RU2846625C1 - Antiviral pharmaceutical composition - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions relates to the use of a pharmaceutical composition for treating or preventing, by intranasal administration, coronavirus infection in a human caused by OC43 or SARS-CoV-2 or SARS-CoV-1 and a method of treating such an infection. Use of a pharmaceutical composition comprising a pharmaceutically effective amount of fucoidan, for treating or preventing, by intranasal administration, coronavirus infection in a human, wherein said composition is a nasal spray, fucoidan has a sulphate content of 20 % to 40 % and has an average molecular weight of 50 kDa to 250 kDa, determined by size-exclusion chromatography with a mobile phase containing 150 mM NaCl and having pH of 6, and said coronavirus infection is an infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1. Method of treating or preventing coronavirus infection, comprising the intranasal administration to a mammalian subject in need thereof of a pharmaceutical composition containing a pharmaceutically effective amount of fucoidan, wherein said composition is a nasal spray, fucoidan has a sulphate content of 20 % to 40 % and has an average molecular weight from 50 kDa to 250 kDa, determined by exclusion chromatography with a mobile phase containing 150 mM NaCl and having pH of 6, and said coronavirus infection is an infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1. Use of a pharmaceutical composition comprising a pharmaceutically effective amount of fucoidan to prevent the spread of coronavirus in a human population, wherein subjects of said population are tested for presence of infection caused by OC43 or SARS-CoV-2, and infected subjects are treated by intranasal administration of said pharmaceutical composition, wherein said composition is a nasal spray, fucoidan has a sulphate content of 20 % to 40 % and has an average molecular weight from 50 kDa to 250 kDa, determined by exclusion chromatography with a mobile phase containing 150 mM NaCl and having pH of 6, and said coronavirus infection is an infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1. Use of a pharmaceutical composition comprising a pharmaceutically effective amount of fucoidan to prevent the spread of coronavirus in a human population, wherein subjects who have a high risk of infection have been identified are treated by the intranasal administration of said pharmaceutical composition, where said composition is a nasal spray, fucoidan has sulphate content from 20 % to 40 % and has an average molecular weight from 50 kDa to 250 kDa, determined by size exclusion chromatography with a mobile phase containing 150 mM NaCl and having pH of 6, and said coronavirus infection is an infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1.

EFFECT: said use and method are effective for treating or preventing, by intranasal administration, a human coronavirus infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1.

27 cl, 11 dwg, 7 tbl, 4 ex

Description

AREA OF TECHNOLOGY

The present invention relates to pharmaceutical compositions for use in methods for the treatment and prevention of respiratory tract infection caused by coronaviruses, in particular coronaviruses OC43, SARS-CoV-1 and SARS-CoV-2. The present invention also relates to the control of the spread of infection caused by coronaviruses, in particular coronaviruses OC43 and SARS-CoV-2.

LEVEL OF TECHNOLOGY

Acute respiratory viral infection (ARVI) is an acute respiratory tract infection that manifests itself as catarrhal inflammation of the upper respiratory tract and is accompanied by high fever, runny nose, sneezing, coughing, and sore throat. The disease can proceed with varying degrees of severity. It can pass on its own, but it can also proceed severely, causing complications or even leading to the death of the patient. ARVI can be caused by more than 200 types of respiratory viruses. Various types of human coronavirus account for approximately 15% of ARVI in humans. Coronaviruses have an enveloped virus genome, represented by single-stranded positive-sense RNA, 26 to 32 thousand bp in length. Based on phylogenetic similarity, they are divided into four categories/genera: α (e.g. 229E and NL-63), β (e.g. SARS-CoV-2, SARS-CoV, MERS-CoV, and OC43), γ, and δ. SARS-CoV-2 has been reported to share 79% sequence identity with SARS-CoV, however, some regions of the SARS-CoV-2 genome show greater or lesser conservation compared to SARS-CoV. Human coronaviruses include viruses such as OC43, which generally cause less severe disease. Highly pathogenic coronaviruses include SARS-CoV, MERS-CoV, and SARS-CoV-2, which can cause severe symptoms, critical illness, and complications with high mortality, such as severe acute respiratory syndrome (SARS) and acute respiratory distress syndrome (ARDS).

Different families of respiratory viruses have very different modes of infection and reproduction. Current antiviral drugs for the treatment of ARVI target key molecules involved in DNA or RNA binding, endocytosis, fusion (decapsidation), synthesis and replication, assembly and release of viruses. Therefore, they are effective against a specific family of viruses that use these target molecules. Even within the same family and virus species, effectiveness may vary due to different mechanisms, genotypes and expression of target proteins. Due to congenital or acquired mutations, different virus strains may be less susceptible or even resistant to drug treatment. However, patients often do not have the opportunity to test for a viral infection, so it is not always possible to choose a suitable antiviral drug. This is due to the fact that today, most ARVI tests require a nasal or oropharyngeal swab to be taken in a medical facility, and the resulting sample is analyzed in a laboratory. The testing capacity of a healthcare facility, the laboratory capacity and the testing equipment used are limited. The need for testing is particularly difficult to meet in a pandemic. It is known that asymptomatic and pre-symptomatic carriers of the virus can contribute to the spread of the virus, so there is a need for a treatment that can be used as a preventive treatment or at least at the onset of the first symptoms, regardless of the test results. However, preventive treatment with antiviral drugs is not widely used, in particular due to potential side effects, the inevitable development of drug resistance and high cost. Currently available antiviral drugs are usually administered systemically, for example orally or intravenously. As a result, concentrations of the drug in the systemic circulation are achieved, which can lead to side effects. Therefore, local treatment of ARVI that effectively stops and destroys the virus at the site of initial infection - on the mucous membrane of the respiratory tract - is highly desirable. There is also a need for pharmaceutical compositions that can be used to prevent ARVI in humans, which are easy to use and have a low risk of side effects.

A novel coronavirus was first identified as a pathogen causing acute respiratory viral infection in humans in December 2019. The pathogen was later named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the disease caused by the virus in humans was named coronavirus disease 2019 (COVID-19). The virus, which can spread from person to person, has spread worldwide, leading to its classification as a pandemic in March 2020, and poses a serious global health threat. Clinical trials are underway to evaluate the effectiveness of modifications of approved or pending antiviral agents for the treatment of SARS-CoV-2 or COVID-19 infection. However, the effectiveness of these candidates has not yet been established, and there is currently no prophylactic treatment. One antiviral agent awaiting approval for this new use is the antiviral drug remdesivir, which is a prodrug of an adenosine nucleoside triphosphate analogue. It has been proposed as an experimental drug for the treatment of COVID-19, as it has shown promising results against MERS-CoV and SARS-CoV-1 in previous animal studies. Pizzorno et al. (Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia, bioRxiv, 02.04.2020, DOI: 10.1101/2020.03.31.017889) found that remdesivir reduced relative viral production in reconstituted human airway epithelial cells infected with SARS-CoV-2. Multiple clinical trials have been initiated to determine whether intravenous remdesivir has clinical benefit in the treatment of COVID-19. The drug received emergency use authorization from the U.S. Food and Drug Administration (FDA) for the treatment of hospitalized adults and children with severe COVID-19 in May 2020 and is the only drug approved for the treatment of COVID-19 as of the filing date of this patent application.

Liu Y et al. (Viral dynamics in mild and severe cases of COVID-19, The Lancet, Volume 395, Issue 10223, 15-21 February 2020, Pages 507-513, DOI: 10.1016/S1473-3099(20)30232-2) noted that viral load in nasopharyngeal swab samples was generally high and viral shedding was prolonged in patients with severe COVID-19. The authors suggested that SARS-CoV-2 viral load in the nasopharynx may be a useful marker for assessing the severity and prognosis of the disease.

Hou, Y.J. et al. (SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract, Cell 11447 (2020), doi: https://doi.Org/10.1016/j.cell.2020.05.042) suggested that nasal cells are the predominant initial site of respiratory tract infection by SARS-CoV-2, and that the virus may enter the lower respiratory tract via aspiration of nasal secretions or mucus containing high viral loads.

Fucoidan, a marine-derived polysaccharide, has been tested against different families, genera, species and strains of respiratory viruses, with conflicting results.

WO 2009/027057 describes antiviral compositions comprising a sulfated polysaccharide and discloses that carrageenan and fucoidan inhibit parainfluenza virus 3-mediated cell death in HNep cells when the cells were inoculated in the presence of the polysaccharides, with carrageenan being more effective than fucoidan (Example 13). Similar results were obtained in the same experiment after inoculation with H3N2 influenza virus, with iota-carrageenan having the best protective effect at most concentrations (Example 14). This document also shows that pre-treatment of the virus with fucoidan is unable to reduce plaque formation of H5N1 avian influenza virus in MDCK cells (Example 15).

WO 2011/100805 describes methods for inhibiting a virus of the orthomyxoviridae family, comprising contacting the virus or a cell infected with the virus with an effective amount of a composition comprising a sulfated polysaccharide with an average molecular weight of 4000 daltons or more. Compositions based on sulfated polysaccharides reduce the cytopathic effect (CPE) of influenza A virus type H1N1, strain California/07/2009, in MDCK cells, but are not effective in the same experiment against influenza A virus type H3N2, strain Brisbane/10/2007. The applicants of WO 2011/100805 reported in a press release dated 5 March 2020 that in previous studies they had noted low activity of their commercially available fucoidan product against coronaviruses compared to the activity observed against other viruses such as influenza, herpes, human metapneumovirus and respiratory syncytial virus (https://www.marinova.com.au/news/fucoidan-and-novel-coronavirus/).

Wang et. al. (Wang, W., Wu, J., Zhang, X., et al. Inhibition of Influenza A Virus Infection by Fucoidan Targeting Viral Neuraminidase and Cellular EGFR Pathway. Sci Rep 7, 40760 (2017). DOI: 10.1038/srep40760) found that fucoidan from Kjellmaniella crassifolia reduces CPE and plaque formation in MDCK cells after infection with different H1N1 strains (PR8, Cal09, and TX09) as well as the H3N2 Minnesota strain. Furthermore, fucoidan was found to bind to the neuraminidase (NA) protein of the H1N1 (Cal09) and H3N2 (Minnesota) subtypes using surface plasmon resonance (SPR) analysis.

The authors suggest that fucoidan may inhibit the entry and release process of influenza A virus by directly binding to influenza A NA.

Thus, fucoidan is effective only against certain virus families, and it is impossible to predict the effectiveness against a specific virus. Even within a family, the response varies greatly between species and even strains. Although a mechanism of action of fucoidan against influenza A virus has been proposed, the results of studies of fucoidan formulations against different influenza A virus species and strains have been contradictory. In addition, the proposed mechanism is limited to influenza A viruses, since it depends on a target molecule specific to this family.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a pharmaceutical composition comprising fucoidan for use in a method of treating or preventing, by intranasal administration, coronavirus infection in humans. The inventors of the present invention have found that fucoidan administered to the nasal epithelium of humans reduces the replication of viruses belonging to the coronavirus family. This is unexpected and contrary to the results previously reported by the applicants of WO 2011/100805. Without being limited by any particular theory, the inventors believe that fucoidan, which contains sulfate groups having a negative charge in solution and physiological environment, interacts with positively charged surface structures, such as surface proteins of the virus with a net positive charge, or with surface proteins of the virus bearing a positive charge. In viruses that have a net positive charge in the receptor-binding domain (RBD) of their receptors that bind to host cells, binding of fucoidan sulfate groups to these positively charged moieties can interfere with the virus's ability to attach to and enter the human host cell. For example, fucoidan can bind to the receptor-binding domain (RBD) of the S1 domain of the spike protein (S protein) of coronavirus OC-43, SARS-CoV-1, and SARS-CoV-2, which have a net positive charge.

Thus, the pharmaceutical compositions of the present invention are effective for treating coronavirus infection in humans. The pharmaceutical compositions provide a reduction in the viral load or titer of the virus in a subject. Both parameters can be determined using methods known in the art, such as quantitative real-time polymerase chain reaction (RT-PCR). In one embodiment, the treatment reduces the nasal viral load in a subject. In one embodiment, the nasal viral load is determined using RT-PCR performed on a nasal swab sample from the subject. Since high nasal SARS-CoV-2 viral load is a risk factor for developing severe forms of COVID-19, reducing the nasal viral load can reduce the risk of developing severe symptoms of COVID-19. Severe COVID-19 can be characterized as the occurrence of shortness of breath, a respiratory rate of 30 or more breaths per minute, blood oxygen saturation of 3% or less, the ratio of the partial pressure of arterial oxygen to the oxygen content of inspired air (PaO ₂ :PiO ₂ ) less than 300 mmHg, or the presence of infiltrates in more than 50% of the lung field (Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72,314 cases from the Chinese Center for Disease Control and Prevention. JAMA 2020; 323:1239-1242). High viral load of coronaviruses leads to a more severe inflammatory response in the patient. Excessive immune response is the cause of the complications mentioned in the background section. Thus, reducing nasal viral load may also prevent the severity of COVID-19 and the development of ARDS or SARS. In addition, reducing nasal viral load may reduce the risk of accumulated nasal secretions being aspirated into the lungs and causing lower respiratory tract infection with SARS-CoV-2, which is consistent with the findings of Hou, YJ et al (cited above).

The present invention includes a method of treating COVID-19, comprising a method of detecting SARS-CoV-2 viral RNA in a sample obtained from a subject and, if viral RNA is detected, the step of treating COVID-19 as described herein. A method for detecting SARS-CoV-2 viral RNA in a sample obtained from a subject may employ a CRISPR diagnostic assay (e.g., using the DECTECTR™ assay using Casl2 (Chen et al., Science 360, 436–439 (2018) and, in particular, for detecting SARS-CoV-2 Broughton et al., Nat Biotechnol (2020) https://doi.org/10.1038/s41587-020-0513-4), or using the SHERLOCK™ assay using Casl3 (Gootenberg et al., Science 356: 438–442 (2017)). Alternatively, a method for detecting SARS-CoV-2 viral RNA in a sample obtained from a subject may employ a reverse transcription polymerase chain reaction (RT-PCR) assay or other diagnostic methods known in the art.

The pharmaceutical compositions of the present invention are also suitable for preventing coronavirus infection in humans. To prevent coronavirus infection, the pharmaceutical compositions of the present invention are administered intranasally to subjects who have not been tested for coronavirus infection or to subjects who have been tested for coronavirus infection and the result was negative. In specific embodiments, the subject is in a high-risk category (as defined herein), is a healthcare worker, or is in close contact with a patient infected with SARS-CoV-2 (as defined herein). These pharmaceutical compositions are effective and suitable for preventing coronavirus infection because, when used preventively, they prevent the virus from infecting nasal epithelial cells, are not harmful to the nasal epithelium, and are convenient to use intranasally. Since fucoidan is approved for use as a dietary supplement at doses of up to 250 mg per day, the compositions of the present invention are considered safe for oral administration, even when swallowed, and have a low risk of adverse events.

In one embodiment, the coronavirus infection is an infection caused by OC43, SARS-CoV-1 or SARS-CoV-2. As found by the inventors, the compositions of the present invention are effective in reducing viral replication of OC43 and SARS-CoV-2 in the nasal epithelium. A similar effect is expected for SARS-CoV-1. The inventors have shown in the experiments cited that the negatively charged sulfate groups of fucoidan interact with positively charged surface fragments of the virus, such as surface proteins of the virus. An example of such a positively charged surface fragment is the RBD of the S1 domain of the pin protein of OC43, SARS-CoV-1 and SARS-CoV-2, which have a net positive charge of +6 in the case of OC-43 and SARS-CoV-2 and +2 in the case of SARS-CoV-1. Therefore, the antiviral activity of the compositions of the present invention observed in studies on OC-43 and SARS-CoV-2 can be extrapolated to SARS-CoV-1.

The infection may also be a co-infection between a coronavirus and another type of respiratory virus. In another embodiment, the infection is a co-infection between a rhinovirus and a coronavirus. In one embodiment, the coronavirus is S ARS-CoV-2.

In one embodiment, the coronavirus infection is an infection caused by SARS-CoV-2. Methods for identifying subjects infected with SARS-CoV-2 are known in the art and are used in current clinical practice. In one embodiment, testing of samples, such as nasal and/or pharyngeal swab samples, using high-throughput sequencing or real-time reverse transcription polymerase chain reaction (RT-PCR) analysis can be used to identify subjects with active SARS-CoV-2 infection. Alternatively, the CRISPR-based diagnostics mentioned above can be used.

According to the present invention, the treatment includes intranasal administration of the pharmaceutical compositions according to the present invention. In one embodiment, the pharmaceutical composition is administered to the nasal epithelium of the subject. Thus, the pharmaceutical composition can be administered locally to the cells of the nasal epithelium. Such administration includes nasal administration of solutions, gels, creams, powders or foams. The administration can be carried out by instillation using a pipette, instillation from a dropper bottle or spraying the pharmaceutical composition in the form of a spray into the nostrils, spraying in the form of an aerosol or fine spray of the composition and inhalation of the sprayed composition, or by applying the composition to the nasal epithelium.

Also provided are methods for treating or preventing coronavirus infection, comprising intranasally administering to a mammalian subject in need thereof a pharmaceutically effective amount of a pharmaceutical composition containing fucoidan. Preferably, the subject is a human.

In one embodiment, the composition is a nasal spray, and intranasal administration thus is administration of a nasal spray. Administration of a nasal spray includes inserting a nozzle into a nostril, activating a spray mechanism that expels and sprays the composition through the nozzle into the nostril, possibly with simultaneous inhalation, and repeating these same steps with the second nostril. The resulting droplets or particles are then deposited on the nasal epithelium.

The natural beating of the cilia of the ciliated epithelium transports the compositions to the adjacent epithelium of the nasopharynx, oropharynx and laryngopharynx. Therefore, the administration of a nasal spray allows the compositions of the present invention to be delivered to the initial point of infection with coronaviruses - the epithelial cells of the upper respiratory tract. This is possible without the intervention of a health worker and without medical assistance, and is achieved using a conventional, easy-to-use device. Nasal sprays are characterized by very good user compliance.

Fucoidan in the context of the present invention is to be understood as polysaccharides of marine origin containing L-fucose monomers, which are partially sulfated, and occur and can be extracted from various species of brown seaweed or brown algae (i.e. class Phaeophyceae). Extracts from brown algae, including extracts containing fucoidan, are being investigated for their various biological activities. As discussed in the section "prior art", fucoidan has been investigated for antiviral activity in vitro with conflicting results. Fucoidan is not harmful to the mucous membrane of the respiratory tract, as shown in the example. Information on the chemical structure of fucoidan is given in the definitions. Fucoidan can be obtained from any species of brown algae, such as Undaria pinnatifida or Fucus vesiculosus.

In one embodiment, the pharmaceutical composition is an aqueous solution. Aqueous solutions are preferred because fucoidan is compatible with water as a carrier and is stable in aqueous formulations. Aqueous solutions are particularly preferred because they are compatible with the spray mechanism and materials of most types of commercially available nasal spray devices. Aqueous solutions are also preferred due to the low risk of side effects.

In one embodiment, the pharmaceutical composition comprises 0.1% to 2.7%, more preferably 0.25 to 1.8%, most preferably 0.5 to 1% (w/w) of fucoidan. These amounts can dissolve well in aqueous vehicles and form stable aqueous solutions, which is preferred for intranasal administration. At fucoidan concentrations above 2.5% (w/w), solubility becomes more problematic. In addition, pharmaceutical compositions containing these amounts have an appearance and odor acceptable for nasal administration.

In another embodiment, the concentration of fucoidan in the pharmaceutical composition may be from about 20,000 μg/mL to about 100 μg/mL, 500 μg/mL, 1250 μg/mL, or 2500 μg/mL. For example, the concentration of fucoidan may be 2500 μg/mL, 1250 μg/mL, 500 μg/mL, or 100 μg/mL. In one embodiment, the concentration of fucoidan is from about 20,000 μg/mL to about 1250 μg/mL.

In one embodiment, the pharmaceutical composition comprises a sodium chloride solution. Sodium chloride can be used as a tonicity adjusting agent. Adjusting the osmolality of pharmaceutical compositions for nasal administration is preferred to maintain the integrity of the nasal epithelium and is well known in the art. Sodium chloride is preferred because the ions of this salt are ubiquitous in the human body and intranasal administration of sodium chloride solutions does not cause any side effects. In addition, the sodium chloride solution is also safe for oral administration and has an acceptable taste and odor. This is important in terms of compliance with treatment, since after nasal administration, especially administration of a nasal spray, as discussed above, the administered dose of the composition may end up in the pharyngeal region of the subject and may contact the taste buds before swallowing. In addition, it is preferred because it is compatible with fucoidan.

In one embodiment, the aqueous solution is isotonic. Isotonic solutions are preferred because when applied to the nasal epithelium, they will not impart osmotic pressure, negative or positive, to the epithelium. Therefore, they are very gentle carriers for the fucoidan compositions of the present invention, maintaining the integrity of the nasal epithelium.

Preferably, the isotonic aqueous solution is sodium chloride solution, but other agents may be used to adjust osmolality.

It should be understood that in addition to the ingredients specifically mentioned above, the compositions described herein may include other agents conventionally used for nasal administration, such as preservatives, pH adjusters, viscosity modifiers, wetting agents, solvents, solubilizers, decongestants such as alpha-sympathomimetics, essential oils such as peppermint oil, or cooling agents such as menthol.

In one embodiment, the pharmaceutical composition is administered intranasally two or three times a day to each nostril of the subject. The inventors have found that administering two doses within 24 hours, eight hours apart, is more effective in reducing viral replication than administering a single dose within 24 hours, and therefore, administering three times a day is expected to further enhance the efficacy. Consumers are accustomed to using the nasal spray three times a day or every 6-8 hours, and more frequent use would be less convenient for the consumer.

In one embodiment, the pharmaceutical composition is administered at a dose of 3.75 mg to 10.15 mg of fucoidan per nostril of the subject per administration. In an alternative embodiment, the pharmaceutical composition is administered at a dose of 1 mg to 4 mg of fucoidan per nostril of the subject per administration. In another embodiment, the pharmaceutical composition is administered at a dose of about 1.4 mg of fucoidan per nasal spray. Preferably, each administration includes two sprays per nostril of the subject. Therefore, preferably, the pharmaceutical composition is administered at a dose of about 2.8 mg of fucoidan per nostril per administration. Thus, the total dose per administration is preferably 5.8 mg of fucoidan. Preferably, the pharmaceutical composition is administered three to four times per day. This dose is considered effective in vivo.

In one embodiment, the fucoidan is fucoidan extracted from Undaria pinnatifida. Fucoidan is extracted from Undaria pinnatifida as described in the literature as sulfated galactofucan, since it contains a high amount of galactose, such as 40 mol% or more, of the total amount of neutral sugars. Undaria pinnatifida is a preferred source of fucoidan for the pharmaceutical compositions of the present invention, since it provides fucoidans with a high sulfate content and is widely available.

In one embodiment, the fucoidan has an average molecular weight of 150 kDa and a lower limit of the molecular weight range of 10 kDa. In one embodiment, the fucoidan has an average molecular weight of greater than 7 kDa. In one embodiment, the fucoidan has an average molecular weight of greater than 10 kDa. In one embodiment, the fucoidan has an average molecular weight of 10 kDa to 500 kDa. In one embodiment, the fucoidan has an average molecular weight of 50 kDa to 250 kDa. In one embodiment, the fucoidan has an average molecular weight of 50 kDa to 120 kDa. In another embodiment, the fucoidan has an average molecular weight of 70 kDa to 105 kDa. Such a high average molecular weight provides a complex structure of the polysaccharide having a large surface area for interaction with the surface of the virus. These large molecules are flexible in their configuration and can wrap around the virus surface, allowing multiple binding between the viral surface proteins and the sulfate groups of fucoidan. In addition, fucoidans with an average molecular weight greater than 70 kDa were effective in reducing the viral genome copy number for both OC43 and SARS-CoV-2 (Example 1, 2, and 3), and exhibited binding activity to the viral S1 and RBD proteins. Setting the lower molecular weight limit to 10 kDa resulted in a higher yield of large polymer chains.

Fractions with a molecular weight of less than 7.2 kDa are less preferred. It is therefore advisable to use fucoidan fractions with an average molecular weight of more than 7.2 kDa, more preferably more than 10 kDa. It is preferable to use fucoidan with a high molecular weight (MW) with the specified lower MW limit, since high MW polymers are not expected to enter the systemic bloodstream through the mucous membranes. Even if a dose of the composition is swallowed and enters the gastrointestinal tract, it is expected that the entry into the systemic bloodstream will be insignificant, since high molecular weight polymers are not absorbed in the intestine. Therefore, no systemic effects are expected upon intranasal administration of the pharmaceutical compositions according to the invention. This is particularly preferable, since the risk of side effects is reduced.

Preferably, the average molecular weight is determined by size exclusion chromatography (SEC) with a mobile phase containing 150 mM NaCl and having a pH of 6.

In one embodiment, the fucoidan has a sulfate content of 20% to 40%. This high sulfate content is accompanied by a high negative charge. In another embodiment, the fucoidan has a sulfate content of greater than 27%. More preferably, the sulfate content is 27% to 35%. It is believed that the large number of negatively charged residues provides particularly good interaction and binding with viral surface proteins, particularly in the case of viruses with surface proteins with a net positive charge. An example of such a positively charged viral surface protein is the RBD of the S1 domain of the OC43 spike protein, which has a net positive charge of +6. Without being limited by any particular theory, it is believed that multiple salt bridges are formed between the positively charged viral surface proteins and the sulfate groups of the fucoidan, resulting in stable binding of the surface proteins to the fucoidan moiety. This binding may even result in a conformational change in the surface protein, altering its function. If the positively charged surface protein is a viral surface protein that is required for binding to the host cell, binding of fucoidan to this surface protein may inactivate it and prevent binding to the receptor and entry into the host cell. The sulfate content of a fucoidan sample can be determined by a gravimetric method known in the art, such as acid hydrolysis followed by oxidation and subsequent precipitation of barium sulfate. The resulting sulfate mass is then related to the mass of the fucoidan sample to obtain the sulfate content w/w.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: OC43 virus genome copy number in copies/mL determined in apical culture media over time, depending on intervention.

FIG. 2: SARS-CoV-2 viral genome copy number in % determined in apical culture media over time, depending on intervention.

FIG. 3: Genome copy number of OC43 virus in copies/ml determined in apical culture media over time, upon treatment with Vesta fucoidan.

FIG. 4: The copy number of the OC43 virus genome in copies/mL determined in apical culture media over time upon treatment with Jiwan fucoidan.

FIG. 5: Genome copy number of OC43 virus in copies/ml determined in apical culture media over time, upon treatment with Nutra Green fucoidan.

FIG. 6: Isothermal titration microcalorimetry (ITM) experiment: results of 1-fucoidan analysis in the S1 domain of the spike protein.

FIG. 7: ITC experiment: results of 2-fucoidan analysis in the S1 domain of spike protein.

FIG. 8: Thermodynamic contributions of 1 μM fucoidan binding to 4 μM SARS-CoV-2 spike protein S1 domain.

FIG. 9: ITC experiment: 3-fucoidan receptor binding domain (RBD) assay results.

FIG. 10: ITC experiment: results of 4-fucoidan analysis in RBD.

FIG. 11: Thermodynamic contributions of 2 μM fucoidan binding to 5 μM SARS-CoV-2 spike protein S1 domain.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

The following definitions define the meanings of specific terms in the context of the present invention unless otherwise indicated in the specific context.

The term fucoidan refers to marine-derived polysaccharides containing L-fucose monomers, partially sulfated, and extracted from edible species of brown seaweed or kelp (Phaeophyceae). The chemical structure of fucoidans is varied and complex. In addition to L-fucose, other monosaccharides such as D-fucose, mannose, galactose, glucose, and xylose may be present. The configuration of the monosaccharides may vary. Glucuronic acid may also be present. The degree of sulfation, that is, how many hydroxyl residues in the polysaccharide are sulfated, may vary. The substitution pattern of the sulfate groups, that is, which hydroxyl groups in the monosaccharide are sulfated, may also vary. The monosaccharides may also be partially O-acetylated. The polymer may be linear or branched. The average molecular weight (MW) of fucoidan may also vary. The average MW has been reported to be between 4 kDa and 5 MDa. The average MW of fucoidan can be determined by size exclusion chromatography (SEC) such as gel permeation chromatography (GPC). There are many types and grades of fucoidan on the market, and they are within the scope of the present invention. For example, fucoidan derivatives such as mechanically, chemically or enzymatically processed fucoidan are on the market, and they are within the scope of the present invention.

Solution/solubilization/dissolution includes molecular dispersed and colloidal dispersed solutions.

An isotonic solution is one that has an osmolarity close to the osmolarity of human plasma, that is, from 250 to 350 mOsm/l.

Saline solution is 0.9% w/v NaCl in purified water.

The degree of sulfation is the calculated percentage of monosaccharide units containing a sulfate substituent. The degree of sulfation can be calculated from elemental analysis data by obtaining the percentage of carbon and sulfur atoms in a sample and relating it to the number of carbon atoms in the galactose or fucose monosaccharide (six) and the degree of acetylation determined for the sample, obtained for example by ¹ H-NMR.

The sulfate content is the calculated mass percentage of sulfate groups relative to the mass of the fucoidan sample. It can be determined by a gravimetric method known in the art, for example by acid hydrolysis followed by oxidation and subsequent precipitation of barium sulfate. The resulting mass of sulfate is then related to the mass of the fucoidan sample to obtain the mass/mass sulfate content.

High-risk categories for SARS-COV-2 infection include the following: individuals aged 60 years or older; smokers; individuals with a chronic medical condition including heart disease, lung disease, diabetes, cancer, or high blood pressure; individuals with immunodeficiency, such as individuals undergoing treatment for cancer or autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and inflammatory bowel disease; individuals who have had a transplant; and individuals with HIV infection.

Close contacts of a patient infected with SARS-CoV-2 are defined as individuals who live in the same household as the patient, have had direct or physical contact with the patient, or have been within two metres of the patient for more than 15 minutes at or after the time the patient first reported symptoms.

EXAMPLES

Example 1

This example describes an in vitro experiment in which the effect of the pharmaceutical compositions of the present invention on the viral genome copy number and indices of epithelial integrity and inflammation in a model of human cells infected with the human coronavirus OC43 was studied. Unless otherwise stated, the culture medium in the experiments was MucilAir culture medium (product number EP04MM, available from Epithelix Sari, Geneva, Switzerland), which is a ready-to-use, chemically characterized, serum-free culture medium. Unless otherwise stated, incubations were performed at 34°C, 5% _CO2 , and 100% humidity.

Cellular model

The in vitro model used in this experiment was a standardized nasal human 3D epithelial model called MucilAir, manufactured by Epithelix Sari, Switzerland. This model has functional characteristics similar to those of epithelium in vivo, such as mucus production, mucociliary clearance, and secretion of cytokines and chemokines, and is therefore a suitable model for the development of new antiviral pharmaceutical compositions (B. Boda et al., Antiviral drug screening by assessing epithelial functions and innate immune responses in human 3D airway epithelium model, Antiviral Research 156 (2018) 72–79, https://doi.Org/10.1016/j.antiviral.2018.06.007 and A. Pizzorno et al., Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia, bioRxiv preprint, 2020, doi: https://doi.org/10.1101/2020.03.31.017889). The mature cell model of MucilAir consists of basal cells, ciliated cells, and mucus cells. The ratio of these different cell types is the same as that observed in vivo. The cell model used in the experiment was prepared as follows. Airway cells were obtained from patients who had undergone nasal biopsy. Human airway epithelial cells were isolated, amplified, and expanded in volume by two passages to maintain the physiological characteristics of the cells.

Cells were seeded on a semiporous membrane (Costar Transwell, 0.4 μm pore size) and cultured at the air-liquid interface for differentiation. Airway epithelium was reconstituted from a mixture of human airway cells from 14 individual healthy donors to reduce inter-donor variation (called MucilAir-Pool method). Reconstituted airway epithelium was cultured at the air-liquid (AL) interface in culture medium in 24-well plates with 6.5 mm Transwell inserts (Cat. #3470, Corning Incorporated, Tweksbury, USA). Cell cultures were maintained for 34 days at the air-liquid interface at the start of the experiments and were fully differentiated at the start of the experiment. A total of 21 inserts were used in the experiment. Three days before the start of the experiment, all cell culture inserts used were checked for integrity and quality in a three-step quality assessment procedure. First, they were visually inspected under a conventional inverted microscope. The pass criteria were visible ciliary beat and a uniform and homogeneous appearance. Second, all inserts were verified to have physiological mucus production by measuring the refractive index of the apical surface of the inserts. Third, the trans epithelial electrical resistance (TEER) was measured as described later in the text. The pass criterion was a TEER value greater than 200 ^Ω.cm2 . All inserts met the specified quality assessment criteria. Each insert was then washed apically with culture medium on the same day to remove accumulated mucus and cellular debris to minimize the risk of interference with the studies.

Compositions

The compositions used in Example 1 are shown in Table 1.

The test compositions were prepared by mixing fucoidan powder with saline (0.9% w/v NaCl in purified water) at a concentration of 10 mg/mL and dissolving by vortexing. This solution was further diluted with saline (0.9% w/v NaCl in purified water) by volume to obtain test compositions with concentrations of 5 mg/mL and 1.25 mg/mL. For each dose, 10 μL of the test composition was pipetted onto the apical side of the cell model to obtain a dose of 12.5 μg, 50 μg, or 100 μg fucoidan.

The experimental drug GS-441524 was provided by Epithelix and was used as a positive control for inhibition of viral replication. GS-441524 is the active metabolite of the prodrug remdesivir. The drug was dissolved in 100% DMSO to obtain a 50 mM stock solution. It was diluted with medium to a final concentration of 25 μM (and 0.05% DMSO). 500 μL of GS-441524 solution was added to the basal well 1 hour before virus inoculation. Previous studies have confirmed that this amount of DMSO does not affect the parameters measured in this experiment. The basal medium was changed every day, and the same dose of GS-441524 solution was then added to fresh basal culture medium. As a negative control, 10 μl of saline was applied to the apical side of the cell cultures. The positive control for cytotoxicity was Triton X-100 (polyethylene glycol tert-octylphenyl ether), a nonionic surfactant often used in biochemistry to permeabilize or lyse cell membranes. 50 μl of 10% (v/v) in saline was pipetted onto the apical side of the inserts. Triton X-100 induces massive LDH release in reconstituted human airway epithelium and was used as a 100% cytotoxicity sample.

Virus inoculation

Coronavirus OC43 was isolated from a clinical specimen in 2014 as described by Essaidi-Laziosi et al., 2017. Virus stock solutions for experiments were prepared in the MucilAir cell model by collecting apical washes with culture medium. Amounts obtained over several days were pooled and quantified by qPCR; aliquots were taken and stored at -80°C. Cell cultures were inoculated on day 1 of the experiment. Before infection, the apical side of the inserts used in the experiment was washed once for 10 min with culture medium. 100 µl of culture medium containing ¹⁰⁵ viral particles coated on the apical side of the cultures (virus concentration was ¹⁰⁶ /ml) were inoculated and incubated for 3 h at 34°C, 5% _CO2 . The virus solution used for inoculation was re-quantified by qPCR and showed a viral genome copy number of 1.1 x 10 ⁶ /ml. Uninfected blanks were exposed to 100 µl of virus-free culture medium at the apical side for 3 h at 34°C, 5% CO ₂ . Unbound viruses were washed with culture medium after 3 h of incubation using three rapid wash steps. Residual viruses after the 3 washes were collected by a 20-min apical wash and quantified by qPCR to establish baseline viral growth at subsequent time points. New viral particles produced by replication in infected cell cultures were collected by 20-min apical washes at 24, 48, 72, and 96 h post-inoculation and quantified by qPCR.

Test parameters

The number of copies of the viral genome is a direct indicator of the number of viral particles present in the sample. In the experiment, the number of copies of the viral genome in cell cultures treated with the studied compositions, positive control (GS-441524) and negative control (one without treatment, one treated with culture medium) were compared to establish the effect of the studied compositions on the OC43 virus. To obtain the number of copies of the viral genome, 20 μl of 200 μl of apical wash fluid were used to extract viral RNA using the QIAamp Viral RNA kit (Qiagen) to obtain 60 μl of eluted RNA. Viral RNA was then quantified by quantitative RT-PCR (QuantiTect Probe RT-PCR, Qiagen) using 5 μl of viral RNA with Mastermix and two OC43-specific primers and a probe with FAM-TAMRA reporter-quencher dyes. Four dilutions of known OC43 RNA concentrations and a RT-PCR control were used, and the plates were read on the Chromo4 PCR Detection System from Bio-Rad. Cycle threshold (Ct) data were plotted on a dilution-corrected standard curve and presented as genome copy number/ml on graphs.

Trans-epithelial electrical resistance (TEER) is a dynamic parameter that reflects the state of the epithelium and is usually between 200 and 600 ^Ohm.cm2 . An increase in TEER reflects the blockade of the ion channel activity of the MucilAir cell model. In some cases, a marked decrease in TEER values can be observed, but with a value still above 100 ^Ohm.cm2 , indicating the activation of ion channels.

Disruption of cell junctions or holes in the epithelium result in TEER values below 100 ^ohm.cm2 . When the epithelium is damaged, a decrease in TEER will be due to an increase in LDH release or a decrease in cell viability. In this example, TEER was measured after adding 200 μl of culture medium to the apical compartment of the inserts (i.e., during the washing step). TEER was measured using an EVOMX volt-ohm meter (World Precision Instruments UK, Stevenage) for each condition. Resistance values (ohms) were converted to TEER ( ^ohm.cm2 ) using the following formula: TEER ( ^ohm.cm2 ) = (resistance value (ohms) - 100 (ohms)) × 0.33 ( ^cm2 ), where 100 ohms is the membrane resistance and 0.33 ^cm2 is the total epithelial surface area in one insert. The TEER measurement was performed as follows. 200 µl of culture medium was added to the apical surface of each insert. The EVOMX was turned on. The electrode was rinsed with 70% ethanol and the EVOMX screen displayed -1. The electrode was then rinsed with culture medium and the EVOMX screen displayed 0.00. The EVOMX then measured the resistance (Ohms) in ohm mode.

Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is rapidly released into the basal culture medium upon rupture of the plasma membrane. In this experiment, it served as a parameter indicating the cytotoxic effect of the applied compositions. It was measured using the Cytotoxicity LDH Assay Kit-WST (Dojindo, CK12-20), which was read using a plate reader to measure the optical density of the samples at 490 nm. The samples were the basal medium collected from all inserts at T48 h and T96 h. The following equation was used to determine the percentage of cytotoxicity (A = optical density values): Cytotoxicity (%) = (A _X (determined optical density of the sample) - A _L (optical density of the low control) / A _H (optical density of the high control) - A _L (low control)) * 100. The high control was the positive control for cytotoxicity (apical treatment with 10% Triton X-100). Triton X-100 causes extensive LDH release and corresponds to 100% cytotoxicity. The low control was the basal culture medium from fresh MucilAir inserts. The negative controls (untreated and vehicle) show low daily basal LDH release <5% due to the physiological cell turnover rate in MucilAir.

The ciliary beat frequency (CBR) is a parameter that shows whether the nasal epithelium is healthy and whether it performs its physiological function of mucus transport. CBR was measured using a special device designed for this purpose.

The system consists of three parts: a Sony XCD V60 camera connected to an Olympus BX51 microscope, a PCI bus, and a specific software package. The ciliary beat frequency is expressed in Hz. For the measurements, a cell culture insert was placed under the microscope and 256 images were taken at a high frequency (125 frames per second) at 34°C. The CBR was then calculated using the appropriate software. It should be noted that the CBR values may fluctuate depending on parameters such as temperature, mucus viscosity, or the liquid applied to the apical surface of the cell model, and the observed values should be compared with the control condition in each experiment.

Mucociliary clearance (MCC) is a result of synchronized ciliary beating and is a parameter that shows whether the nasal epithelium is healthy and performs its physiological function of mucus transport. MCC was monitored using a Sony XCD-U100CR camera connected to an Olympus BX51 microscope with a 5x objective. Polystyrene microbeads with a diameter of 30 μm (Sigma, 84135) were added to the apical surface of cell cultures. Microbead movements were monitored on video at a rate of 2 frames per second for 30 images at 34°C. Three movies were taken per insert. The average velocity of granule movement (μm/s) was calculated using ImageProPlus 6.0 software.

Description of the experiment

The experimental design was as shown below in Table 2. T denotes the measurement time point, where the number denotes the number of hours after the start of the experiment; for example, T24h denotes the time point 24 hours after the start of the experiment.

At the beginning of the experiment, T0h, the inserts were washed with 200 μl of culture medium for 10 min at 34°C and the inserts were transferred to a new culture plate containing 500 μl of culture medium per well. According to the experimental design, the inserts were either treated with 10 μl of the test composition at one of the three indicated concentrations apically, 10 μl of the negative control apically, the positive control at the basal side, or not treated at all. All inserts were incubated for 1 h.

At T1h, all inserts except blanks were inoculated as described above. All inserts were incubated for 3 h.

At T4h, all inserts were washed with three rapid washes of 200 μl of culture medium at 34°C to remove viral inoculum and unbound viruses. Then, 200 μl of culture medium was added apically to all inserts and the inserts were left for 20 min at 34°C. This apical fluid was then removed and stored at -80°C until viral copy number determination. According to the experimental design, inserts were either treated with 10 μl of one of the three indicated concentrations of the test composition apically, 10 μl of the negative control apically, or not treated. All inserts were incubated for 5 h.

At T8h, according to the experimental design, the inserts were either treated with 10 μl of the test composition at one of the three indicated concentrations apically, 10 μl of the negative control apically, or not treated. All inserts were incubated for 16 h.

At T24h, 200 μl of culture medium was added apically to all inserts and the inserts were left for 20 min at 34°C. During this time, the TEER of all inserts was measured. The apical fluid was then removed and stored at -80°C until viral copy number determination. According to the experimental design, inserts were either treated with 10 μl of the test composition at one of the three specified concentrations apically, 10 μl of the negative control apically, or not treated. The basal growth medium was removed and stored. All inserts were transferred to new culture plates containing 500 μl of culture medium per well. According to the experimental design, positive control inserts were again treated with the positive control on the basal side. All inserts were incubated for 8 h.

At T32h, according to the experimental design, the inserts were either treated with 10 μl of the test composition at one of the three indicated concentrations apically, 10 μl of the negative control apically, or not treated. All inserts were incubated for 16 h.

At T48h, 200 μl of culture medium was added apically to all inserts and the inserts were left for 20 min at 34°C. During this time, the TEER of all inserts was measured. This apical wash was removed and stored at -80°C until viral copy number was determined. For the LDH assay, 50 μl of basal culture medium from all inserts was used. All inserts were transferred to new culture plates containing 500 μl of culture medium per well. According to the experimental design, inserts were either treated with 10 μl of the test composition at one of the three indicated concentrations apically, 10 μl of the negative control apically, untreated, or re-treated with the positive control at the basal side. All inserts were incubated for 8 h.

At T56h, according to the experimental design, the inserts were either treated with 10 μl of the test composition at one of the three indicated concentrations apically, 10 μl of the negative control apically, or not treated. All inserts were incubated for 16 h.

At T72h, 200 μl of culture medium was added apically to all inserts and the inserts were left for 20 min at 34°C. During this time, the TEER of all inserts was measured. The apical wash was then removed and stored at -80°C until viral copy number was determined. According to the experimental design, inserts were either treated with 10 μl of the test composition at one of the three indicated concentrations apically, 10 μl of the negative control apically, or untreated. The basal growth medium was removed and stored. All inserts were transferred to new culture plates containing 500 μl of culture medium per well. According to the experimental design, positive control inserts were again treated with the positive control on the basal side. All inserts were incubated for 8 h.

At T80h, according to the experimental design, the inserts were either treated with 10 μl of the test composition at one of the three indicated concentrations apically, 10 μl of the negative control apically, or not treated. All inserts were incubated for 16 h.

At T96h, the ciliary beat frequency (CBR) and mucociliary clearance (MCC) were measured in all inserts. 200 μl of culture medium was added apically to all inserts and the inserts were left for 20 min at 34°C. During this time, the TEER of all inserts was measured. The apical wash fluid was then removed and stored at -80°C until viral copy number was determined. The basal culture medium was removed and stored. For LDH analysis, 50 μl of basal culture medium from all inserts was used.

Results

Data on the number of OC43 virus genome copies were expressed as log 10 (((copies/mL) + 1). The number 1 was added to include values of 0 copies/mL. Repeated measures analysis of variance (ANOVA) with composition and time as factors was used in the analysis, based on the study design and data from 24 to 96 hours. Since repeated measurements for the same inserts were correlated and showed different variability, Proc Mixed in SAS ^® for Windows, Version 9.4 (Cary, NC; USA) with an unstructured covariance matrix was used in the analysis. The assumption of normal distribution was tested by analysis of residuals. The analysis was considered statistically significant at a p-value ≤ 0.05. To account for the statistically significant composition x time interaction term in hours and to control for the effect of multiple comparisons at the significance level of 0.05, each composition was compared with the negative control using Dunnett's test for each day. To assess whether a three-log (log 10) reduction was achieved for the formulations compared to the negative control, the lower bounds of the one-sided 95% Dunnett-corrected confidence intervals were obtained for the difference from the negative control (i.e., negative control formulation). JMP ^® Statistical Discovery™, Version 14.0.0 (Cary, NC, USA) was used to summarize the results in tables and graphs.

Reduction in the number of copies of the virus genome

The OC43 replication curves for the negative control, Test. 12.5, Test. 50, Test. 100 and positive control inserts are shown in Figure 1 and Table 3.

When the viral genome copy number was below the detection limit of the assay, it was reported as 0.00 in the table and expressed as log 10 (((copies/mL) + 1) in the graphs. The number 1 was added to include 0.00 copies/mL values in the graphs. As can be seen from these results and Fig. 1, OC43 showed good viral replication in the negative control, indicating that this cell model is suitable for investigating the efficacy of the test compositions against OC43. Compared with the negative control, treatment with the positive antiviral control and the test composition of fucoidan at all three concentrations statistically significantly reduced the apical genome copy number in the cell model at each time point (T24h - T96h). This reduction relative to the negative control for the test compositions was statistically significant by more than three log scales on days 1-4, except for Test. 12.5 at T72h. In addition, on days 1-4 The apical genome copy number for all three concentrations of the test composition was numerically less than or equal to the corresponding number for the positive antiviral control, with the exception of Test. 12.5 at T48h.

To enter the cell, coronaviruses use the membrane-bound spike protein to bind to a host cell surface receptor. For example, the receptor-binding domain (RBD) of the S1 domain of the SARS-CoV-2 spike protein (S protein) binds to the cellular receptor ACE2, while the RBD of the OC-43 S protein binds to 9-O-acetylated sialic acid. Without being bound by any particular theory, the present inventors believe that fucoidan, which contains sulfate groups that are negatively charged in solution and physiological environments, interacts with positively charged surface proteins such as the receptor-binding domain (RBD) of the S1 domain of the SARS-CoV-2 spike protein (S protein) and OC-43, thereby preventing the virus from entering the human epithelial cell. Although the studies described in the examples were performed for the OC43 coronavirus, the results obtained can be extrapolated to other coronavirus strains that have a similar net positive charge in the RBD of the spike protein. In particular, similar results were expected when studying SARS-CoV-2 and SARS-CoV-1. The OC43 RBD contains amino acids 327-541, containing 21 basic amino acids and 15 acidic amino acids. The SARS-CoV-2 RBD contains amino acids 318-541, containing 22 basic amino acids and 16 acidic amino acids. The net charge for both RBDs is +6. The negatively charged sulfate groups of the fucoidan polymer can form salt bridges with these positively charged amino acid residues in the RBD, and this polymer structure can coat the surface of the virus and interact with multiple S proteins on the surface of the virus and thus block its interaction with glycosaminoglycans or epithelial cell receptors. The RBD can even undergo a conformational change upon binding to fucoidan, resulting in the inactivation of the S protein for receptor binding. Inactivated virus particles cannot infect epithelial cells and cannot replicate.

TEER:

In all cell culture inserts, TEER values were between 800 and 300 ^Ohm.cm2 at all measurement times, which is within the normal range for this cell model, except for the positive controls for cytotoxicity, for which TEER values were less than 100 ^Ohm.cm2 . The results obtained indicate that the studied compositions do not affect the integrity of the human nasal airway epithelial tissue. This indicates that these pharmaceutical compositions can be used in vivo as nasal spray compositions for humans without adverse effects on the nasal epithelium.

Cytotoxicity:

In accordance with the cytotoxicity measurement scheme described above, LDH levels for Triton X-100 treated inserts were set equal to 100% cytotoxicity, and LDH levels measured in all other inserts were normalized to this value. All cell culture treated inserts (except the positive control) showed cytotoxicity of less than 5% at all time points measured. This result indicates that the compositions do not have cytotoxic effects and can be used in vivo as nasal spray compositions in humans without adverse effects on the nasal epithelium.

Ciliary beat frequency (CBR):

The FBR in the blank (non-infected) inserts was 8.5 Hz, which was within the normal range for the cell model. The negative control, positive control, Test. 12.5, Test. 50, and Test. 100 each showed a significant increase in FBR to approximately 15 Hz. This may be a response of the cell model to viral infection or may be due to the repeated apical addition of fluid to these inserts.

Mucociliary clearance (MCC):

For the negative, uninfected control (blank), the bead velocity was 4 μm/s at 34°C, which is unexpected and outside the normal range for this cell model (normal range: 40-50 μm/s). Repeated exposure to apical fluid or OC43 infection increased mucociliary clearance to normal values, with the OC43-infected insert treated with the apical negative control being significantly different from the blank. The test formulations did not significantly alter mucociliary clearance compared to the negative control.

Example 2:

To confirm that the test compositions are also effective against SARS-CoV-2, the experiment of Example 1 was repeated using SARS-CoV-2 (French circulating strain) as the viral inoculum. SARS-CoV-2 was inoculated at a theoretical multiplicity of infection (MOI) of 0.1. To prepare the test compositions, Undaria pinnatifida powder (Vesta UP, from Vesta Ingredients, Inc., lot VIFD190724) was dissolved in 0.9% NaCl at two concentrations: 5 mg/mL and 10 mg/mL. 10 μL was applied to tissue samples for 2 hours and 24 hours with 2 exposures on day 0 and 1 exposure on day 1 and day 2. The same cell model was used as for Example 1, which underwent the same quality control. The positive control for antiviral activity was remdesivir (MedChemExpress, HY-104077), which was diluted in DMSO and used at a concentration of 5 μM (the final DMSO concentration was 0.05%) in the basolateral medium. The reference antiviral drug was added one hour after virus inoculation and changed every day. The negative control was the vehicle in both infected and uninfected inserts. The viral genome copy number was measured by Taqman RT-PCR after 72 hours for each of the two inserts (results are shown in Table 4).

Virus inoculation

The SARS-CoV-2 strain used in the study was isolated by direct inoculation of VeroE6 cell monolayers with a nasal swab specimen obtained at Bichat Claude Bernard Hospital, Paris. After characteristic cytopathic effect was observed in more than 50% of the cell monolayer, supernatants were collected and immediately stored at -80°C. The complete viral genome sequence was obtained using Illumina MiSeq sequencing technology and deposited under the name BetaCoV/France/IDF0571/2020. Virus stocks were titrated with 50% tissue culture infective dose (TCiD50/ml) in VeroE6 cells using the Reed & Muench statistical method. Before infection, the apical side of the MucilAir™ cultures was washed twice for 10 minutes. Inoculation was with 150 µl at a theoretical multiplicity of infection (MOI) of 0.1 (TCID50 50,000 for an average of 500,000 cells in MucilAirTM) applied to the apical side of the cultures for 1 hour at 34°C, 5% _CO2 . Uninfected controls were also exposed to 150 µl of culture medium on the apical side for 1 hour. Unbound viruses were removed after an incubation period of one hour. New virus particles were collected by 10-minute apical washes (200 µl) at 48 and 72 hours post-inoculation and quantified by qRT-PCR.

At the beginning of the experiment, T1h, all inserts were transferred to a new culture plate containing 700 µl of MucilAir™ culture medium per well. All inserts were washed twice with 200 µl of OptiMEM™ culture medium for 10 min at 37°C. Inserts were then treated with 10 µl of the test composition at one of the two indicated concentrations apically or 10 µl of the negative control apically. All inserts were incubated for 1 h (37°C; 5% CO ₂ ; 100% humidity).

At 10 h, the inserts were inoculated with virus (150 µl in OptiMEM™ culture medium) and incubated (37°C; 5% _CO2 ; 100% humidity) for 1 h.

At T1h, the viral inoculum was removed. The inserts were then transferred to a new culture plate containing 700 µl of MucilAir™ culture medium per well. Remdesivir was added to the basal culture medium as a positive control. The remaining inserts were then treated with 10 µl of the test composition at one of the two indicated concentrations apically or 10 µl of the negative control apically. All inserts were incubated (37°C; 5% _CO2 ; 100% humidity) for 23 h.

At T24h, the inserts were transferred again to a new culture plate containing 700 µl of MucilAir™ culture medium per well. Remdesivir was again added to the basal culture medium as a positive control. The remaining inserts were then treated apically with 10 µl of the test composition at one of the two indicated concentrations or with 10 µl of the negative control. All inserts were incubated (37°C; 5% _CO2 ; 100% humidity) for 24 h.

At T48h, 200 µl OptiMEM™ culture medium was added apically to all inserts and the inserts were left for 10 min at 37°C. This apical fluid was then removed. The inserts were again transferred to a new culture plate containing 700 µl MucilAir™ culture medium per well. Remdesivir was again added to the basal culture medium as a positive control. The remaining inserts were then treated with 10 µl of the test composition at one of the two indicated concentrations apically or 10 µl of the negative control apically. All inserts were incubated (37°C; 5% _CO2 ; 100% humidity) for 24 h.

At T72h, 200 µl of OptiMEM™ culture medium was added apically to all inserts and the inserts were left for 10 min at 37°C. This apical fluid was then removed and stored at -80°C until virus copy number determination.

To determine the number of copies of the viral genome, RNA was extracted from 20 µl of apical wash (from T48h and T72h) using the ^QIAamp® Viral RNA Extraction Kit (Qiagen) to obtain 60 µl of eluted RNA. RNA was quantified using the QuantiTect Probe RT-PCR Kit (Qiagen) (5 μl RNA from 60 μl) and two ORFlb-nsp14-specific primers (5'-TGGGGYTTTACRGGTAACCT-3' (SEQ-ID NO: 1); 5'-AACRCGCTTAACAAAGCACTC-3' (SEQ-ID NO: 2)) and a SARS-CoV-2 probe (5'-FAM-TAGTTGTGATGCWATCATGCACTAGACTAG-TAMRA-3' (SEQ-ID NO: 3)) developed by the School of Public Health/University of Hong Kong (Leo Poon, Daniel Chu and Malik Peiris). Samples were analyzed using the StepOnePlus™ Real-Time PCR System (Applied Biosystems). Ct data were determined and relative changes in gene expression were calculated using the 2-ACt method, expressing the result as a fold decrease relative to the mean value for infected vehicle-treated inserts.

Statistical analysis

Data were expressed as mean ± standard error of the mean. Differences between three or more groups were tested by one-way ANOVA with Dunnett's multiple comparison post hoc test or Kruskal-Wallis nonparametric test with Dunn's post hoc tests using Prism 6 GraphPad software (La Jolla, USA). Differences between two groups were tested by Student's t-test or Mann-Whitney nonparametric test. P values <0.05 were considered statistically significant.

Results

The antiviral control, remdesivir, was effective in reducing the apical copy number of the ARS-CoV-2 S genome. The inhibition magnitude was 5.6 log. In addition, Vesta UP formulation reduced the apical copy number of the SARS-CoV-2 genome in MucilAir™ at both time points by 5.1 and 5.3 log10 at 10 mg/mL and 5 mg/mL, respectively. This result supports the extrapolation of the results obtained for coronavirus OC43 in Example 1. Fucoidan formulations were shown to be effective in reducing the copy number of the ARS-CoV-2 S genome in nasal epithelium.

The results of genome copy number measurements are given in Table 4 and presented graphically in Fig. 2.

Example 3

To determine whether the molecular weight of the fucoidan affects the potency, the experiment of Examples 1 and 2 was repeated using coronavirus OC43 as the virus inoculum and using three different commercially available fucoidans from Undaria pinnatifida extract. The same cell model and quality control were used. The same positive and negative controls were also used as in Example 1. Virus inoculation was performed as in Example 1. The average molecular weight of the three extracts was determined by size exclusion chromatography (SEC) with a mobile phase containing 150 mM NaCl and having a pH of 6. The SEC result is shown in Table 5 below.

Two additional batches of Vesta UP (not used in the cell model experiment) were characterized by SEC and showed that their average molecular weights were 100.6 kDa and 102.7 kDa.

The test formulations were prepared by dissolving Undaria pinnatifida powders in 0.9% NaCl to obtain a stock solution of 5 mg/mL, which was then diluted to a second test concentration of 1.25 mg/mL. Each dose was 10 μL apically, corresponding to 50 μg and 12.5 μg of Undaria extract per cell model insert. Three doses were applied on day 0, and two doses were applied on days 1, 2, and 3.

At the beginning of the experiment, T0h, all inserts were washed with 200 µl MucilAir™ culture medium for 10 min at 34°C and transferred to a new culture plate containing 700 µl MucilAir™ culture medium per well. Inserts were then treated with 10 µl of the test composition at one of the two indicated concentrations apically, or 10 µl of the negative control apically, or 500 µl of GS-441524 basally as a positive control. All inserts were incubated for 1 h (34°C; 5% _CO2 ; 100% humidity).

At T1h, inserts were inoculated with virus (100 µl in MucilAir™ culture medium) and incubated (34°C; 5% _CO2 ; 100% humidity) for 3 h.

At T4h, all inserts were washed three times with culture media. All inserts were then washed with 200 µl of MucilAir™ culture medium for 10 min at 34°C. This apical fluid was then removed and stored at -80°C until virus copy number determination. Inserts were then treated with 10 µl of the test formulation at one of the two indicated concentrations apically or 10 µl of the negative control apically. All inserts were incubated (34°C; 5% _CO2 ; 100% humidity) for 4 h.

At T8h, the inserts were again treated with 10 μl of the test composition at one of the two indicated concentrations apically or with 10 μl of the negative control apically. All inserts were incubated (34°C; 5% _CO2 ; 100% humidity) for 16 h.

At T24h, all inserts were washed with 200 µl MucilAir™ culture medium for 20 min at 34°C. This apical fluid was then removed and stored at -80°C until virus copy number determination. The inserts were then transferred to a new culture plate containing 500 µl MucilAir™ culture medium per well. 500 µl GS-441524 was again added to the basal culture medium as a positive control. The remaining inserts were then treated with 10 µl of the test formulation at one of the two indicated concentrations apically or 10 µl of the negative control apically. All inserts were incubated (34°C; 5% _CO2 ; 100% humidity) for 8 h.

At T32h, the inserts were again treated with 10 μl of the test composition at one of the two indicated concentrations apically or with 10 μl of the negative control apically. All inserts were incubated (34°C; 5% _CO2 ; 100% humidity) for 16 h.

At T48h, all inserts were washed with 200 µl MucilAir™ culture medium for 20 min at 34°C. This apical fluid was then removed and stored at -80°C until virus copy number determination. The inserts were then transferred to a new culture plate containing 500 µl MucilAir™ culture medium per well. 500 µl GS-441524 was again added to the basal culture medium as a positive control. The remaining inserts were then treated with 10 µl of the test formulation at one of the two indicated concentrations apically or 10 µl of the negative control apically. All inserts were incubated (34°C; 5% _CO2 ; 100% humidity) for 8 h.

At T56h, the inserts were again treated with 10 μl of the test composition at one of the two indicated concentrations apically or with 10 μl of the negative control apically. All inserts were incubated (34°C; 5% _CO2 ; 100% humidity) for 16 h.

At T72h, all inserts were washed with 200 μl MucilAir™ culture medium for 20 min at 34°C. This apical fluid was then removed and stored at -80°C until virus copy number determination. The inserts were then transferred to a new culture plate containing 500 μl MucilAir™ culture medium per well. 500 μl GS-441524 was again added to the basal culture medium as a positive control. The remaining inserts were then treated with 10 μl of the test formulation at one of the two indicated concentrations apically or 10 μl of the negative control apically. All inserts were incubated (34°C; 5% _CO2 ; 100% humidity) for 8 h.

At T80h, the inserts were again treated with 10 μl of the test composition at one of the two indicated concentrations apically or with 10 μl of the negative control apically. All inserts were incubated (34°C; 5% _CO2 ; 100% humidity) for 16 h.

At T96h, all inserts were washed with 200 μl MucilAir™ culture medium for 20 min at 34°C. The apical fluid was then removed and stored at -80°C until viral copy number determination. To determine viral genome copy number, RNA was extracted from 20 μl apical washes using the QIAamp ^® Viral RNA kit (Qiagen) to yield 60 μl eluted RNA. Viral RNA was quantified by quantitative RT-PCR (QuantiTect Probe RT-PCR, Qiagen) using 5 μl viral RNA Mastermix and two OC43-specific primers and a probe with FAM-TAMRA reporter-quencher dyes. Four dilutions of a known concentration of the OC43 N2 gene-containing plasmid and RT-PCR control were used, and the plates were read on a Chromo4 PCR Detection System from Bio-Rad. Cycle threshold (Ct) data were plotted on a dilution-corrected standard curve and represented on graphs as genome copy number per ml. Data were expressed as mean ± standard deviation and assumed to be normally distributed. Differences between three or more groups were tested by one-way or two-way ANOVA with Dunnett's multiple comparison post hoc test using Prism 6 GraphPad software (La Jolla, USA). Differences between two groups were tested by Student's t-test. P values < 0.05 were considered statistically significant.

Results

The results are shown in Figures 3-5. Fucoidan from Vesta and Jiwan was effective in reducing the number of viral genome copies in the experiment at all time points in a dose-dependent manner. These results indicate that an average molecular weight greater than 7 kDa is preferable to achieve antiviral activity.

Example 4:

To confirm the theory of electrostatic interaction between fucoidan and SARS-CoV-2 proteins, an experiment using isothermal titration microcalorimetry (ITM) was conducted. ITM can be used to study the thermodynamics of specific host-guest interactions in biology and molecular chemistry. It is a solution-based technique that allows specific measurement of the affinity and thermodynamic driving forces behind binding interactions. ITM involves titrating an aqueous solution of the test compound in a suitable buffer with a solution of viral proteins in an identical buffer medium. To confirm the theory of electrostatic binding, both enthalpy and entropy were measured for each interaction. According to polyelectrolyte theory, when the interaction between positively charged proteins and negatively charged polysaccharides is mainly due to ionic interactions, the entropy change is expected to be positive (release of ions from the protein) and the enthalpy change is expected to be negative. If the entropy change is negative and/or the enthalpy change is positive, then this is an indication of non-ionic interactions involved in the bonding.

Materials

The proteins used were purchased from Peak Proteins. The RBD of SARS-CoV-2 spike protein aa319-541 with a C-terminal 6His tag was used at a concentration of 0.924 mg/mL in phosphate-buffered saline (PBS). The S1 domain of SARS-CoV-2 spike protein aa14-685 with a C-terminal Avi-6His tag was used at a concentration of 0.5 mg/mL in PBS. Heparin (CAS #9041-08-1) was used as a positive control for binding. Sodium heparin (Santa Cruz #sc-203075, lot #130321) was used. The buffer used was PBS (1.32 mM _Na3HPO4 , 0.3 mM _NaH2PO4 , 23.8 mM NaCl). The measurements were performed _in a MicroCal PEAQ-ITC. MicroCal _PEAQ -ITC Analysis Software Version 1.30 was used for the analysis. The fucoidan used was Vesta UP from the same lot as used in Example 3 and characterized by SEC (see Table 5). In a separate ITC experiment (data not shown), NG UP from Nutra Green was used. However, no heat change was observed when titrating the compound against S1 protein or the RBD domain. Therefore, it was concluded that there was no binding to this compound, which confirms the results for the Nutra Green fucoidan from Example 3. Therefore, data for Nutra Green fucoidan are not shown.

The table below shows which combinations of compounds were tested in ITC:

Way

Viral proteins were diluted to the appropriate concentration in PBS. The ITC reference well was filled with PBS according to the ITC software protocol, avoiding air bubbles. The ITC sample well was filled with protein solution or buffer (200 μL) according to the ITC software protocol, avoiding air bubbles. Excess solution was removed from the well. Fucoidan or heparin was dissolved in PBS. The ITC syringe was filled with fucoidan solution, heparin solution or buffer (40 μL) according to the ITC software protocol, avoiding air bubbles. Then the syringe was placed in the sample well and the assay was started.

The instrument parameters were as follows: Temperature: 10°C, Feedback mode: High, Reference power: 10 μcal/sec, Injection volume: 1×0.4 μl and 12×3 μl, Initial delay: 60 sec, Interinjection interval: 100 sec, Stirring speed: 750 revolutions per minute (rpm).

After each analysis, both the sample cell and syringe were cleaned using the automated cleaning protocols in the ITC software. Cells were cleaned using the Wash method (wash with detergent, then rinse with water). Syringes were cleaned using the Rinse method (rinse with water, then dry with methanol).

Results

Assay 1: An air bubble was observed in the sixth through eighth sample injections, resulting in large peaks. Regardless, the peak areas (except peak 8) could be plotted against the molar ratio and a curve was generated. This resulted in an affinity value of 10 nM, which was similar to the results of preliminary experiments of 14 nM using 2 μM fucoidan (these initial experiments were performed to refine the method and are not fully described herein). Figure 6 shows these results for Assay 1.

Assay 2: Figure 7 shows the results of Assay 2, which had no air bubbles/sharp peaks. Here, all the data points could be plotted and the resulting affinity value was 9.2 nM. The thermodynamic contributions to the binding of fucoidan to the S1 protein were also plotted (Figure 8). This suggests that the binding event is driven solely by enthalpic forces (negative kcal/mol) with a large positive entropic contribution. As discussed earlier, this suggests that the binding is driven primarily by ionic interactions. Assays 3 and 4: Figure 9 shows the results of Assay 3, which was the first assay using the RBD protein. The experiment resulted in an affinity value of 7.2 nM for fucoidan to the RBD. Figure 10 shows the results of Assay 4, which yielded an affinity value of 6.3 nM. The thermodynamic contributions to the binding of fucoidan to the RBD protein were also plotted (Figure 11). This suggests that the binding event is driven solely by enthalpic forces (negative kcal/mol) with a large positive entropic contribution.

Thus, the NTC experiment showed that fucoidan does indeed bind to the SARS-CoV-2 S1 protein and the SARS-CoV-2 RBD protein, and that this binding is mainly due to ionic interactions.

The results of the NTC experiment are summarized in Table 7. KD is the dissociation constant, ΔН is the change in enthalpy, TΔS is the change in entropy.

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Claims (27)

1. The use of a pharmaceutical composition containing a pharmaceutically effective amount of fucoidan for the treatment or prevention, by intranasal administration, of a coronavirus infection in humans, characterized in that said composition is a nasal spray, fucoidan has a sulfate content of 20% to 40% and has an average molecular weight of 50 kDa to 250 kDa, determined by size exclusion chromatography with a mobile phase containing 150 mM NaCl and having a pH of 6, and said coronavirus infection is an infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1. 2. Use of a pharmaceutical composition according to claim 1, characterized in that said coronavirus infection is an infection caused by SARS-CoV-2. 3. The use of a pharmaceutical composition according to any of the preceding claims, characterized in that said coronavirus infection is an infection caused by a coronavirus that electrostatically binds to fucoidan. 4. Use of a pharmaceutical composition according to any of the preceding claims, characterized in that said coronavirus infection is an infection caused by a coronavirus that has a net positive charge in the receptor-binding region. 5. Use of a pharmaceutical composition according to claim 4, characterized in that said coronavirus infection is an infection caused by a coronavirus that has a total positive charge of at least 5 in the receptor-binding region of the spike protein. 6. The use of a pharmaceutical composition according to any of the preceding paragraphs, characterized in that said pharmaceutical composition is an aqueous solution. 7. Use of a pharmaceutical composition according to any of the preceding claims, characterized in that said pharmaceutical composition contains from 0.1% to 2.7% (w/w) fucoidan. 8. Use of a pharmaceutical composition according to any of the preceding claims, characterized in that said pharmaceutical composition contains from 0.25% to 1.8% (w/w) fucoidan. 9. Use of a pharmaceutical composition according to any of the preceding claims, characterized in that said pharmaceutical composition contains from 0.5% to 1% (w/w) fucoidan. 10. Use of a pharmaceutical composition according to paragraphs 6-9, characterized in that said pharmaceutical composition contains a sodium chloride solution. 11. Use of a pharmaceutical composition according to any of paragraphs 6-10, characterized in that said aqueous solution is isotonic. 12. The use of a pharmaceutical composition according to any of the preceding claims, characterized in that said pharmaceutical composition is administered intranasally two or three times a day into each nostril of the subject. 13. The use of a pharmaceutical composition according to any of the preceding claims, characterized in that said pharmaceutical composition is administered in a dose of 1 mg to 4 mg of fucoidan into each nostril of the subject per administration. 14. Use of a pharmaceutical composition according to any of the preceding claims, characterized in that the fucoidan is an extract of Undaria pinnatifida . 15. Use of a pharmaceutical composition according to any of the preceding claims, characterized in that fucoidan has an average molecular weight of 50 kDa to 120 kDa, determined by size exclusion chromatography with a mobile phase containing 150 mM NaCl and having a pH of 6. 16. Use of a pharmaceutical composition according to claim 15, characterized in that fucoidan has an average molecular weight of 70 kDa to 105 kDa, determined by size-exclusion chromatography with a mobile phase containing 150 mM NaCl and having a pH of 6. 17. The use of a pharmaceutical composition according to any of the preceding claims, characterized in that the fucoidan has an average molecular weight of 150 kDa and a lower limit of the range of molecular weight values of 10 kDa. 18. Use of a pharmaceutical composition according to any of the preceding claims, characterized in that said pharmaceutical composition is administered to the nasal epithelium of a subject. 19. The use of a pharmaceutical composition according to any of the preceding claims, characterized in that said composition is administered in the form of two doses every 24 hours, wherein said two doses are administered at an interval of eight hours. 20. The use of a pharmaceutical composition according to any of the preceding claims, characterized in that said method of treatment includes reducing the viral load in the subject. 21. Use of a pharmaceutical composition according to claim 20, characterized in that the viral load is determined using RT-PCR carried out on a sample of a swab from the subject's nose. 22. The use of a pharmaceutical composition according to any of the preceding claims, characterized in that said treatment comprises reducing the number of copies of the viral genome in the subject. 23. The use of a pharmaceutical composition according to any of the preceding claims, characterized in that said treatment includes reducing the risk of developing severe symptoms of COVID-19. 24. Use of a pharmaceutical composition according to any of the preceding claims, characterized in that said treatment includes preventing the progression of the severity of COVID-19. 25. A method for treating or preventing a coronavirus infection, comprising intranasally administering to a mammalian subject in need thereof a pharmaceutical composition containing a pharmaceutically effective amount of fucoidan, characterized in that said composition is a nasal spray, fucoidan has a sulfate content of 20% to 40% and has an average molecular weight of 50 kDa to 250 kDa, determined using size exclusion chromatography with a mobile phase containing 150 mM NaCl and having a pH of 6, and said coronavirus infection is an infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1. 26. The use of a pharmaceutical composition containing a pharmaceutically effective amount of fucoidan for preventing the spread of coronavirus in a human population, wherein subjects of said population are tested for the presence of an infection caused by OC43 or SARS-CoV-2, and infected subjects are treated by intranasal administration of said pharmaceutical composition, characterized in that said composition is a nasal spray, fucoidan has a sulfate content of 20% to 40% and has an average molecular weight of 50 kDa to 250 kDa, determined using size exclusion chromatography with a mobile phase containing 150 mM NaCl and having a pH of 6, and said coronavirus infection is an infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1. 27. The use of a pharmaceutical composition containing a pharmaceutically effective amount of fucoidan for preventing the spread of coronavirus in a human population, wherein subjects identified as having a high risk of infection are treated by intranasal administration of said pharmaceutical composition, characterized in that said composition is a nasal spray, fucoidan has a sulfate content of 20% to 40% and has an average molecular weight of 50 kDa to 250 kDa, determined using size exclusion chromatography with a mobile phase containing 150 mM NaCl and having a pH of 6, and said coronavirus infection is an infection caused by OC43 or SARS-CoV-2 or SARS-CoV-1.