WO2023180567A1 - Cyclodextrins for use in coronavirus infection therapy - Google Patents

Abstract

The present invention relates to cyclodextrins for use in preventing, inhibiting the progression or treating Coronavirus infections, administered intranasally. Pharmaceutical compositions comprising cyclodextrins and combinations of cyclodextrins are provided. Kits including cyclodextrins, combinations of cyclodextrin, and their pharmaceutical composition are also described.

Description

Cyclodextrins for use in Coronavirus infection therapy

Field of the Invention

The present invention relates to cyclodextrins for use in preventing, inhibiting the progression or treating Coronavirus infections, administered nasally. Pharmaceutical compositions comprising cyclodextrins and combinations of cyclodextrins are provided. Kits including cyclodextrins, combinations of cyclodextrins, and their pharmaceutical compositions are also described.

Background of the Invention

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus which has caused a respiratory disease pandemic that has now spread across the world (Chen N, et al., Lancet 2020; 395:507-513). To date, remdesivir is the only approved antiviral drug for the specific treatment of this coronavirus infectious disease 2019 or CO VID- 19 (Beigel J, et al. , N Engl J Med 2020; 383: 1813-1826; Grein J, etal., N Engl J Med 2020; 382:2327). However, several drugs are being used in the frontline of clinical management of SARS-CoV-2-infected individuals in hospitals all around the world, to try to avoid the development of the COVID-19 associated pneumonia, which can be fatal. Over 6 million people had died from CO VID-19, and almost 465 million cases have been reported (https://covidl9.who.int, March 2020).

Although different drug regimens are being applied to hospitalized patients, no clinical study has evidenced their efficacy yet. Under this scenario, initiatives launched by the World Health Organization (WHO), such as the SOLIDARITY study that has compared remdesivir, hydroxychloroquine, ritonavir/lopinavir and ritonavir/lopinavir plus B-interferon regimes, have been of critical importance to prioritize the use of the most active compounds (WHO, 2020). Unfortunately, although remdesivir has proven efficacy in randomized controlled trials (Beigel, 2020, supra,' Grein, 2020, supra), a recent update of the WHO clinical trial has failed to detect any effect on overall mortality, initiation of ventilation and duration of hospital stay with any of the antivirals tested (Pan H, BioRxiv 2020; 17, doi: 10.1101/2020.10.15.20209817). Thus, there is an urgent need to identify novel therapeutic approaches for individuals with COVID-19 developing severe disease and fatal outcomes. Summary of the Invention

In one aspect, the present invention refers to cyclodextrin for use in preventing, inhibiting the progression or treating a Coronavirus infection (e.g., SARS-CoV-2 or its variants) in a subject in need thereof, wherein said cyclodextrin is administered nasally or orally, preferably nasally. In some embodiments, the cyclodextrin of the invention is effective in preventing or inhibiting the cellular entry of the Coronavirus. In some embodiments, the cyclodextrin is a P-cyclodextrin (e.g., methyl -P-cyclodextrin).

In another aspect, the present invention refers to a pharmaceutical composition comprising at least one cyclodextrin (e.g., methyl-P-cyclodextrin) or a combination of cyclodextrins according to the invention for use in the prevention, inhibition of progression or treatment of a Coronavirus infection (e.g., SARS-CoV-2 or its variants), wherein said composition is administered nasally or orally, preferably nasally.

In a further aspect, the present invention refers to a method for preventing, inhibiting the progression or treating a Coronavirus infection (e.g., SARS-CoV-2 or its variants) in a subject in need thereof which comprises administering intranasally a therapeutically effective amount of (a) at least one cyclodextrin or a combination thereof or (b) a pharmaceutical composition according to the invention or a combination thereof to the subject. Alternatively, the invention refers to the use of a cyclodextrin for the manufacture of a medicament for use in preventing, inhibiting the progression or treating a Coronavirus infection in a subject in need thereof, wherein said medicament is administered nasally or orally, preferably nasally.

In an additional aspect, the present invention refers to kits comprising (a) at least one cyclodextrin, (b) a combination of cyclodextrins, and/or (c) a pharmaceutical composition comprising at least one cyclodextrin according to the invention or a combination thereof, and a device for the nasal administration.

Brief Description of the Drawings

Figure 1. Antiviral activity of drugs against HCoV-229E and SARS-CoV-2. (A) Dose-response curves (top lines) against HCoV-229E of HpCD were determined by nonlinear regression. Data is shown as mean ± S.E.M. of 3 biological replicates. Cytotoxic effect on MRC-5 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (bottom lines). (B) Cytopathic effect of SARS-CoV-2 on Vero E6 cells exposed to SARS-CoV-2 at 200 TCID50/mL in the presence of increasing concentrations of Hb-CD (hydroxypropyl-P-cyclodextrin) (20 - 0.00026 mM). Non-linear fit to a variable response curve from one representative experiment out of three with two replicates is shown (top lines), excluding data from drug concentrations with associated toxicity. Cytotoxic effect with the same drug concentrations in the absence of virus is also shown (bottom lines). The IC50 value is indicated on each graph. (C) Cytopathic effect of SARS-CoV-2 on Vero E6 cells exposed to different variants of concern of SARS-CoV-2 (all at 200 TCID50/mL) in the presence of increasing concentrations of Hb-CD (20 - 0.00026 mM). Non-linear fit to a variable response curve from one representative experiment out of two with two replicates is shown (top lines), excluding data from drug concentrations with associated toxicity. Cytotoxic effect with the same drug concentrations in the absence of virus is also shown (black bottom lines). (D) Effect Hb-CD on luciferase expression of reporter lentiviruses pseudotyped with SARS-CoV-2 Spike in ACE2 expressing HEK-293T cells. Cells were exposed to fixed amounts of SARS-CoV-2 Spike lentiviruses. Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and standard deviation from two experiments with two replicates each are represented, excluding cytotoxic values. (E) Viral replication of SARS-CoV-2 assessed on CaLu-3 cells in the presence of HbCD. After 24h of incubation, cells were washed, and compounds were added at the same final concentration for 48h. Supernatants were tested for viral release by detecting nucleocapsid concentration. Represented values are normalized to the nucleocapsid concentration by mock-treated cells set at 100%, which corresponds to raw values of 5716 ± 2237 pg/mL (mean ± SD). Mean and standard deviations from four experiments are represented, excluding cytotoxic values.

Figure 2. Members of the Cyclodextrin family inhibit pseudovirus entry in ACE2-293T cells and SARS-CoV-2 infection in pulmonary cells. (A) Relative viral entry of SARS-CoV-2 pseudoviruses in the presence of the indicated cyclodextrins in ACE2 expressing HEK-293T cells. Cells were exposed to fixed amounts of SARS-CoV-2 Spike lentiviruses in the presence of decreasing drug concentrations. Values show luciferase expression of the reporter lentiviruses pseudotyped with SARS-CoV-2, normalized to the luciferase expression of mock-treated cells (set at 100%). Mean and standard deviation from two experiments with two replicates each are represented, excluding cytotoxic values. (B) Relative viral replication of D614G (C) or Omicron (D) SARS-CoV-2 variant was assessed on CaLu-3 cells in the presence of the indicated cyclodextrins. After 24h of adding virus and drugs at the indicated concentrations, cells were washed and compounds were added at the same final concentration for additional 48h. Then supernatants were tested for viral release by detecting SARS-CoV-2 nucleocapsid concentration by ELISA. Values are normalized to the nucleocapsid concentration by mock-treated cells (set at 100%), which reached 5716 ± 2237 pg/mL (mean ± SD). Mean and standard deviation from three experiments are represented, excluding cytotoxic values.

Figure 3. Cyclodextrins inhibit SARS-CoV-2 replication by interfering with viral fusion via cholesterol depletion. (A) MP™ activity measured in presence of increasing concentrations of methyl-P-CD (M0CD) or GC376 as positive inhibitor. Results are represented as the percentage of inhibition of M^pro activity in the absence of drugs. (B) Lipidomic measurement of plasma membranes from Calu3 cells treated or not with M0CD for 2h at 37°C and 5% CO2.

Figure 4. Methyl-P-cyclodextrin inhibit SARS-CoV-2 replication in a human nasal epithelial model. (A) Schematic representation of the HNE model used, showing the apical side, the basal medium and the cells cultured in the air-liquid interphase. (B) SARS- CoV-2 replication in the HNE model in the presence of 2.5mM M0CD (methyl- - cyclodextrin) either on the apical side (dark triangle) or on the basal medium (grey triangle), or 2.5 uM Remdesivir (square) on the basal medium, without drugs (bottom dots), or in the absence of virus (top dots). SARS-CoV-2 was added to the apical side for Ih, extensively washed afterwards, and nucleocapsid concentration was measured by ELISA at 24, 48 and 72 hpi. Results from one experiment.

Figure 5. Cytotoxicity of drugs tested against SARS-CoV-2. (A top) Cytotoxicity of indicated drugs at decreasing concentrations in ACE2 expressing HEK-293T cells. At 48 h cells were lysed with the Glo Luciferase system (Promega) and luminescence was measured with a plate reader giving relative light units (RLUs). Cytotoxic concentrations are indicated with an arrow. Mean and standard deviation from one experiment with two replicates is represented. (B bottom) Cytotoxicity of indicated drugs at decreasing concentrations on Calu-3. After 24h of adding virus and drugs at the indicated concentrations, cells were washed and drugs were added at the same final concentration for an additional 48h. Then cells were lysed with the Glo Luciferase system (Promega) and luminescence was measured with a plate reader giving relative light units (RLUs). Cytotoxic concentrations are indicated with an arrow. Mean and standard deviation from one experiment with two replicates is represented. (C) Cytotoxicity of indicated cyclodextrins at decreasing concentrations in ACE2 expressing HEK-293T cells. At 48 h cells were lysed with the Glo Luciferase system (Promega) and luminescence was measured with a plate reader giving relative light units (RLUs). Cytotoxic concentrations are indicated with an arrow. Mean and standard deviation from two experiments with two replicates each are represented. (D) Cytotoxicity of indicated cyclodextrins at decreasing concentrations on Calu-3. After 24h of adding virus and drugs at the indicated concentrations, cells were washed and drugs were added at the same final concentration for an additional 48h. Then cells were lysed with the Gio Luciferase system (Promega) and luminescence was measured with a plate reader giving relative light units (RLUs). Cytotoxic concentrations are indicated with an arrow. Mean and standard deviation from one experiment with two replicates is represented. Mean and standard deviation from three experiments are represented. (E) Cytopathic effect of M0CD, on the apical side or the basal medium, and Remdesivir as control, in the human nasal epithelial model (Mucilair™). At 72h cells were lysed with the Gio Luciferase system (Promega) and luminescence was measured with a plate reader giving relative light units (RLUs). One experiment is represented.

Figure 6. Methyl-P-cyclodextrin nasal application inhibits SARS-CoV-2 replication in a hamster model. Hamsters were intranasally administered with or without methyl- -cyclodextrin at 100 mM and challenged with a SARS-COV-2 Nanoluciferase reporter virus. Control uninfected animals were also assayed. (A-B) At 1 or 2 days postinfection, nasal turbinates and lungs collected from euthanized animals were lysed with the Nano-glo Luciferase system (Promega) and luminescence was measured with a plate reader in relative light units (RLUs). (C-D) At 1 or 2 days post-infection, nasal turbinates and lungs collected from euthanized animals were analyzed for viral RNA presence in inverted CTs by qPCR.

Detailed Description of the Invention

The present invention discloses that cyclodextrins, and in particular, methyl- - cyclodextrin, prevent or inhibit the cellular entry of Coronavirus in pulmonary epithelial cells when administered intranasally. Thus, this new application of cyclodextrins could be useful in preventing, inhibiting the progression, or treating coronavirus infection, such as that by SARS-CoV-2 and its variants or by alpha-coronavirus, such as HCoV-229E.

A list of effective compounds with proven antiviral efficacy in vitro to halt SARS- CoV-2 replication was prepared by the inventors. The compounds were analyzed depending on their expected mechanism of action, to identify candidates tackling diverse steps of the viral life cycle. SARS-CoV-2 entry requires viral binding and spike protein activation via interaction with the cellular receptor ACE2 and the cellular protease TMPRSS2 at the plasma membrane (Hoffmann M, et aL, Cell 2020; 181 :271-280). Interference with either of these ligands has proven to decrease SARS-CoV-2 infectivity (Monteil V, et al., Cell 2020;181(4):905-913; Hoffmann, 2020, supra), and therefore, inhibitors targeting viral entry may prove valuable. In addition, SARS-CoV-2 enters into the cells via endocytosis and accumulates in endosomes where cellular cathepsins can also prime the spike protein and favor viral fusion upon cleavage (Simmons G, et al., Proc Natl Acad Sci 2005; 102: 11876- 11881; Mingo R, et al., J Virol 2015; 89:2931-2943; Hoffmann, 2020, supra), providing additional targets for antiviral activity. Once SARS-CoV-2 fuses with cellular membranes, it triggers viral RNA release into the cytoplasm, where polyproteins are translated and cleaved by proteases (Song Z, et al., Viruses 2019; 11 : 59). This leads to the formation of an RNA replicase — transcriptase complex driving the production of several negative — stranded RNA via both replication and transcription (Song, 2019, supra). Numerous negative- stranded RNAs transcribe into messenger RNA genomes, allowing for the translation of viral nucleoproteins, which assemble in viral capsids at the cytoplasm (Song, 2019, supra). These capsids then bud into the lumen of endoplasmic reticulum (ER)-Golgi compartments, where viruses are finally released into the extracellular space by exocytosis. Potentially, any of these viral cycle steps could be targeted with antivirals, thus the inventors searched for these compounds as well.

Finally, as the most effective antiviral treatments are usually based on combined therapies that tackle distinct steps of the viral life cycle, the active compounds were also tested in combination. These combinations may be critical to abrogate the potential emergence of resistant viruses and to increase antiviral activity, enhancing the chances to improve clinical outcome. However, surprisingly, methyl-P-cyclodextrin showed the most promise in preventing the viral entry of SARS-CoV-2 and its variants in pulmonary epithelial cells, in a nasal epithelium model and even in preventing infection in an in vivo hamster model, when administered nasally.

1. Definitions of general terms and expressions

The term “comprising” or “comprises”, as used herein, discloses also “consisting of’ according to the generally accepted patent practice.

The term “Coronavirus”, as used herein, refers to any member of the Coronaviridae viral family. The Coronaviridae family includes single-stranded RNA viruses, about 120 nanometers in diameter. The family is divided in two subfamilies: Letovirinae and Coronavirinae. The Coronavirinae subfamily comprises the Alphacoronavirus (e.g., human coronavirus 229E (HCoV-229E), Betacoronavirus (e.g., human coronavirus HKU1, human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), human coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV or HCoV-EMC, the causative agent of MERS), severe acute respiratory syndrome coronavirus (SARS-CoV, the causative agent of SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the causative agent of COVID-19), Deltacoronavirus, and Gammacoronavirus genus. Coronaviruses can also infect non-human subjects such as, for example, cattle (e.g., bovine coronavirus (BCV), cats (e.g., feline coronavirus (FCoV), dogs (e.g., canine coronavirus (CCoV), pigs (e.g., porcine coronavirus HKU15, porcine epidemic diarrhea virus (PED or PEDV), rabbits (e.g., rabbit enteric coronavirus), and birds (e.g., infectious bronchitis virus (IBV), turkey coronavirus (TCV)). There are more than 40 species of Coronaviruses. Examples of SARS-CoV-2 functionally equivalent variants include, but are not limited to B. l (D614G) isolated in Spain in March 2020 (EPI ISL 510689); and 4 VOC isolated in Spain from January to February 2021 : Alpha or B. l.1.7 (EPI ISL 1663567), Beta or B. l.351 (originally detected in South Africa; EPI ISL 1663571), Gamma or P. l (originally detected in Brazil; EPI ISL 1831696) and Delta or B. l.617.2 (originally detected in India; EPI ISL 3342900).

The term “cyclodextrin”, as used herein, refers to cyclic oligosaccharides made up of a number of dextrose units of (a-l,4)-linked a-D-glucopyranose. These cyclic structures contain a lipophilic central cavity and a hydrophilic outer surface. Cyclodextrins are made up of six, seven or eight dextrose units (i.e., a-, P-, and y-cyclodextrins, respectively). Cyclodextrins interact with hydrophobic drug molecules to form inclusion complexes. Examples of P-cyclodextrins include, but are not limited to, sulfobutylether-P-cyclodextrin, 2-hydroxypropyl-P-cyclodextrin (herein as Hb-CD or as 2HP-Beta-CD), and methyl-P- cyclodextrin (herein as MpCD or as Methyl- Beta-CD).

The expression “functionally equivalent variant”, as used herein, refers to a polynucleotide resulting from the modification, deletion or insertion or one or more bases and which substantially preserves the activity of its reference polynucleotide. Functionally equivalent variants contemplated in the context of the present invention, include polynucleotides showing at least 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% of similarity or identity with polynucleotide sequences of the SARS-CoV-2 or its variants B.l (D614G), 4 VOC, B. l.1.7, B.l.351, P.l, or B.1.617.2. The degree of identity or similarity between two polynucleotides is determined by using computer-implemented algorithms and methods that are widely known in the art.

The terms “identical” or percent “identity” in the context of two or more nucleic acids refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same when compared and aligned (introducing gaps, if necessary) for maximum correspondence. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art which can be used to obtain alignments nucleotide sequences. Examples of algorithms suitable for determining sequence similarity include, but are not limited to, the BLAST, Gapped BLAST, and BLAST 2.0, WU-BLAST-2, ALIGN, and ALIGN-2 algorithms (Altschul S, et al., Nuc. Acids Res. 1977; 25:3389-3402, Altschul S, et al., J. Mol. Biol. 1990; 215:403-410, Altschul S, et al., Meth. Enzymol. 1996; 266:460- 480, Karlin S, et al., Proc. Natl. Acad. Sci. USA 1990; 87:2264-2268, Karlin S, et al., Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877, Genentech Corp, South San Francisco, CA, US, https://blast.ncbi.nlm.nih.gov/Blast.cgi, March 2022). Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for instance, by the Smith-Waterman local homology algorithm, by the Needleman-Wunsch homology alignment algorithm, by the Pearson-Lipman similarity search method, by computerized implementations of these algorithms or by manual alignment and visual inspection (Smith T, et al., Adv. Appl. Math. 1981; 2:482-489, Needleman S, et al., J. Mol. Biol. 1970; 48:443-453, Pearson W, et al., Lipman D, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448, the GAP, BESTFIT, FASTA and TFASTA programs, Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, USA; Ausubel F, etal., Eds., Short Protocols in Molecular Biology, 5th Ed. (John Wiley and Sons, Inc., New York, NY, USA, 2002)).

The term “kit”, as used herein, refers to a product containing the different reagents necessary for carrying out the uses and methods of the invention which is packed so as to allow their transport and storage. Materials suitable for packing the components of the kit include crystal, plastic (e.g., polyethylene, polypropylene, polycarbonate), bottles, vials, paper or envelopes.

The term “methyl-P-cyclodextrin” or “MpCD” or “Methyl -Beta-CD”, as used herein, refers to a P-cyclodextrin heptasaccharide, soluble in water, with 1310 Da (average) molecular weight (CAS [128446-36-6]). M|3CD is used for improving the water-solubility and bioavailability of drugs.

The expression “pharmaceutically acceptable carrier”, as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible with the cyclodextrins of the invention.

The terms “prevent,” “preventing” and “prevention”, as used herein, refer to inhibiting the inception or decreasing the occurrence of a disease in a subject. The prevention may be complete (e.g., the total absence of pathological cells in a subject). The prevention may also be partial, such as, for example, lowering the occurrence of pathological cells in a subject. Prevention also refers to a reduced susceptibility to a clinical condition. Within the context of the present invention, the terms “prevent,” “preventing” and “prevention”, refer specifically to averting or reducing the probability of that a subject develops a Coronavirus infection (e.g., SARS-CoV-2 or its variants) disease or any of the consequences related to a Coronavirus infection (e.g., pulmonary distress).

The term “subject”, as used herein, refers to an individual or animal, such as a human, a nonhuman primate (e.g., chimpanzees and other apes and monkey species); farm animals, such as birds, fish, cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals including rodents, such as mice, rats, and guinea pigs. The term does not denote a particular age or sex. The term “subject” encompasses an embryo and a fetus. In some embodiments, the subject is a human.

The term “therapeutically effective amount”, as used herein, refers to the dose or amount of a cyclodextrin or combination thereof according to the present invention or the pharmaceutical compositions of the present invention that produce a therapeutic response or desired effect in a subject.

The term “topical”, and the related term “topically” as used herein refer to any administration of a cyclodextrin (e.g., M|3CD) or combination thereof according to the present invention or a pharmaceutical composition comprising said cyclodextrin or combination by applying the cyclodextrin, combination or pharmaceutical composition to a particular place on or in the body, such as the skin or a mucous membrane. Topical administration includes, but is not limited to, the aural, cutaneous, nasal, transdermal, urethral, vaginal, and urethral routes of administration. The term “treat” or “treatment”, as used herein, refers to the administration of at least one cyclodextrin, a combination of cyclodextrin or a pharmaceutical composition according to the present invention for controlling the progression of a disease after its clinical signs have appeared. Control of the disease progression is understood to mean the beneficial or desired clinical results that include, but are not limited to, reduction of the symptoms, reduction of the duration of the disease, stabilization of pathological states (specifically to avoid additional deterioration), delaying the progression of the disease, improving the pathological state and remission (both partial and total). The control of progression of the disease also involves an extension of survival compared with the expected survival if treatment was not applied. Within the context of the present invention, the terms “treat” and “treatment” refer specifically to stopping or slowing the consequences (e.g., pulmonary distress) of a Coronavirus infection (e.g., SARS-CoV-2 or its variants) in a subject afflicted with such syndrome or disease. “Treatment” can also mean prolonging survival of a subject afflicted with a Coronavirus infection as compared to the expected survival of the subject if the subject does not receive any of the cyclodextrin or pharmaceutical compositions according to the present invention.

2. Cyclodextrins

In a first aspect, the present invention refers to cyclodextrin for use in preventing, inhibiting the progression or treating a Coronavirus infection in a subject in need thereof, wherein said cyclodextrin is administered nasally or orally, preferably nasally. In a preferred embodiment of the first aspect, cyclodextrin is administered as sole active ingredient or, if combined with other active ingredients, said other active ingredients are other than hydroxytyrosol, niclosamide or quercetin.

In a preferred embodiment, cyclodextrin is used for the prevention of a coronavirus infection. In some embodiments, the cyclodextrin of the invention is effective in preventing or inhibiting the cellular entry of the Coronavirus. In some embodiments, the target cell is an epithelial cell, preferably a nasal epithelial cell. In some embodiments, the epithelial cell is a pulmonary cell. In some embodiments, the pulmonary cell is an alveolar cell. In some embodiments, the alveolar cell is a Type I or Type II cell. In some embodiments, the alveolar cell is a macrophage.

In some embodiments, the cyclodextrins of the invention comprise a-, P-, y- cyclodextrin or a combination thereof. In some embodiments, the P-cyclodextrin of the invention comprise sulfobutylether-P-cyclodextrin, 2-hydroxypropyl -P-cyclodextrin, methyl-P-cyclodextrin or a combination thereof. In a preferred embodiment, the cyclodextrin is at least one P-cyclodextrin. Preferably the P-cyclodextrin is hydroxypropyl P-cyclodextrin or methyl-P-cyclodextrin, or a combination thereof. Preferably, the cyclodextrin is methyl- P-cyclodextrin.

In some embodiments, the virus is a member of Coronaviridae family or a combination thereof. In some embodiments, the virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a functionally equivalent variant thereof. In some embodiments, the SAR-CoV-2 variants comprise B.l (D614G), 4 VOC, B.l.1.7, B.1.351, P.1, B.1.617.2, a combination thereof, or a functionally equivalent variant thereof. In another embodiment, the virus is alphacoronavirus HCoV-229E (see figure 2B). The present invention is thus useful in preventing viral infection in very different coronavirus, such as alpha and beta coronavirus.

3. Pharmaceutical compositions

In a second aspect, the present invention refers to a pharmaceutical composition comprising at least one cyclodextrin or a combination of cyclodextrins according to the invention for use in the prevention, inhibition of progression or treatment of a Coronavirus infection (e.g., SARS-CoV-2 or its variants) wherein said composition is administered nasally or orally, preferably nasally. In a preferred embodiment of the second aspect, cyclodextrin is the sole active ingredient in the composition or, if combined with other active ingredients, said other active ingredients are other than hydroxytyrosol, niclosamide or quercetin. In a preferred embodiment, the composition is used for the prevention of a coronavirus infection. In a preferred embodiment of the second aspect, the composition is in the form of a nasal spray, nasal solution or nasal suspension. The compositions of the first aspect are administered into the nasal cavity. Pharmaceutical compositions for use according to the present invention typically comprise an effective amount of at least on cyclodextrin or a combination thereof and at least one pharmaceutical acceptable carrier. The preparations may be prepared in a manner known in the art, which usually involves mixing the at least one cyclodextrin according to the invention with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary, under aseptic conditions. See US6372778, US6369086, US6369087, US6372733 and Remington: The Science and Practice of Pharmacy, 21st Ed. (Pharmaceutical Press, Philadelphia, PA, USA, 2011). Generally, for pharmaceutical use, the compounds may be formulated as a pharmaceutical preparation comprising at least one compound and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.

The pharmaceutical preparations of the present invention are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Preferably, the compositions of the present invention are packaged in a suitable device for nasal administration of the composition, Such as in the form of a nasal solution, drops, spray, powder inhaler, aerosol, etc.

A “therapeutically effective amount” of the cyclodextrin or combination thereof would generally be administered to the subject in need thereof.

Depending upon the manner of introduction, the cyclodextrin or combinations thereof of the invention may be formulated in a variety of ways. Formulations containing one or more cyclodextrin can be prepared in various pharmaceutical forms including, but not limited to, granules, tablets, capsules, powders, suspensions, emulsions, creams, gels, ointments, salves, lotions or aerosols. In some embodiments, these formulations are employed in solid dosage forms suitable for simple, and preferably oral, administration of precise dosages. Solid dosage forms for oral administration include, but are not limited to, tablets, soft or hard gelatin or non-gelatin capsules, and caplets. However, liquid dosage forms, such as solutions, syrups, suspension, and shakes can also be utilized. In some embodiments, the formulation is administered topically. Suitable topical formulations include, but are not limited to, lotions, ointments, creams, and gels. In some embodiments, the topical formulation is a gel. In all embodiments, the formulation is administered orally or intranasally.

Formulations containing one or more of the cyclodextrin of the present invention may be prepared using a pharmaceutically acceptable carrier composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. As generally used herein “carrier” includes, but is not limited to, diluents, binders, lubricants, disintegrators, fillers, pH modifying agents, preservatives, antioxidants, solubility enhancers, and coating compositions.

If desired, the composition may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents or preservatives.

The concentration of the compound(s) to carrier and/or other substances may vary from about 0.5 to about 100 wt % (weight percent). For oral use, the pharmaceutical formulation will generally contain from about 5 to about 100% by weight of the active material. For other uses, the pharmaceutical formulation will generally have from about 0.5 to about 50 wt % of the active material.

The cyclodextrin, combinations and pharmaceutical compositions of the invention can be administered adjunctively with other active compounds. These compounds include but are not limited to analgesics, antipyretics, antidepressants, antiepileptics, antihistamines, antimigraine drugs, antimuscarinics, antivirals, anxiolytics, sedatives, hypnotics, antipsychotics, bronchodilators, anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics, electrolytes, gastro-intestinal drugs, muscle relaxants, nutritional agents, vitamins, parasympathomimetics, stimulants, anorectics, and anti-narcoleptics. “Adjunctive administration”, as used herein, means the compounds can be administered in the same dosage form or in separate dosage forms with one or more other active agents.

4. Methods of treatment and prevention

In a further aspect, the present invention refers to a method for preventing, inhibiting the progression or treating a Coronavirus infection in a subject in need thereof which comprises orally or nasally, preferably intranasally administering a therapeutically effective amount of (a) at least one cyclodextrin or a combination thereof or (b) a pharmaceutical composition according to the invention or a combination thereof to the subject. Alternatively, the invention refers to the use of a cyclodextrin for the manufacture of a medicament for use in preventing, inhibiting the progression or treating a Coronavirus infection in a subject in need thereof, wherein said medicament is administered orally or nasally, preferably nasally. In some embodiments, the subject is human.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art. The amount of active agent which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active agent which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, this amount will range from about 0.001% to about 90% of active agent, preferably from about 0.005% to about 70% and, most preferably, from about 0.01% to about 30%.

Actual dosage levels of cyclodextrin, combinations of cyclodextrin and pharmaceutical compositions according to the present invention may be varied for attaining the desired therapeutic response in a subject. If desired, the therapeutically effective daily dose the cyclodextrin, combinations of cyclodextrin and pharmaceutical compositions according to the present invention may be administered as two, three, four, five, six or more sub-doses applied separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for an active agent of the invention to be administered alone, it is preferable to administer said agent as a pharmaceutical composition.

5. Kits

In an additional aspect, the present invention refers to kits comprising, according to the invention, (a) at least one cyclodextrin, (b) a combination of cyclodextrins, and/or (c) a pharmaceutical composition comprising at least one cyclodextrin or a combination thereof, and a device for nasal administration. The kits of the invention are used for preventing, inhibiting the progression or treating Coronavirus infections (e.g., SARS-CoV-2 or its variants) in a subject in need thereof. The components of the kits of the invention may be optionally packed in suitable containers and be labeled for preventing, inhibiting the progression or treating Coronavirus infections. The components of the kits may be stored in unit or multi-dose containers as a solid or aqueous, preferably sterile, solution. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port. The kits may further comprise more containers comprising a pharmaceutically acceptable carrier. They may further include other materials desirable from a commercial and user standpoint, including, but not limited to, buffers, diluents, filters, or other active agents. The kits can contain instructions customarily included in commercial packages of diagnostic and therapeutic products that contain information, for example, about the indications, usage, dosage, manufacture, administration, contraindications or warnings concerning the use of such diagnostic and therapeutic products.

Having now generally described the invention, the same will be more readily understood through reference to the following general procedures, which are provided by way of illustration and are not intended to be limiting of the present invention, unless specified. General Procedures

1. Cell Cultures

Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco’s modified Eagle medium, (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 pg/mL streptomycin, and 2 mM glutamine (Invitrogen Corp., Waltham, MA, USA). HEK-293T (ATCC repository) were maintained in DMEM with 10% fetal bovine serum, 100 lU/mL penicillin and 100 pg/mL streptomycin (Invitrogen Corp., Waltham, MA, USA). HEK-293T overexpressing the human ACE2 (Integral Molecular Co., Philadelphia, PA, USA) and maintained in DMEM with 10% fetal bovine serum, 100 lU/mL penicillin and 100 pg/mL streptomycin, and 1 pg/mL of puromycin (Invitrogen Corp., Waltham, MA, USA). TMPRSS2 human plasmid (OriGene Co., Rockville, MD, USA) was transfected using X-tremeGENE HP Transfection Reagent (Merck KGaA, Darmstadt, DE) on HEK- 293 T overexpressing the human ACE2 and maintained in the previously described media containing 1 mg/ml of geneticin (Invitrogen Corp., Waltham, MA, USA) to obtain TMPRSS2/ACE2 HEK-293T cells. CaLu-3 cells were maintained in DMEM with 10% fetal bovine serum, 100 lU/mL penicillin and 100 pg/mL streptomycin.

2. Virus isolation and sequencing

SARS-CoV-2 variants were isolated from clinical nasopharyngeal swabs in Vero E6 cells, as previously described (Rodon J, et al.. Front Pharmacol 2021; 12:646676). Viral stocks were grown in Vero E6 cells and supernatants were collected and stored at -80°C until use.

The following SARS-CoV-2 variants with deposited genomic sequence at the GISAID repository (http://gisaid.org, March 2022) were tested: B.l (D614G) isolated in Spain in March 2020 (EPI ISL 510689); and 4 VOC isolated in Spain from January to February 2021 : Alpha or B. l.1.7 (EPI ISL 1663567), Beta or B. l.351 (originally detected in South Africa; EPI ISL 1663571), Gamma or P. l (originally detected in Brazil; EPI ISL 1831696) and Delta or B.1.617.2 (originally detected in India; EPI ISL 3342900).

Viral variants were titrated at U dilutions on Vero E6 cells using the same luminometric assay described for antiviral testing. Thus, for all VOCs, we used equivalent infectious units inducing 50 % of viral induced cytopathic effect were used.

Assays with HCoV-229E: Cells and virus. MRC5 cells (ATCC CCL-171) were grown in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% inactivated fetal bovine serum (FBS, Biological Industries), 4 mM glutamine (Sigma- Aldrich), 1 x non- essential amino acid solution (Sigma-Aldrich), 100 U/ml penicillin and 100 pg/ml streptomycin (both from Sigma-Aldrich). For preparing virus stocks, human coronavirus 229E (HCoV-229E; ATCC VR-740) was propagated in MRC-5 cells as described (Mesel- Lemoine et al., 2012, J Virol 86, 7577-87.) with a modification that is maintaining cell cultures at 35°C. Viral titer was calculated as 50% tissue culture infective dose (TCID50). Briefly, MRC-5 cells in a 96-well plate at 80% confluency were inoculated with a serial dilution of the viral stock, from 10-1 to 10-8. The plate was incubated at 33°C and with 5% CO2 during 5 days and then fixed with 4% paraformaldehyde (PF A) at room temperature (RT) during 20 minutes. Monolayers were processed by indirect immunofluorescence (IF) using specific antibodies against the HCoV-229E nucleocapsid (N) protein (see below), and titer calculated as described (Ramakrishnan, 2016, World J. Virol. 5, 85-6; Reed and Muench, 1938, Am. J. Hyg. 27, 493-497). Cytotoxicity Assay. A stock solution of each compound was prepared. Hydrophobic compounds were dissolved in dimethyl sulfoxide (DMSO) or ethanol (as recommended by the manufacturer) and stored at -20°C. To evaluate the viability of cell cultures when treated with the compounds, we performed an MTT (3- [4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, which measures the mitochondrial dehydrogenase activity of living cells (Shearman et al., 1994, Proc. Natl. Acad. Sci. U. S. A. 91, 1470-4; Tolosa et al., 2015, General Cytotoxicity Assessment by Means of the MTT Assay, pp. 333-348. https://doi.org/10.1007/978-l-4939-2074-7_26). MRC-5 cells were cultured in a 96-well plate until 80% confluency was reached. Serial dilutions of the compounds in DMEM with 10% FBS were added in triplicate and in a final volume of 100 pl/well. After 24 h, 5 mg/ml of MTT reagent (Sigma- Aldrich) was added to a final concentration of 0.5 mg/ml. Incubation was maintained during 4 h at 37°C with 5% CO2 for metabolization of the reagent and then 100 pl of lysis buffer (10 % SDS, 0.01 M HC1 and 85% isopropanol) was added and incubation maintained for 30 minutes on an orbital shaker protected from light. Plates were read at 570 nm and the background (measured at 690 nm) was subtracted. Data ware calculated from three independent replicates.

Antiviral Activity. The effect of the compounds on viral infection was determined by IF as follows: 20,000 MRC5 cells/well were seeded in duplicates per drug and condition in 96- wells flat-bottom plates. When 80% confluency was reached, they were adsorbed with HCoV-229E in DMEM media without FBS at MOI= 0.1 PFU/cell, and incubated for Ih at 37°C. The inoculum was then removed and a serial dilution of the compounds in DMEM supplemented with 1% FBS was added. A duplicate of the infected cells without any drug treatment served as control. After 24h, cells were fixed with 4% PF A for 20 min and washed three times with PBS. Cells were permeabilized with 0.25% saponin in PBS for 10 min and then treated 30 min with blocking buffer (IxPBS with 0.25% saponin and 2% FBS). Cells were then incubated 1 h with a rabbit antibody specific for the HCoV-229E nucleocapsid (N) protein (Ingenasa) diluted 1 :200 in blocking buffer. After 3 washes with PBS, cells were incubated 45 min with an anti-rabbit secondary antibody conjugated with Alexa fluor 488 (Invitrogen) diluted 1 :500 in blocking buffer and washed three times with PBS. Finally, cell nuclei were labeled 20 min with 4',6-diamidino-2-phenylindole (DAPI) diluted 1 :200 in blocking buffer and cells then washed 3 times with PBS. Images were obtained with a Leica DMi8 S widefield epifluorescence microscope and processed with Image J software. Data were normalized by setting the positive infection control as 100% of infection. Inhibition data were plotted as dose-effect curves fitted to a nonlinear regression model in GraphPad Prism v 9.4 software. The IC50 was calculated with Quest Graph™ IC50 Calculator (https://www.aatbio.com/tools/ic50-calculator). All experiments were replicated three times.

3. Pseudovirus production

HIV-1 reporter pseudoviruses expressing SARS-CoV-2 Spike protein and luciferase were generated using two plasmids. pNL4-3.Luc.R-.E- was obtained from the NTH AIDS repository. SARS-CoV-2. SctA 19 was generated (GeneArt, ThermoFisher Scientific Corp., Waltham, MA, USA) from the full protein sequence of SARS-CoV-2 spike with a deletion of the last 19 amino acids in C-terminal, human-codon optimized and inserted into pcDNA3.4-TOPO (Ou X, et al., Nat Commun 2020; 11 : 1620). Spike plasmid was transfected with X-tremeGENE HP Transfection Reagent (Merck KGaA, Darmstadt, DE) into HEK-293T cells, and 24 hours post-transfection, cells were transfected with pNL4- 3.Luc.R-.E-. Supernatants were harvested 48 hours later, filtered with 0.45 pM (Millex Millipore, Merck KGaA, Darmstadt, DE) and stored at -80°C until use. The p24gag content of all viruses was quantified using an ELISA (PerkinElmer, Inc., Waltham, MA, USA) and viruses were titrated in HEK-293T overexpressing the human ACE2.

4. Pseudoviral entry inhibition assay

HEK-293T overexpressing the human ACE2 and TMPRSS2 were used to test compounds at the concentrations indicated. A constant pseudoviral titer was used to pulse cells in the presence of the drugs. At 48 h post-inoculation, cells were lysed with the Gio Luciferase system (Promega Corp., Madison, WI, USA). Luminescence was measured with an EnSight Multimode Plate Reader (PerkinElmer, Inc., Waltham, MA, USA).

5. Antiviral activity with SARS-CoV-2

Increasing concentrations of the indicated antiviral compounds were added to Vero E6 cells. Immediately after, equivalent infectious units of SARS-CoV-2 variants were added to the cells to achieve a 50 % of cytopathic effect. Untreated non-infected cells and untreated virus-infected cells were used as negative and positive controls of infection, respectively. To detect any drug-associated cytotoxic effect, Vero E6 cells were equally cultured in the presence of increasing drug concentrations, but in the absence of virus. Cytopathic or cytotoxic effects of the virus or drugs were measured 3 days after infection, using the CellTiter-Glo luminescent cell viability assay (Promega Corp., Madison, WI, USA). Luminescence was measured in a Fluoroskan Ascent FL luminometer (ThermoFisher Scientific Corp., Waltham, MA, USA).

Viral replication of SARS-CoV-2 was also assessed on CaLu-3 cells in the presence of the compounds U18666A, OSW-1, HbCD, Phytol and Remdesivir as control. Compounds were incubated for 15 minutes before adding the SARS-CoV-2 virus at MOI of 0.33. After 24h of incubation at 37°C and 5% CO2, cells were washed with PBS and compounds were added at the same final concentration for 48h. The amount of SARS-CoV-2 nucleoprotein released to the supernatant was measured with SARS-CoV-2 nucleocapsid protein High- Sensitivity Quantitative ELISA (ImmunoDiagnostics, Ltd., Toronto, CA) according to the manufacturer’s protocol. The cytopathic effect on CaLu-3 cells was assessed with Cell Titer-Gio Assay with a Fluoroskan Ascent FL luminometer at the time of supernatant collection.

6. IC50 calculation and statistical analysis

Response curves of compounds or their mixes were adjusted to a non-linear fit regression model, calculated with a four-parameter logistic curve with variable slope. Cells not exposed to the virus were used as negative controls of infection and were set as 100% of viability to normalize data and calculate the percentage of cytopathic effect. All analyses and figures were generated with the GraphPad Prism v8.0b Software.

7. SARS-CoV-2 antiviral activity in a human nasal epithelial model.

To assess the MpCD antiviral activity in a physiological model, we used a human nasal airway epithelium (HNE) model (Mucilair™, Epithelix)) and the specific medium provided. Inserts were cultured at the air liquid interface, mimicking in vivo nasal tissue. In this model we added 2.5 mM MpCD on the apical side or on the basal medium, or 2 pM Remdesivir on the basal medium, or left cultures untreated. SARS-CoV-2 D614G was added at an MOI=0,01 to the apical side of each inset for 1 hour at 37°C and 5% CO2. Apical side was then extensively washed with PBS and incubated for 24 hours. MpCD on the apical side was washed and not replaced thereafter. Drugs added on the basal medium were present during the whole experiment and replaced at 48 hours pi. Viral content was measured collecting the apical side of insets after adding 300 pl/well of PBS at 24, 48 and 72 hours pi. SARS-CoV-2 nucleocapsid concentration was measured by ELISA. The cytopathic effect was measured 72 hours pi using the Cell Titer-Gio Assay to measure ATP released by living cells on a DL Ready Luminoskan (ThermoScientific).

Remdesivir 0.51 ± 100

0.71

OSW-1 ~ 0.002 ± 0.007

68.41

Hb-CD 847.09 ± 20000

2240.10

U18666A ~ 0.17 ± 20

539.43

Phytol ~ 140.90 ± 100-200

402.29

Bortezomib - 0.4958 0.8

FLI06 0.0151 4-6.25

Mdivi-1 - 0.8317 20

PIK93 - 20

0.005508

CI976 0.2997 100

Valinomycin NA 0.32 Dehydrocostus - 0.8439 100 lactone

Digitoxin - 1.524 1.56

Wortmanin 0.941 25

Terpinolene - 2.305 10000

Gingenoside - 1.403 50

L-arginine - 0.000 NA a-Terpineol - 23.67 NA

Table 1. List of drugs tested in Vero E6 cells infected with SARS-CoV-2, for which the IC50 of the viral cytopathic effect is provided, or uninfected for which the first drug- cytotoxic concentration is provided. SD, standard deviation; NA, non-applicable.

8. SARS-CoV-2 antiviral activity in an in vivo hamster model.

Nine-week-old golden Syrian hamsters (Janvier) were divided into 3 groups in a sex- balanced ratio (50% females): GO (n=4; untreated and uninfected animals), G1 (n=8; untreated and SARS-CoV-2-NanoLuc infected animals), G2 (Beta Methyl CD-treated and SARS-CoV-2-NanoLuc infected animals). Animals were inoculated by intranasal instillation with 50 mM Beta Methyl CD or PBS (100 pL/individual, 50 pL for each nostril), for the treated and untreated groups, respectively. Then, animals were inoculated by intranasal instillation with 10^A3 SARS-CoV-2 NanoLuc replicative competent reporter virus (n=16) resuspended in 50 mM Beta Methyl CD (for the treated animals) or PBS (for the untreated animals; 100 pL/individual, 50 pL for each nostril). The four remaining hamsters were intranasally inoculated with PBS (100 pL/individual, 50 pL for each nostril) and used as negative controls. For virological examinations, 4 hamsters per group (G1-G2) and 2 control hamsters (GO) were euthanised on days 1 and 2 dpi.

Oral swabs were collected from all animals before the challenge and at the euthanasia. At necropsy, samples from nasal turbinate and lung were taken and placed in individual microfuge tubes containing 500 pL of DMEM (GIBCO) supplemented with 1% penicillin-streptomycin (PS) (GIBCO) and a single zinc-plated, steel, 4.5-mm bead. Samples were homogenized at 30 Hz for 2 min using a TissueLyser II (QIAGEN GmbH, Hilden, Germany) and centrifuged for 30 s at 11,000 rpm. Supernatants were analyzed for Nanoluciferase content with the Nano-Gio Luciferase Assay System (Promega) at a 1 :1 sampleluciferase ratio and assessed on a Fluoroskan Ascent FL luminometer (ThermoFisher). Samples were stored at -70° C for further analysis.

Viral RNA was extracted from samples using the IndiMag pathogen kit (Indical Bioscience) on a Biosprint 96 workstation (QIAGEN) according to the manufacturer’s instructions. RT-PCR used to detect viral gRNA is based on the one published by Corman et al. Eurosurveillance; 2020;25:2000045, with minor modifications to adapt it to the AgPath-ID One- Step RT-PCR Kit (Life Technologies). The primers and probes used, and their final concentration are the following: forward: 5'-

ACAGGTACGTTAATAGTTAATAGCGT-3' [400 nM], reverse: 5'-

ATATTGCAGCAGTACGCACACA-3' [400 nM] probe: 5'-FAM

ACACTAGCCATCCTTA CTGCGC TTCG-TAMRA-3 ' [200 nM], Thermal cycling was performed at 55°C for 10 min for reverse transcription, followed by 95°C for 3 min and then 45 cycles of 94°C for 15 s, 58°C for 30 s.

Example 1. H0CD show anti- HCoV-229E and SARS-CoV-2 activity in Vero E6 cells and Caln 3 cells

To test the antiviral activity of selected compounds, MRC-5 cells were infected with HCoV- 229E and treated in parallel with increasing concentrations of the selected compounds. Drug cytotoxicity was first measured, and safe drug-doses were used to determine the percentage of infected cells by immunofluorescence using an antibody specific for the HCoV-229E nucleocapsid protein. With these assays, we calculated the concentration of compound required to inhibit 50% of the virus (IC50), and the concentration for the 50% cytotoxic effect (CC50). HP-P-cyclodextrin (HpCD) inhibited HCoV-229E infection at non-toxic concentrations (Fig. 1A).

Vero E6 cells were exposed to SARS-CoV-2 in the presence of increasing concentrations of HPCD. After three days, the cytopathic effect of the virus and the cytotoxic effect of the drugs on cells were analyzed and the IC50 value calculated for each drug at non-cytotoxic concentrations. Results showed HPCD had antiviral activity on these cells (Fig. IB). The antiviral activity of was similar when tested for different variants of concern of SARS-CoV- 2 and comparable to the control Remdesivir (Fig. 1C). We therefore assessed the capability of H0CD to inhibit the entry of SARS-CoV-2 pseudovirus in ACE2 expressing HEK-293T cells. Cells were exposed to fixed amounts of lentiviruses pseudotyped with SARS-CoV-2 spike in the presence of decreasing drug concentrations. Results showed that at drug concentrations without any cytotoxic effect (Fig 5), H0CD inhibited the entry of pseudovirus (Fig. ID). Cytopathic effect of the drugs in the absence of virus is shown Fig. S9A. These findings with the functional pseudoviral assay suggest the mechanism of action of cyclodextrins is reducing viral fusion.

We next tested SARS-CoV-2 antiviral activity in pulmonary Calu-3 cells as a more physiological and relevant cellular model. After incubating these cells with the drugs and the virus for 24h, the virus was washed away and compounds were added at the same concentration for 48h. The amount of SARS-CoV-2 nucleoprotein released to the supernatant was then measured by ELISA and the cytopathic effect of the drugs in the absence of virus in Calu-3 cells was assessed by luminescence (Fig. 5). Only HP-0-CD at 10 mM was able to effectively inhibit SARS-CoV-2 viral release into the supernatant of Calu-3 cells as observed for control remdesivir, while no inhibition was observed for the other compounds (Fig. IE) at non-cytotoxic concentrations. Hence, HP-0-CD resulted as the most promising candidate to block SARS-CoV-2 replication on pulmonary cells.

Example 2: Other members of the Cyclodextrin family also inhibit SARS-CoV-2 infection in pulmonary cells

Given the well-known safety profiles of different types of 0-cyclodextrins, that have been used as excipients in the pharmaceutical industry for decades (Lachowicz et al., 2020 Curr. Drug Targets 21, 1495-1510), we next aimed to test whether other cyclodextrins could hold the potential to inhibit viral replication. To test if other cyclodextrins could inhibit SARS-Cov-2 pseudoviral entry, we performed studies in ACE2 expressing HEK-293T cells. At no cytotoxic concentrations (Fig. 2A), seven cyclodextrins inhibited the pseudoviral SARS-CoV-2 entry, being the most potent 0, HP-0, HP-y and methyl-0-CDs.

Next, we assayed if the most active cyclodextrins inhibiting pseudoviral fusion could also block SARS-CoV-2 replication in pulmonary Calu3 cells. The quantification of SARS- CoV-2 nucleoprotein released to the supernatant detected by ELISA revealed that 0-CDs, HP-0-CDs, and methyl-0-CDs were able to inhibit the SARS-CoV-2 viral activity on Calu- 3 cells, both for the D614G and for the Omicron BA.1 variant of concern (Fig. 2B) at no cytotoxic concentrations (Fig 5). These results further highlight the potential of the 0 cyclodextrin family as antivirals against SARS-CoV-2.

Example 3: Cyclodextrins inhibit SARS-CoV-2 replication by interfering with viral fusion via cholesterol depletion We had previously found that 0-CD are in the catalytic pocket of the M^pro of SARS-

CoV-2 by molecular modelling (Table 2). This finding pointed to a possible explanation for the antiviral activity detected. All cyclodextrins, including a, 0, y, HP- a, HP-0, HP-y and methyl-0-CDs, were screened by molecular modeling against the SARS-CoV-2 M^pro. Since is possible to inhibit the action of M^pro by targeting two allosteric sites - PDB codes 7AXM and 7 AGA- (Gunther et al., 2021, Science 80, 372, 642-646), we expanded the chemical space of search into the designed models. Theory predicts that cyclodextrins can bind both active and allosteric Mpro sites, although significant dissimilarities appear depending on the size and nature of the macrocycle (Table 2). To test whether this interaction occurred in vitro, the Mpro activity was measured in presence of increasing concentrations of methyl-0- CD (M0CD). While M0CD did not show activity, active GC376 control inhibited Mpro in a dose dependent manner (Fig. 3 A).

Table 2. Energies for the top-ranked cyclodextrins against the active site and the two allosteric sites of M^pro.

We next explored alternative antiviral mechanisms of action of cyclodextrins. Given the well-known capacity of cyclodextrins to extract cholesterol from biological membranes (Lopez et al., 2011, PLoS Comput. Biol. 7, el002020), we performed a lipidomic analysis focusing on different lipids associated to cholesterol enriched domains in biological membranes. Calu-3 cells treated with increasing concentrations of MDCD showed a reduction of free and ester cholesterol along with sphingomyelin, and did not affect phosphatidylcholine (Fig. 3B). These results further confirm the capacity of cyclodextrins to alter the composition of cholesterol enriched domains actively involved in viral fusion processes. Taken together, these experiments along the pseudoviral fusion assays highlight the potential of cyclodextrins to inhibit SARS-CoV-2 replication by interfering with viral fusion via cholesterol depletion.

Example 4: Methyl-P-cyclodextrin inhibit SARS-CoV-2 replication in a human nasal epithelial model

Finally, we aimed to test the antiviral effect of M0CD in a more physiologically relevant model. We used a human nasal epithelial (HNE) model which mimics the characteristics of in vivo nasal tissue. In this HNE model we added M0CD, either on the apical side or the basal medium of the culture (Fig. 4A), just before adding SARS-CoV-2 to the apical side for 1 hour. Apical side was then extensively washed and incubated for 72h. Remdesivir was also added to the basal medium and other inserts were left untreated. Viral replication was monitored at 48 and 72 hpi by measuring SARS-CoV-2 nucleocapsid concentration by ELISA. SARS-CoV-2 released to the apical supernatant picked at 48 hpi and decreased at 72 hpi (Fig. 4B). Initial M0CD addition to the apical side reduced viral replication by 74% at 48 hpi (Fig. 4B), while constant presence of M0CD in the basal side almost had no effect (11%) as opposed to the condition with remdesivir that completely blocked it (Fig. 4B). The cytopathic effect measured at 72 hours pi indicated the HNE cells were alive with all the tested conditions (Fig.5E). These results further emphasize the potential use of M0CD as a prophylactic antiviral in nasal sprays against SARS-CoV-2. Example 5: Prophylaxis treatment with Methyl-Beta-CD protected treated animals from infection:

To test the capacity of Methyl- Beta-CD to prevent SARS-CoV-2 infection in vivo, we tested its prophylactic activity in hamsters. Sixteen animals were pre-treated with Beta-Methyl -CD (n=8) or PBS (n=8) intranasally before challenging via the same route with a SARS-CoV-2 reporter virus that produces nano-luciferase upon infection. Eight animals (four per group and per day) were euthanized at day 1 and 2 post infection and compared to uninfected controls (two animals euthanized per day) measuring the amount of nanoluciferase content in nasal turbinates and lungs. At day 1, the four infected untreated animals had detectable nanoluciferase levels both at the nasal turbinates and lungs, while no nanoluciferase signal was detected on Methyl- Beta-CD treated animals. Similar results were obtained at day 2 post-infection, where higher levels of nanoluciferase were detected in the nasal turbinates and lungs of untreated infected animals, and no sign of infection was detected on Methyl- Beta-CD treated animals. These results were further confirmed by qPCR at days 1 and 2 post-infection in nasal turbinates and lungs. Prophylaxis treatment with Beta-Methyl -CD protected treated animals from infection.

Claims

Claims

1. Cyclodextrin for use in preventing, inhibiting the progression or treating a Coronavirus infection in a subject in need thereof, wherein said cyclodextrin is administered nasally or orally, preferably nasally.

2. Cyclodextrin for use according to claim 1, wherein the cyclodextrin is effective in preventing or inhibiting the cellular entry of the Coronavirus in a nasal epithelial cell.

3. Cyclodextrin for use according to any of the preceding claims, wherein the cyclodextrin is a a-, P-, y-cyclodextrin or a combination thereof.

4. Cyclodextrin for use according to any of the preceding claims, wherein the cyclodextrin is a P-cyclodextrin.

5. Cyclodextrin for use according to any of the preceding claims, wherein the P- cyclodextrin is hydroxypropyl P-cyclodextrin or methyl-P-cyclodextrin, preferably is methyl-P-cyclodextrin.

6. Cyclodextrin for use according to any of the preceding claims, wherein the virus is a member of Coronaviridae family or a combination thereof.

7. Cyclodextrin for use according to any of the preceding claims, wherein the virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a functionally equivalent variant thereof, or HCoV-229E.

8. Cyclodextrin for use according to claim 7, wherein the SAR-CoV-2 variant is B.l (D614G), 4 VOC, B.l.1.7, B.l.351, P.l, B.1.617.2 or a combination thereof.

9. A pharmaceutical composition comprising a therapeutically effective amount of at least one cyclodextrin for use in preventing, inhibiting the progression or treating a Coronavirus infection in a subject in need thereof, wherein said composition is administered nasally or orally, preferably nasally.

10. The pharmaceutical composition according to claim 9, wherein said composition is in the form of a nasal spray, nasal solution or nasal suspension.

11. A method for preventing, inhibiting the progression or treating a Coronavirus infection in a subj ect in need thereof which comprises administering intranasally a therapeutically effective amount of (a) at least one cyclodextrin or a combination thereof or (b) a pharmaceutical composition according to claim 11 or a combination thereof to the subject.

12. The use of a cyclodextrin for the manufacture of a medicament for use in preventing, inhibiting the progression or treating a Coronavirus infection in a subject in need thereof, wherein said medicament is administered nasally or orally, preferably nasally.

13. The methods and uses according to any of the preceding claims wherein the subject is human.

14. A kit comprising at least one cyclodextrin or pharmaceutical composition for use according to any of the preceding claims, a device for nasal administration and instructional materials for their handling.