultaneously or successively.

[0126] 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.

[0127] 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, 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, liraglutide or gefitinib or a salt, solvate or combination thereof is used in subjects with a high risk, e.g. a high probability of developing disease.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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

[0132] In Vitro Testing

[0133] Peritoneal immune cells as well as peripheral blood mononuclear cells (PBMC) provide an optimal in vitro test system to copy, as a surrogate system, the sensor functions of the innate immunity against infectious agents and, in the case of PBMC, to provide the link to adaptive immunity in cell culture. The clearance and control of viruses in the body depends on the cascade-like orchestrated functions of the innate and adaptive immune system and its efficiency in overcoming viral infection. The innate immune response (Innate immunity/first line of defense mechanisms) to the majority of viral infections essentially comprises NK (Natural Killer) cells and type I interferons (IFN) as key regulators. In addition to Type I IFNs, which are mainly produced by plasmocytoid dendritic cells (pDCs), high levels of IL12 and IL18 produced by conventional DCs may play a role. Besides T cells and mast cells, other myeloid cell types such as macrophages or neutrophil granulocytes contribute to orchestrating the early response to viral infections. The composition of immune cells in the PD effluate represents a representative population for the representation of the Innate Immunity, and allows a read-out in a highly-abundant biological waste material (typically 90% monocytes/macrophages, 5% lymphocytes, 3% neutrophil granulocytes, 2% others). In several preliminary studies this circumstance was exploited to develop a clinically applicable assay of ex-vivo stimulated cytokine release for testing peritoneal immune competence (Herzog et al. Sci Rep 2017, PMID: 28740213) and successfully used as primary parameter of an interventional randomized controlled phase II study in patients undergoing peritoneal dialysis (PD) (Vychytil et al. Kidney Int 2018, 94(6) 1227-1237, PMID: 30360960). Alternative stimulants (e.g. viral antigens, live virus or pseudo-viral substances such as poly:IC) can also be used in the present assay. This assay has been adapted for viral infections (stimulation conditions, read-out) and thus represents a novel tool. In combination, these well-established in vitro test systems form a read-out system that has proven to be a valuable tool for the analysis of immunological activity profiles against bacterial and viral agents both for the innate immunity and for the interface to the adaptive immunity (Sadeghi et al. J Infect Dis 2007 PMID:17191175; Sadeghi et al PLoS ONE 2016, PMID:27695085; Wisgrill et al J Leukoc Biol 2016 PMID:26965638; Wisgrill et al J Leukoc Biol 2019 PMID:31211458). This immune-profiling system is applied to a SARS-CoV-2 infection model.

[0134] Culture and Stimulation of Cells

[0135] PBMCs/ml (1×10.sup.6) were cultured in AIM-V cell culture medium (Thermo Fisher Scientific), supplemented with IL-3 (10 ng/ml; PeproTech, Rocky Hill, N.J., USA). Cells were stimulated with SARS-CoV2 proteins (spike or nucleocapsid) or left untreated (mock) for 4-6 or, 20-22 hrs. Cultures were incubated in a humidified 5% CO.sub.2 environment at 37° C. For intracellular cytokine experiments, 1.5 M monensin was added additionally after 2 h to all wells to block intracellular protein transport. After 8, 20, or 40 h, cells were harvested on ice, and supernatants were frozen at −80° C. for further cytokine analysis with ELISA (Schüller S. et al. (J. Leukocyte Biol., 93, 2013, 781-787).

[0136] Cytokine Release Assay

[0137] Examples are based on well-established in-vitro challenge model (infectious encounter with primary human immune cells) as described above. In order to characterize the ‘inflammatory signatures’ driven by SARS-CoV2, multiplex protein analyzes (qualitative and quantitative) and the molecular patterns were analyzed using multi-omics applications.

[0138] Peripheral blood mononuclear cells (PBMC) were used as a surrogate system for the sensor functions of the bodies innate immune system against the infectious agent SARS-CoV2 and the first-line-of-defense immunological activity profile is monitored using the harvested cell culture supernatants and frozen cells, respectively. To this end, a well-established assay system was further developed by using commercially available SARS-CoV2 antigens (spike protein and nucleocapsid protein) and the PBMC from SARS-CoV2 naive donors were incubated with these surrogate infectious challenges. The cell culture supernatants were qualitatively and quantitatively examined for inflammatory and regulatory proteins, mainly cytokines. Furthermore, this cell culture approach was expanded in order to test immunomodulation with approved therapeutic agents, which previously had been defined using a digital algorithm as substance screen for anti-inflammatory agents.

[0139] Interleukin 6 was defined as one of the most relevant read-out parameters, since the COVID-19-associated inflammation is also monitored in sick patients using IL-6. In addition, further inflammatory and anti-inflammatory/regulatory factors were measured in few selected samples, in order to define additional parameters possibly relevant during immunomodulation for ongoing larger experiments. These extended inflammatory/regulatory signatures shall be used for eventual future clinical studies.

[0140] FIG. 1 shows release of interleukin 6 (IL-6) in response to nucleocapsid (N) stimulation or Mock stimulation as control. Immune modulation by gefitinib (GEF) alone was stronger than by dexamethasone (DEX) alone. The strongest immunomodulatory effect was observed in combination of GEF and DEX.

[0141] FIG. 2 shows release of further inflammatory and anti-inflammatory/regulatory factors, such as interleukin 17 (IL-17), tumor necrosis factor beta (TNF-beta) and IL-21 in response to nucleocapsid (N) stimulation or Mock stimulation as control. Immune modulation by gefitinib (GEF, 5 μM), liraglutide (LIR, 2 μg/ml), and dexamethasone (DEX, 5 μM) alone as well as in combination is shown relative to stimulation with the positive control resiquimod (R848, 200 ng/ml).

[0142] FIG. 3 shows release of interleukin 6 (IL-6) in response to spike (S), spike trimer (St) or Mock stimulation as control. Immune modulation by gefitinib (GEF, 0.5 μM and 5 μM) alone is shown in comparison to pomalidomide (POM, 100 ng/ml) as an example of a compound that was not identified with the digital substance screen, and which does not show immune modulation in this assay.

Example 2

[0143] Transcription Fingerprinting by 3′ RNA-Sequencing

[0144] NGS Library Preparation (Lexogen QuantSeq 3′mRNA-Seq)

[0145] The amount of total RNA was quantified using the Qubit 4.0 Fluorometric Quantitation system (Thermo Fisher Scientific, Waltham, Mass., USA) and the RNA integrity number (RIN) was determined using the 2100 Bioanalyzer instrument (Agilent, Santa Clara, Calif., USA). RNA-seq libraries were prepared with the QuantSeq 3′ mRNA-Seq Library Prep Kit (FWD) for Illumina (Lexogen, Vienna, Austria). Library concentrations were quantified with the Qubit 4.0 Fluorometric Quantitation system (Life Technologies, Carlsbad, Calif., USA) and the size distribution was assessed using the 2100 Bioanalyzer instrument (Agilent, Santa Clara, Calif., USA). For sequencing, samples were diluted and pooled into NGS libraries in equimolar amounts.

[0146] Next-Generation Sequencing and Raw Data Acquisition

[0147] Expression profiling libraries were sequenced on HiSeq 3000/4000 instruments (Illumina, San Diego, Calif., USA) following a 50-base-pair, single-end recipe. Raw data acquisition (HiSeq Control Software, version 3.4.0.38) and base calling (Real-Time Analysis Software, version 2.7.7) was performed on-instrument, while the subsequent raw data processing off the instruments involved two custom programs. In a first step, base calls were converted into lane-specific, multiplexed, unaligned BAM files suitable for long-term archival. In a second step, archive BAM files were demultiplexed into sample-specific, unaligned BAM files.

[0148] Transcriptome Analysis

[0149] NGS reads were mapped to the Genome Reference Consortium GRCh38 assembly via “Spliced Transcripts Alignment to a Reference” (STAR) utilising the “basic” Ensembl transcript annotation from version e100 (April 2020, Dobin et al., Bioinformatics, 2013, 29(1), 15-21) as reference transcriptome. STAR was run with options recommended by the ENCODE project. Aligned NGS reads overlapping Ensembl transcript features were counted with the Bioconductor (version 3.12) GenomicAlignments (version 1.26.0) package via the summarizeOverlaps function in Union mode, taking into account that the Quant-seq protocol leads to sequencing of the second strand so that all reads needed inverting before counting. Transcript-level counts were aggregated to gene-level counts and the Bioconductor DESeq2 (1.30.0, Love M I, et al (2014), Genome Biology, 15, 550.) package was used to test for differential expression based on a model using the negative binomial distribution.

[0150] Results

[0151] FIG. 4 shows that those genes from the COVID-19 model that were up- or down-regulated 2-fold in the comparison of SARS-CoV2 nucleocapsid (i.e. upon stimulation with nucleocapsid (N) protein) vs Mock are “normalized” in their expression by both GEF and LIR.

[0152] For this purpose, the genes that were at least 2-fold up- or 2-fold down-regulated in the comparison of N vs Mock were extracted and compared to N+GEF vs N as well as N+LIR vs N for the same genes, respectively.

[0153] The boxplots show that the regulation was clearly reversed in its direction, e.g. the up-regulated genes at time 4 h N vs Mock were strongly down-regulated in the comparisons N-LIR vs N and N-GEF vs N).

[0154] Many of the genes represented in the proprietary molecular disease pathophysiology model are in fact reversed in their regulation compared to stimulation with SARS-CoV2 nucleocapsid. Genes that were upregulated in the comparison of nucleocapsid (N) stimulation vs Mock were downregulated through the effect of gefitinib (GEF) and/or liraglutide (LIR) or combinations with dexamethasone (DEX). Genes that were downregulated in the comparison of nucleocapsid (N) stimulation vs Mock were upregulated by GEF and/or LIR and/or or combinations with DEX.