ANTIVIRAL PHARMACEUTICAL COMPOSITION
20230270802 · 2023-08-31
Inventors
Cpc classification
A61K36/03
HUMAN NECESSITIES
A61K31/737
HUMAN NECESSITIES
International classification
A61K36/03
HUMAN NECESSITIES
A61K31/737
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
Pharmaceutical compositions are described comprising fucoidan for use in a method for the treatment or the prevention, by intranasal administration, of coronavirus infection in a human. Particularly, the viral infection is OC43 or SARS-CoV-1 or SARS-CoV-2 infection. There are also described Pharmaceutical compositions comprising fucoidan for use in a method for prevention coronavirus spread in a human population, wherein subjects of the population are tested for infection with OC43 or SARS-CoV-1 or SARS-CoV-2 and infected subjects are treated with intranasal administration of the Pharmaceutical composition.
Claims
1. Pharmaceutical composition comprising fucoidan for use in a method for the treatment or the prevention, by intranasal administration, of coronavirus infection in a human.
2. Pharmaceutical composition for use according to claim 1, wherein the coronavirus infection is an infection with OC43 or SARS-CoV-2 or SARS-CoV-1.
3. Pharmaceutical composition for use according to claim 2, wherein the coronavirus infection is an infection with SARS-CoV-2.
4. Pharmaceutical composition for use according to claim 1, wherein the coronavirus infection is an infection with a coronavirus that binds electrostatically to fucoidan.
5. Pharmaceutical composition for use according to claim 1, wherein the coronavirus infection is an infection with a coronavirus that has a positive net charge in a receptor binding region.
6. Pharmaceutical composition for use according to claim 5, wherein the coronavirus infection is an infection with a coronavirus that has a positive net charge of at least 5 in a in a receptor binding region of the spike protein.
7. Pharmaceutical composition for use according to claim 1, wherein the composition is a nasal spray.
8. Pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is an aqueous solution.
9. Pharmaceutical composition for use according to claim 1, wherein the composition comprises 0.1% to 2.7% (w/w) fucoidan.
10. Pharmaceutical composition for use according to claim 1, wherein the composition comprises 0.25% to 1.8% (w/w) fucoidan.
11. Pharmaceutical composition for use according to claim 1, wherein the composition comprises 0.5 to 1% (w/w) fucoidan.
12. Pharmaceutical composition according to claim 8, wherein the pharmaceutical composition comprises a sodium chloride solution.
13. Pharmaceutical composition for use according to claim 8, wherein the aqueous solution is isotonic.
14. Pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered intranasally two or three times daily into each nostril of a subject.
15. Pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered in a dose of 1 mg to 4 mg fucoidan into each nostril of a subject per administration.
16. Pharmaceutical composition for use according to claim 1, wherein the fucoidan is an extract of Undaria pinnatifida.
17. Pharmaceutical composition for use according to claim 1, wherein the fucoidan has an average molecular weight from 50 kDa to 120 kDa, determined by size exclusion chromatography, with a mobile phase comprising 150 mM NaCl and having pH 6.
18. Pharmaceutical composition for use according to claim 17, wherein the fucoidan has an average molecular weight from 70 kDa to 105 kDa, determined by size exclusion chromatography, with a mobile phase comprising 150 mM NaCl and having pH 6.
19. Pharmaceutical composition for use according to claim 1, wherein the fucoidan has an average molecular weight of 150 kDa and a lower molecular weight cut-off of 10 kDa.
20. Pharmaceutical composition for use according to claim 1, wherein the fucoidan has a sulfate content of 20% to 40%.
21. Pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered to nasal epithelium of a subject.
22. Pharmaceutical composition for use according to claim 1, wherein the pharmaceutical composition is administered as two doses every 24 hours, the two doses being administered eight hours apart.
23. Pharmaceutical composition for use according to claim 1, wherein the method of treatment comprises reduction of viral load in a subject.
24. Pharmaceutical composition for use according to claim 23, wherein the viral load is determined by RT-PCR, performed on a nasal swab specimen from the subject.
25. Pharmaceutical composition for use according to claim 1, wherein the method of treatment comprises reducing viral genome copy number in a subject.
26. Pharmaceutical composition for use according claim 1, wherein the method of treatment comprises reducing the risk of severe symptoms of COVID-19.
27. Pharmaceutical composition for use according to claim 1, wherein the method of treatment comprises preventing progressing of severity of COVID-19.
28. A method of treating or preventing coronavirus infection, comprising intranasally administering to a mammalian subject in need thereof a pharmaceutically effective amount of a pharmaceutical composition comprising fucoidan.
29. Pharmaceutical composition comprising fucoidan for use in a method of preventing coronavirus spread in a human population, wherein subjects of the population are tested for infection with OC43 or SARS-CoV-2 and infected subjects are treated with intranasal administration of the pharmaceutical composition.
30. Pharmaceutical composition comprising fucoidan for use in a method of preventing coronavirus spread in a human population wherein subjects who have been identified as being at a high risk of infection are treated with intranasal administration of the pharmaceutical composition.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0047] The following definitions provide the meaning that should be given to specific terms in the context of the present disclosure, unless specified otherwise in specific context.
[0048] The term fucoidan specifies marine polysaccharides comprising L-fucose monomers, partially sulfated, and extracted from edible species of brown seaweed or brown algae (Phaeophyceae). The chemical structure of fucoidans is diverse and complex. Besides L-fucose, other monosaccharides such as D-fucose, mannose, galactose, glucose and xylose can be present. The configuration of the monosaccharides can vary. Glucuronic acid may also be present. The degree of sulfation, i.e. how many of the hydroxy residues of the polysaccharide are sulfated, can vary. The sulfate substitution pattern, i.e. which hydroxy groups of a monosaccharide is sulfated, can also vary. The monosaccharides can further be partly O-acetylated. The polymer can be linear or branched. The average molecular weight (MW) of fucoidan can also vary. Average MW of 4 kDa to 5 MDa have been reported. The average MW of fucoidan can be determined by size exclusion chromatography (SEC) such as gel permeation chromatography (GPC). Many forms and grades of fucoidan are commercially available and are encompassed herein. For example, derivatives of fucoidan, such as mechanically, chemically or enzymatically treated fucoidan, are available commercially and are encompassed herein.
[0049] Solution/ solubilising/ dissolving encompasses molecular disperse and colloidal disperse solutions.
[0050] Isotonic is a solution that has an osmolarity close to that of human plasma, i.e. from 250 to 350 mOsm/L.
[0051] Normal saline is 0.9% w/v NaCl in purified water.
[0052] The degree of sulfation is the calculated percentage of monosaccharide units that have a sulfate substitution. The degree of sulfation may be calculated from elementary analysis to obtain the percentages of carbon and sulfur atoms in a probe and putting them in relation to an amount of carbon atoms in a galactose or fucose monosaccharide (six) and the degree of acetylation determined from that probe, obtained for example with .sup.1H-NMR.
[0053] The sulfate content is the calculated weight percentage of sulfate groups in relation to the weight of a specimen of fucoidan. It may be determined by a gravimetric method known in the art, for example acidic hydrolysis, followed by oxidation and subsequent precipitation of barium sulfate. The determined weight for sulfate is then put in relation to the weight of the specimen of fucoidan to give the sulfate content in w/w.
[0054] High risk categories for SARS-COV-2 infection include the following: subjects of 60 years of age and over; smokers; subjects having a chronic medical condition including heart disease, lung disease, diabetes, cancer or high blood pressure; immunocompromised subjects such as subjects undergoing treatment for cancer or autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and inflammatory bowel disease, subjects having a transplant and HIV positive individuals.
[0055] Close contacts of a patient infected with SARS-CoV-2 are defined as subjects living in the same household as the patient, as having had direct or physical contact the patient, or having remained within two metres of the patient for longer than 15 minutes on or after the date on which symptoms were first reported by the patient.
EXAMPLES
Example 1
[0056] The example describes an in vitro experiment that examined the effect of the pharmaceutical compositions of the invention on viral genome copy number and indicators of epithelia integrity and inflammation in a human cell model infected with human coronavirus OC43. Unless indicated otherwise, in the experiment, culture medium was MucilAir culture medium which is a ready-to-use, chemically defined, serum-free culture medium (product number EP04MM, can be purchased at Epithelix Sàrl, Geneva, Switzerland). Unless specified otherwise, incubation was at 34° C., 5% CO.sub.2 and 100% humidity.
Cell Model
[0057] The in vitro model used for the experiment was a standardised nasal human 3D epithelial model called MucilAir, marketed by Epithelix Sàrl, Switzerland. The model has functional characteristics similar to in vivo epithelia, such as mucus production, mucociliary clearance, and secretion of cytokines and chemokines, and therefore is a suitable model to support 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 MucilAir cell model is composed of basal cells, ciliated cells and mucus cells. The proportion of these various cell types is preserved compared to what one observes in vivo. The cell model used for the experiment was obtained as follows: Airway cells were obtained from patients undergoing nasal biopsy. Human airway epithelial cells were isolated, amplified and expanded by two passages to preserve the physiological characteristics of the cells. The cells were seeded onto a semi-porous membrane (Costar Transwell, pore size 0.4 .Math.m) and cultured at the air-liquid interface for differentiation. Airway epithelia was reconstituted from a mixture of human airway cells of 14 individual healthy donors to lessen differences between donors (technique called MucilAir-Pool). The reconstituted airway epithelia was cultured at the air-liquid interface (ALI) in culture medium in 24-well plates with 6.5-mm Transwell inserts (cat #3470, Corning Incorporated, Tweksbury, USA). The cell cultures were stored 34 days at the air-liquid interface at start of experiments and were fully differentiated at the start of the experiment. A total number of 21 inserts was used in the experiment. Three days before the start of the experiment, the integrity and quality of all used cell culture inserts used was ensured by a three-step quality assurance test. Firstly, visual inspection under a conventional inverted microscope was performed. The pass criteria were visible cilia beating and uniform and homogenous appearance. Secondly, it was ensured that all inserts showed physiological mucus production by measuring the refractive index of the apical surface of the inserts. Thirdly, trans epithelial electronic resistance (TEER) was measured as described later in the description. Pass criteria was a TEER value of more than 200 Ω.cm.sup.2. All inserts complied with the quality assurance test. Each insert was then, still on the same day, washed apically with culture medium to remove accumulated mucus and cell debris to minimize the risk of interference with the tests.
Compositions
[0058] The compositions used in Example 1 are specified in Table 1:
TABLE-US-00001 Compositions used in Example 1 Composition Main component Concentration of main component Details of main component Dose and application Test 12.5 Fucoidan 1.25 mg/ml in normal saline Fucoidan powder from Undaria pinnatifida, product Vesta UP, from Vesta ingredients, Inc. Average molecular weight: 150 kDa, purity: 95,68% fucoidan content, sulfate content: 30,04% 10 .Math.l, apically Test 50 As Test 12.5 5 mg/ml in normal saline As Test 12.5 10 .Math.l, apically Test 100 As Test 12.5 10 mg/ml in normal saline As Test 12.5 10 .Math.l, apically Positive control GS-441524 25 .Math.M in DMSO (final conc. of DMSO 0.05%) Molecular weight: 291.26 Da 500 .Math.l, added to basal culture medium, and 10 .Math.l normal saline apically Negative control Normal saline / / 10 .Math.l, apically Positive control for cytotoxicity: Triton X-100 Triton X-100 10% (v/v) in normal saline - 50 .Math.l, apically
[0059] The test compositions were produced by mixing the fucoidan powder with normal saline (0.9% w/v NaCl in purified water) in a concentration of 10 mg/ml and solubilising by vortexing. This solution was further diluted with normal saline (0.9% w/v NaCl in purified water) by volume to obtain the test compositions of the concentrations 5 mg/ml and 1.25 mg/ml. For each dose, a volume of 10 .Math.l of test composition was pipetted onto the apical side of the cell model resulting in a dose of 12.5 .Math.g or 50 .Math.g or 100 .Math.g fucoidan.
[0060] Investigational drug GS-441524 was provided by Epithelix and used as a positive control for inhibition of virus replication. GS-441524 is the active metabolite of the prodrug remdesivir. The drug was dissolved in 100% DMSO to make a stock solution of 50 mM. This was diluted in medium to a final concentration of 25 .Math.M (and 0.05% DMSO). 500 .Math.l of GS-441524 solution were added to the basal well 1 hour before viral inoculation. In previous studies it had been confirmed that this amount of DMSO has no impact on the parameters measured in the experiment. The basal medium was changed every day and the same dose of GS-441524 solution was subsequently added to the fresh basal culture medium. As a negative control, 10 .Math.l normal saline were applied to the apical side of the cell cultures. Positive control for cytotoxicity was Triton X-100 (Polyethylene glycol tert-octylphenyl ether), a non-ionic surfactant often used in biochemical applications to permeabilise or lyse cell membranes. 50 .Math.l at a concentration of 10% (v/v) in normal saline were pipetted onto the apical side of the inserts. Triton X-100 causes a massive LDH release in reconstituted human airway epithelia and was used as 100% cytotoxicity landmark.
Virus Inoculation
[0061] Coronavirus OC43 was isolated from clinical specimen in 2014 as described in Essaidi-Laziosi et al., 2017. Viral stocks for the experiments were produced in the MucilAir cell model by collecting apical washes with culture medium. The production of several days was pooled and quantified by qPCR, aliquoted and stored at -80° C. Inoculation of the cell cultures was performed on day 1 of the experiment. Prior to infection, the apical side of the inserts used for the experiment was washed once for 10 minutes with culture medium. Inoculations were performed with 100 .Math.l of culture medium containing 10.sup.5 viral particles applied to the apical side of the cultures (the virus concentration was 10.sup.6/ml) and incubated for 3 hours at 34° C., 5 % CO.sub.2. The viral solution used for the inoculation was re-quantified by qPCR and confirmed the viral genome copy number of the inoculum to be 1.1×10.sup.6/ml. Non-infected control inserts (“Mock”) were exposed to 100 .Math.l of culture medium without virus on the apical side for 3 hours at 34° C., 5 % CO.sub.2. Unbound viruses were washed away with culture medium after the 3 hours of incubation period by three rapid washing steps. Residual viruses after the 3 washes were collected by a 20 min apical wash and quantified by qPCR to establish a baseline for viral growth at later time points. New viral particles from replication in the infected cell cultures were collected by 20 min apical washes at 24, 48, 72 and 96 hours post-inoculation and quantified by qPCR.
Test Parameters
[0062] Viral genome copy numbers are a direct measure for the number of viral particles present in a sample. In the experiment, viral genome copy numbers of the cell cultures treated with the test compositions, positive control (GS-441524) and negative control (one not treated, one treated with culture medium) were compared to establish the effect of the test compositions on OC43 virus. To obtain the viral genome copy numbers, 20 .Math.l of the 200 .Math.l of apical washing liquid were used for viral RNA extraction with the QIAamp Viral RNA kit (Qiagen), obtaining 60 .Math.l of eluted RNA. Viral RNA was then quantified by quantitative RT-PCR (QuantiTect Probe RT-PCR, Qiagen) using 5 .Math.l of viral RNA with Mastermix and two OC43-specific primers and probe with FAM-TAMRA reporter-quencher dyes. Four dilutions of known concentration of OC43 RNA as well as control for RT-PCR were included and the plates were run on a Chromo4 PCR Detection System from Bio-Rad. Cycle threshold (Ct) data were reported to the standard curve, corrected with the dilution factor and presented as genome copy number per ml on the graphs.
[0063] Trans-epithelial electronic resistance (TEER) is a dynamic parameter that reflects the state of epithelia and is typically between 200 to 600 Ω.cm.sup.2. An increase of the TEER value reflects a blockage of the ion channel activities of the MucilAir cell culture. A notable decrease of the TEER values, but with the value still above 100 Ω.cm.sup.2 can be observed in certain cases and indicates an activation of the ion channels. Disruption of cellular junction or holes in the epithelia result in TEER values below 100 Ω.cm.sup.2. When an epithelium is damaged, a decrease of TEER would be associated with an increase of LDH release or a decrease of the cell viability. In this example, TEER was measured after addition of 200 .Math.l of culture medium to the apical compartment of the inserts (i.e. during the washing step). TEER was measured with an EVOMX volt-ohm-meter (World Precision Instruments UK, Stevenage) for each condition. Resistance values (Q) were converted to TEER (Ω.cm.sup.2) using the following formula: TEER (Ω.cm.sup.2) = (resistance value (Ω) - 100 (Ω)) x 0.33 (cm.sup.2), where 100 Q is the resistance of the membrane and 0.33 cm.sup.2 is the total surface of the epithelium in one insert. The TEER measurement was conducted as follows: 200.Math.l of culture medium was added on apical surface of each insert. The EVOMX was turned on. The electrode was washed with ethanol 70%, and the EVOMX screen shows the value -1. Then, the electrode was washed with culture medium, and the EVOMX screen shows 0.00. Then, the resistance (Q) was measured with the EVOMX in the Ohms measurement function.
[0064] Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme that is rapidly released into the basal culture medium upon rupture of the plasma membrane. In the experiment, it served as a parameter indicating cytotoxic effect of the applied compositions. It was measured using the Cytotoxicity LDH Assay Kit-WST (Dojindo, CK12-20) which was read out using a plate reader to measure the absorbance of the samples at 490 nm. Samples were the basal media collected from all inserts at T48h and T96h. To determine the percentage of cytotoxicity, the following equation was used (A = absorbance values): Cytotoxicity (%) = (A.sub.x (determined absorbance of the sample)-A.sub.L (absorbance of the low control)/A.sub.H (absorbance of the high control)-A.sub.L (low control))*100. The high control was the positive control for cytotoxicity (10 % Triton X-100 apical treatment). Triton X-100 causes a massive LDH release and corresponds to 100 % cytotoxicity. The low control was basal culture medium of fresh MucilAir cell inserts. The negative controls (non-treated and vehicle) show a low daily basal LDH release, <5 %, which is due to physiological cell turnover in MucilAir.
[0065] Cilia beating frequency (CBF) is a parameter that indicates if the nasal epithelia is healthy and carrying out its physiologic function of mucus transport. CBF was measured by a dedicated setup for this purpose. The system consists of three parts: a Sony XCD V60 camera connected to an Olympus BX51 microscope, a PCI card and a specific package of software. The cilia beating frequency is expressed in Hz. For measurement, a cell culture insert was placed under the microscope and 256 images were captured at high frequency rate (125 frames per second) at 34° C. CBF was then calculated using relevant software. It should be pointed out that CBF values may be subject to fluctuations due to parameters such as temperature, mucus viscosity or liquid applied on the apical surface of the cell model and observed values should be compared to a control condition in each experiment.
[0066] Mucociliary clearance (MCC) results from synchronised cilia-beating and also is a parameter that indicates if the nasal epithelia is healthy and carries out its physiologic 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 of 30 .Math.m diameter (Sigma, 84135) were added on the apical surface of the cell cultures. Microbeads movements were video tracked at 2 frames per second for 30 images at 34° C. Three movies were taken per insert. Average beads movement velocity (.Math.m/sec) was calculated with the ImageProPlus 6.0 software.
Experimental Protocol
[0067] The experimental setup was as below in Table 2. T specifies the point in time of a measurement, with the number indicating the number of hours after the start of the experiment, e.g. T24h specifies the time point 24 hours after start of the experiment.
TABLE-US-00002 Experimental setup of Example 1 Name of the test series Number of repeats Viral infection Treatment at T0h T4h T8h T24h T32h T48h T56h T72h T80h Mock 3 no / / / / / / / / / Negative control 3 yes 10 .Math.l 10 .Math.l 10 .Math.l 10 .Math.l 10 .Math.l 10 .Math.l 10 .Math.l 10 .Math.l 10 .Math.l Test 12.5 3 yes 12.5 .Math.g fucoidan 12.5 .Math.g fucoidan 12.5 .Math.g fucoidan 12.5 .Math.g fucoidan 12.5 .Math.g fucoidan 12.5 .Math.g fucoidan 12.5 .Math.g fucoidan 12.5 .Math.g fucoidan 12.5 .Math.g fucoidan Test 50 3 yes 50 .Math.g fucoidan 50 .Math.g fucoidan 50 .Math.g fucoidan 50 .Math.g fucoidan 50 .Math.g fucoidan 50 .Math.g fucoidan 50 .Math.g fucoidan 50 .Math.g fucoidan 50 .Math.g fucoidan Test 100 3 yes 100 .Math.g fucoidan 100 .Math.g fucoidan 100 .Math.g fucoidan 100 .Math.g fucoidan 100 .Math.g fucoidan 100 .Math.g fucoidan 100 .Math.g fucoidan 100 .Math.g fucoidan 100 .Math.g fucoidan Positive (antiviral) control 3 yes GS-441524 basal + 10 .Math.l normal saline apically / / GS-441524 basal + 10 .Math.l normal saline apically / GS-441524 basal + 10 .Math.l normal saline apically / GS-441524 basal + 10 .Math.l normal saline apically / Positive control cytotoxicity (Triton X-100) 3 yes 50 .Math.l apically / / 50 .Math.l apically / 50 .Math.l apically / 50 .Math.l apically /
[0068] At the start of the experiment, T0h, all inserts were washed with 200 .Math.l culture medium for 10 minutes at 34° C. and the inserts were transferred into a new culture plate containing 500 .Math.l of culture medium per well. According to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, with 10 .Math.l negative control apically, with positive control from the basal side or not treated at all. All inserts were incubated for 1 hour.
[0069] At T1h, all inserts except the mock inserts were inoculated as described above. All inserts were incubated for 3 hours.
[0070] At T4h, all inserts were washed with three quick washing steps with 200 .Math.l of culture medium at 34° C. to remove the viral inoculum and unbound viruses. Afterwards, 200 .Math.l of culture medium was added apically to all inserts and inserts were let sit for 20 minutes at 34° C. This apical liquid was then removed and was stored at -80° C. until the virus copy number was determined. According to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, treated with 10 .Math.l negative control apically, or not treated. All inserts were incubated for 5 hours.
[0071] At T8h, according to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, treated with 10 .Math.l negative control apically, or not treated. All inserts were incubated for 16 hours.
[0072] At T24h, 200 .Math.l of culture medium was added apically to all inserts and inserts were let sit for 20 minutes at 34° C. During this time, TEER of all inserts was measured. This apical liquid was then removed and was stored at -80° C. until the virus copy number was determined. According to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, treated with 10 .Math.l negative control apically, or not treated. The basal culture medium was removed and stored. All inserts were transferred to new culture plates containing 500 .Math.l culture medium per well. According to the experimental setup, positive control inserts were again treated with positive control from the basal side. All inserts were incubated for 8 hours.
[0073] At T32h according to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, treated with 10 .Math.l negative control apically, or not treated. All inserts were incubated for 16 hours.
[0074] At T48h, 200 .Math.l of culture medium was added apically to all inserts and inserts were let sit for 20 minutes at 34° C. During this time, TEER of all inserts was measured. This apical washing liquid was removed and was stored at -80° C. until the virus copy number was determined. 50 .Math.l of the basal culture medium of all inserts was used for the LDH assay. All inserts were transferred to new culture plates containing 500 .Math.l culture media per well. According to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, treated with 10 .Math.l negative control apically, or not treated, or were again treated with positive control from the basal side. All inserts were incubated for 8 hours.
[0075] At T56h, according to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, treated with 10 .Math.l negative control apically, or not treated. All inserts were incubated for 16 hours.
[0076] At T72h, 200 .Math.l of culture medium was added apically to all inserts and inserts were let sit for 20 minutes at 34° C. During this time, TEER of all inserts was measured. This apical washing liquid . was then removed and was stored at -80° C. until the virus copy number was determined. According to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, treated with 10 .Math.l negative control apically, or not treated. The basal culture medium was removed and stored. All inserts were transferred to new culture plates containing 500 .Math.l culture medium per well. According to the experimental setup, positive control inserts were again treated with positive control from the basal side. All inserts were incubated for 8 hours.
[0077] At T 80h, according to the experimental setup, inserts were either treated with 10 .Math.l of test composition in one of the three concentrations apically, treated with 10 .Math.l negative control apically, or not treated. All inserts were incubated for 16 hours.
[0078] At T96h, the ciliary beating frequency (CBF) and the mucociliary clearance (MCC) of all inserts were measured. 200 .Math.l of culture medium was added apically to all inserts and inserts were let sit for 20 minutes at 34° C. During this time, TEER of all inserts was measured. This apical washing liquid was then removed and was stored at -80° C. until the virus copy number was determined. The basal culture medium was removed and stored. 50 .Math.l of the basal culture medium of all inserts was used for the LDH assay.
Results
[0079] The OC43 viral genome copy number data were expressed as log 10 ((copies/ml) + 1). The number 1 was added to include values of 0 copies/ml. Based on the study design and data from hours 24 - 96, the analysis used a repeated measures analysis of variance (ANOVA) with composition and time as factors. Since the repeated measurements on the same inserts are correlated and exhibited varied variability, the analysis used Proc Mixed in SAS® for Windows, Version 9.4 (Cary, NC; USA) with an unstructured covariance matrix. The assumption of normality was checked via examination of summaries of residuals. The analysis set statistical significance at p-value ≤ 0.05. Due to the statistically significant composition x hour interaction and to control for the effect of multiple comparisons at the 0.05 significance level, each composition was compared to the negative control using Dunnett’s Test at each day slice. To assess whether compositions achieved a 3 log 10 reduction from negative control, Dunnett-adjusted one-sided 95% lower confidence limits on the difference from negative control (i.e., negative control - composition) were formed. JMP® Statistical Discovery ™, Version 14.0.0 (Cary, NC; USA) was used to summarize the results via tables and graphs.
Reduction of Viral Genome Copy Number
[0080] The OC43 replication curves of the negative control, Test 12.5, Test 50, Test 100 and positive control inserts are given in
TABLE-US-00003 Results of the measurement of the viral genome copy number in Example 1 Mean Standard Deviation Log 10 ((Copies/ml) + 1) Log 10 ((Copies/ml) + 1) Timepoint of measurement Timepoint of measurement Composition T4h T24h T48h T72h T96h T4h T24h T48h T72h T96h Negative control 0.00 5.39 7.77 8.29 7.99 0.00 0.47 0.48 0.36 0.10 Positive antiviral control 0.00 0.00 0.00 5.21 4.69 0.00 0.00 0.00 0.37 0.13 Test 12.5 0.00 0.00 1.96 3.69 4.01 0.00 0.00 1.70 0.26 0.19 Test 50 0.00 0.00 0.00 2.38 3.25 0.00 0.00 0.00 2.07 0.15 Test 100 0.00 0.00 0.00 3.15 3.36 0.00 0.00 0.00 0.18 0.01
[0081] When the viral genome copy number was below the detection limit of the method, it was noted as 0.00 in the table and expressed as log 10 ((copies/ml) + 1) in the graphs. The number 1 was added to include values of 0.00 copies/ml in the graphs. As can be seen from these results and
[0082] Coronaviruses utilise the membrane bound spike protein to bind to a host cell surface receptor to gain cellular entry. For example, the receptor binding domain (RBD) of the S1 domain of the spike protein (S protein) of SARS-CoV-2 binds to the cellular ACE2 receptor, while the RBD of the S protein of OC-43 binds to 9-O-acetlyated sialic acid. Without being bound to a specific theory, the present inventors believe that fucoidan, comprising sulfate groups negatively charged in solution and the physiological environment, interacts with positively charged surface proteins such as the receptor binding domain (RBD) of the S1 domain of the spike protein (S protein) of SARS-CoV-2 and OC-43 and thereby hinders the virus from entering the human epithelial cell. Whilst the tests described in the examples have been carried out with the coronavirus individuum OC43, the results can be extrapolated to other coronavirus strains having a similar positive net charge in the RBD of the spike protein. Particularly, similar results were expected in testing against SARS-CoV-2 and SARS-CoV-1. The RBD of OC43 comprises amino acids 327 to 541, comprising 21 basic amino acids and 15 acidic amino acids. The RBD of SARS-CoV-2 comprises amino acids 318 to 541, comprising 22 basic amino acids and 16 acidic amino acids. The net charge of both RBDs is +6. The negatively charged sulfate groups of the fucoidan polymer can form salt bridges with these positively charged amino acid residues of the RBD, and the polymeric structure can embrace the surface of the virus and interact with a plurality of S proteins on the viral surface and therefore block it from interacting with the epithelial cell glucosaminoglycans or receptors. The RBD may even undergo conformational change upon fucoidan binding, resulting in an inactivation of the S protein for receptor binding. Inactivated viral particles cannot infect the epithelial cells and cannot replicate.
Teer
[0083] All cell culture inserts at all measured timepoints showed TEER values from 800 to 300 Ω.cm.sup.2, which is in the normal range for the cell model, except for the positive controls for cytotoxicity, which showed TEER values below 100 Ω.cm.sup.2. The results show that the test compositions do not influence tissue integrity of human nasal airway epithelia. This indicates that the pharmaceutical compositions may be used in vivo as nasal spray compositions for humans without negative effects on the nasal epithelia.
Cytotoxicity
[0084] As per the measurement scheme used for cytotoxicity explained above, LDH levels of Triton X-100 treated inserts was set to be 100% cytotoxicity, and the LDH levels measured in all other inserts were put in relation to this value. All cell culture inserts treated (except for the positive control) at all measured timepoints showed less than 5% cytotoxicity. This result shows that the compositions do not have cytotoxic effects and may be used in vivo as nasal spray compositions for humans without negative effects on the nasal epithelia.
Cilia Beating Frequency (CBF)
[0085] CBF of the Mock (not infected) inserts was 8.5 Hz which is in the normal range for the cell model. Negative control, positive control, Test 12.5, Test 50 and Test 100, all showed significant increase of CBF to about 15 Hz. This may be the response of the cell model to the viral infection or may be related to the repeated apical liquid addition in these inserts.
Mucociliary Clearance (MCC)
[0086] Negative, uninfected control (mock) showed beads’ velocity of 4 .Math.m/s at 34° C., which is unexpected and out of the normal range of the cell model (normal range: 40-50 .Math.m/s). Repeated exposure to apical liquid, or OC43 infection increased the mucociliary clearance toward normal values, where OC43 infection treated with apical negative control was significantly different from Mock. Test compositions did not change significantly mucociliary clearance compared to negative control.
Example 2
[0087] To confirm that the tested compositions are also effective against SARS-CoV-2, the experiment of Example 1 was repeated with 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., batch VIFD190724) was dissolved in 0.9% NaCl at two concentrations: 5 mg/ml and 10 mg/ml. 10.Math.l were applied on the tissue samples for 2 hrs and 24 hrs in 2 exposures on day 0 and 1 exposure at day 1 and day 2. The same cell model was used as for Example 1, which underwent the same quality control. Positive control for antiviral effect was Remdesivir (MedChemExpress, HY-104077), which was diluted in DMSO and used at 5 .Math.M (final concentration of DMSO was 0.05 %) in the basolateral medium. Reference antiviral was added after one hour of viral inoculation and changed every day. Negative control was vehicle control, both on infected and non-infected inserts. Virus genome copy number was measured with Taqman RT-PCR after 72 hours for two inserts each (results in Table 4).
Virus Inoculation
[0088] The SARS-CoV-2 strain used in the study was isolated by directly inoculating VeroE6 cell monolayers with a nasal swab sample collected from Bichat Claude Bernard Hospital, Paris. Once characteristic cytopathic effect was observable 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 was deposited under the name BetaCoV/France/IDF0571/2020. Viral stocks were titrated by tissue culture infectious dose 50 % (TCID50/ml) in VeroE6 cells, using the Reed & Muench statistical method. Prior to infection, the apical side of the MucilAir™ cultures were washed twice for 10 min. Inoculations were performed with 150 .Math.l at a theoretical multiplicity of infection (MOI) of 0.1 (50 000 TCID50 for an average of 500 000 cells in MucilAirTM), applied to the apical side of the cultures for 1 hour at 37° C., 5 % CO2. Non-infected control was also exposed to 150 .Math.l of culture medium on the apical side for 1 hour. Unbound viruses were removed after one hour of incubation period. New viral particles were collected by 10 min apical washes (200 .Math.l) 48 and 72 hours post-inoculation and quantified by RT-qPCR.
[0089] At the start of the experiment, T-1h, all inserts were transferred into a new culture plate with 700 .Math.l of MucilAir™ culture media per well. All inserts were washed twice with 200 .Math.l OptiMEM™culture medium for 10 minutes at 37° C. Then, inserts were treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated for 1 hour (37° C.; 5% CO.sub.2; 100% humidity).
[0090] At T0h, inserts were inoculated with virus (150 .Math.l in OptiMEM™ culture media and incubated (37° C.; 5% CO.sub.2; 100% humidity) for 1 hour.
[0091] At T1h, viral inoculum was removed. The inserts were then transferred into a new culture plate with 700 .Math.l of MucilAir™ culture medium per well. Remdesivir was added to the basal culture medium for the positive control condition. The rest of the inserts were then treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (37° C.; 5% CO.sub.2; 100% humidity) for 23 hours.
[0092] At T24h, the inserts were again transferred into a new culture plate with 700 .Math.l of MucilAir™ culture medium per well. Remdesivir was again added to the basal culture medium for the positive control condition. The rest of the inserts were then treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (37° C.; 5% CO.sub.2; 100% humidity) for 24 hours.
[0093] At T48h, 200 .Math.l of OptiMEM™ culture media was added apically to all inserts and inserts were let sit for 10 minutes at 37° C. This apical liquid was then removed. The inserts were again transferred into a new culture plate with 700 .Math.l of MucilAir™ culture medium per well. Remdesivir was again added to the basal culture medium for the positive control condition. The rest of the inserts were then treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (37° C.; 5% CO.sub.2; 100% humidity) for 24 hours.
[0094] At T72h, 200 .Math.l of OptiMEM™ culture media was added apically to all inserts and inserts were let sit for 10 minutes at 37° C. This apical liquid was then removed and was stored at -80° C. until the virus copy number was determined.
[0095] For the determination of viral genome copy number, RNA was extracted from 20.Math.l apical wash (from T48h and T72h) with QIAamp® Viral RNA extraction kit (Qiagen), obtaining 60 .Math.l of eluted RNA. RNA was quantified using QuantiTect Probe RT-PCR (Qiagen) kit for RT-qPCR (5.Math.l of RNA out of 60.Math.l) and two ORF1b-nsp14 specific primers (5′-TGGGGYTTTACRGGTAACCT-3′ (SEQ-ID No. 1); 5′-AACRCGCTTAACAAAGCACTC-3′(SEQ-ID No. 2)) and probe (5′-FAM-TAGTTGTGATGCWATCATGACTAG-TAMRA-3′ (SEQ-ID No. 3)) of SARS-CoV-2 designed by the School of Public Health/University of Hong Kong (Leo Poon, Daniel Chu and Malik Peiris). Samples were run on StepOnePlus™ Real-Time PCR System (Applied Biosystems). Ct data were determined and relative changes in gene expression were calculated using the 2-ΔCt method and reported as the fold reduction relative to the mean of vehicle treated infected inserts.
Statistical Analysis
[0096] Data were expressed as mean±standard error of mean. Differences between three or more groups were tested by one-way ANOVA with Dunnett’s multiple comparison post-tests or nonparametric Kruskal-Wallis test with Dunn’s post-tests using Prism 6 GraphPad software (La Jolla, USA). Differences between two groups were tested by Student’s t test or nonparametric Mann-Whitney test. The values P<0.05 were considered statistically significant.
Results
[0097] Antiviral control remdesivir efficiently reduced apical SARS-CoV-2 genome copies. The magnitude of inhibition was 5.6 log. Also the exposure to Vesta UP formulation decreased apical SARS-CoV-2 genome copies on MucilAir™ at both time points, by 5.1 and 5.3 log10 at 10 mg/ml and 5 mg/ml, respectively. The result confirms the extrapolation of the results obtained for coronavirus OC43 in Example 1. It was shown that fucoidan formulations are effective in reducing viral genome copy number of SARS-CoV-2 in nasal epithelia.
[0098] The results of the genome copy number measurements are given in Table 4 and visualized in
TABLE-US-00004 Results of the measurement of the viral genome copy number in Example 2 Sample % of log reduction in genome copy number compared to the average of negative control Negative control 1 4.76E+01 Negative control 2 1.52E+02 Positive control 1 2.19E-04 Positive control 2 2.40E-04 Test compound 10 mg/ml 1 6.00E-03 Test compound 10 mg/ml 2 5.11E-03 Test compound 5 mg/ml 1 1.91E-03 Test compound 5 mg/ml 2 7.99E-03
Example 3
[0099] To find out if the molecular weight of the fucoidan was of significance for the efficacy, the experiment of Example 1 and 2 was repeated using coronavirus OC43 as viral inoculum and with three different commercially available fucoidans from Undaria pinnatifida extract. The same cell model and quality control was used. Also, the same positive and negative controls as in Example 1 were used. Viral inoculation was performed as in Example 1.The average molecular weight of the three extracts were determined by size exclusion chromatography (SEC), with a mobile phase comprising 150 mM NaCl and having pH 6. The result of the SEC is given in Table 5 below.
TABLE-US-00005 Average molecular weight of fucoidans used in Example 3 Name and supplier of Fucoidan used Average molecular weight as determined by SEC [kDa] Vesta UP, from Vesta 73.3 Jiwan UP, from Jiwan 71.9 NG UP, from Nutra Green 7.2
[0100] Two additional batches of Vesta UP (not used in the cell model experiment) were tested with SEC and showed an average molecular weight of 100.6 kDa and 102.7 kDa.
[0101] The test compositions were prepared by dissolving the Undaria pinnatifida powders in 0.9 % NaCl to obtain a stock solution of 5 mg/ml, which were then diluted to the second tested concentration of 1.25 mg/ml. Each dose was 10 .Math.l apically, corresponding to 50 .Math.g and 12. 5 .Math.g 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.
[0102] At the start of the experiment, T0h, all inserts washed twice with 200 .Math.l of MucilAir™ culture media during 10 min at 34° C. and then transferred into a new culture plate with 700 .Math.l f MucilAir™ culture media per well. Then, inserts were treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically, or 500 .Math.ml GS-441524 basally as positive control. All inserts were incubated for 1 hour (34° C.; 5% CO.sub.2; 100% humidity).
[0103] At T1h, inserts were inoculated with virus (100 .Math.l in MucilAir™ culture media and incubated (34° C.; 5% CO2; 100% humidity) for 3 hours.
[0104] At T4h, all inserts were washed with culture media three times. Then all inserts were washed with 200 .Math.l of MucilAir™ culture media during 10 min at 34° C. This apical liquid was then removed and was stored at -80° C. until the virus copy number was determined. The inserts were then treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (34° C.; 5% CO.sub.2; 100% humidity) for 4 hours.
[0105] At T8h, the inserts were again treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (34° C.; 5% CO.sub.2; 100% humidity) for 16 hours.
[0106] At T24h, all inserts were washed with 200 .Math.l of MucilAir™ culture media during 20 min at 34° C. This apical liquid was then removed and was stored at -80° C. until the virus copy number was determined. The inserts were then transferred into a new culture plate with 500 .Math.l of MucilAir™ culture medium per well. 500 .Math.l of GS-441524 was again added to the basal culture medium for the positive control condition. The rest of the inserts were then treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (34° C.; 5% CO.sub.2; 100% humidity) for 8 hours.
[0107] At T32h, the inserts were again treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (34° C.; 5% CO.sub.2; 100% humidity) for 16 hours.
[0108] At t48h, all inserts were washed with 200 .Math.l of MucilAir™ culture media during 20 min at 34° C. This apical liquid was then removed and was stored at -80° C. until the virus copy number was determined. The inserts were then transferred into a new culture plate with 500 .Math.l of MucilAir™ culture medium per well. 500 .Math.l of GS-441524 was again added to the basal culture medium for the positive control condition. The rest of the inserts were then treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (34° C.; 5% CO.sub.2; 100% humidity) for 8 hours.
[0109] At T56h, the inserts were again treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (34° C.; 5% CO.sub.2; 100% humidity) for 16 hours.
[0110] At T72h, all inserts were washed with 200 .Math.l of MucilAir™ culture media during 20 min at 34° C. This apical liquid was then removed and was stored at -80° C. until the virus copy number was determined. The inserts were then transferred into a new culture plate with 500 .Math.l of MucilAir™ culture medium per well. 500 .Math.l of GS-441524 was again added to the basal culture medium for the positive control condition. The rest of the inserts were then treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (34° C.; 5% CO.sub.2; 100% humidity) for 8 hours.
[0111] At T80h, the inserts were again treated with 10 .Math.l of test composition in one of the two concentrations apically, or treated with 10 .Math.l negative control apically. All inserts were incubated (34° C.; 5% CO.sub.2; 100% humidity) for 16 hours.
[0112] At T96h, all inserts were washed with 200 .Math.l of MucilAir™ culture media during 20 min at 34° C. This apical liquid was then removed and was stored at -80° C. until the virus copy number was determined. For the determination of viral genome copy number, RNA was extracted from 20 .Math.l of the apical washes using the QIAamp® Viral RNA kit (Qiagen), obtaining 60 .Math.l of eluted RNA. Viral RNA was quantified by quantitative RT-PCR (QuantiTect Probe RT-PCR, Qiagen) using 5 .Math.l of viral RNA with Mastermix and two OC43 specific primers and probe with FAM-TAMRA reporter-quencher dyes. Four dilutions of known concentration of the plasmid containing N2 gene of OC43, as well as control for RT-PCR were included and the plates were run on a Chromo4 PCR Detection System from Bio-Rad. Ct data were reported to the standard curve, corrected with the dilution factor and presented as genome copy number per ml on the graphs. Data were expressed as mean±standard error of mean and assumed to have normal distribution. Differences between three or more groups were tested by one-way or two-way ANOVA with Dunnett’s multiple comparison post-tests using Prism 6 GraphPad software (La Jolla, USA). Differences between two groups were tested by Student’s t test. The values P<0.05 were considered statistically significant.
Results
[0113] The results are visualized in
Example 4
[0114] To confirm the theory of the electrostatic interaction between fucoidan and SARS-CoV-2-proteins, an isothermal titration microcalorimetry (ITC) experiment has been performed. ITC can be used to investigate the thermodynamics of specific host-guest interactions in biology and molecular chemistry. It is a method performed in-solution, and specifically measures affinity and thermodynamic driving forces behind binding interactions. ITC involves the titration of an aqueous solution of the test compound in an appropriate buffer into a solution of viral proteins in identical buffer conditions. 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 driven mainly by ionic interactions, it is expected that the entropy change is positive (release of ions from the protein) and the enthalpy change is negative. If the entropy change is negative and/or the enthalpy change is positive, then that is an indication of non-ionic interactions being involved in the binding.
Materials
[0115] The used proteins were purchased from Peak Proteins. SARS-CoV-2 Spike RBD aa319-541 with C-terminal 6His tag was used at 0.924 mg/mL in PBS. SARS-CoV-2 Spike S1 domain aa14-685 with C-terminal Avi-6His tag was used at 0.5 mg/mL in PBS. Heparin was used as a positive control for binding (CAS No. 9041-08-1). Heparin sodium salt (Santa Cruz Product # sc-203075, Lot # 130321) was used. Buffer used was PBS (1.32 mM Na.sub.2HPO.sub.4, 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 analysis. Fucoidan used was Vesta UP, the same batch that was used in Example 3 and was characterised by SEC (see Table 5). In a separate ITC experiment (data not reported herein), NG UP from Nutra Green was used. However, no heat changes were observed when titrating the compound into either S1 protein or RBD domain. Thus it was concluded that no binding occurs with this compound, which confirms the findings for Nutra Green fucoidan from Example 3. Therefore, data on Nutra Green fucoidan was not reported.
[0116] The following table shows which compound combinations were tested in which ITC “run”:
TABLE-US-00006 Compounds and concentrations used in Example 2 Run Test compound Protein 1 1 .Math.M fucoidan 4 .Math.M SARS-CoV-2 Spike S1 domain 2 1 .Math.M fucoidan 4 .Math.M SARS-CoV-2 Spike S1 domain 3 2 .Math.M fucoidan 5 .Math.M SARS-CoV-2 RBD domain 4 2 .Math.M fucoidan 5 .Math.M SARS-CoV-2 RBD domain
Method
[0117] The viral proteins were diluted to the appropriate concentration in PBS. The ITC reference cell was filled with PBS following the ITC software protocol, avoiding the introduction of any air bubbles. The ITC sample cell was filled with protein solution or buffer (200 .Math.l), following to the ITC software protocol, avoiding the introduction of any air bubbles. Any excess solution was removed from the cell. Fucoidan or heparin were solubilised in PBS. The ITC syringe was filled with Fucoidan solution, heparin solution, or buffer (40 .Math.l) following the ITC software protocol, avoiding the introduction of any air bubbles. The syringe was then placed into the sample cell and the run started.
[0118] Instrument parameters were as follows: Temperature: 10° C., Feedback mode: High, Reference Power: 10 .Math.cal/sec, Injection volume: 1 × 0.4 .Math.L and 12 × 3 .Math.L, Initial delay: 60 sec, Interval between injections: 100 sec, Stirring Speed: 750 rotations per minute (RPM).
[0119] After each run both the sample cell and syringe were cleaned using the automated cleaning protocols in the ITC software. Cell cleaning was performed using the ‘Wash’ method (washing with detergent, then rinsing with water. Syringe cleaning was performed using the ‘Rinse’ method (rinsing with water then drying with methanol).
Results
[0120] Run 1: An air bubble was observed in the sixth to eighth injections, resulting in large spikes. Regardless of this, the peak areas (except peak 8) could be plotted against molar ratio, and a curve fitted. This resulted in an affinity reading of 10 nM which was similar to the results of preliminary experiments, which resulted in 14 nM using 2 .Math.M fucoidan (these initial experiments were performed to refine the method and are not fully reported herein).
[0121] Run 2:
[0122] The ITC experiment thus did show that fucoidan binds to SARS-CoV-2 Spike S1 protein and SARS-CoV-2 RBD protein, and that the binding is mainly due to ionic interactions.
[0123] The results of the ITC experiment are summarised in Table 7. KD is the dissociation constant, OH is the change in enthalpy and TΔS is the change in entropy.
TABLE-US-00007 Results of Runs 1 to 4 Run KD (nM) ΔH/ TΔS 1 10.1 -1.08 2 9.17 -1.25 3 7.15 -1.08 4 6.30 -1.10