WO2022167925A1

WO2022167925A1 - Association or composition comprising stabilised lactoferrin

Info

Publication number: WO2022167925A1
Application number: PCT/IB2022/050853
Authority: WO
Inventors: Giancarlo RUOCCO; Alberto BOFFI
Original assignee: Disruptive Technological Advances In Life Science Srl; Fondazione Istituto Italiano Di Tecnologia
Priority date: 2021-02-04
Filing date: 2022-02-01
Publication date: 2022-08-11
Also published as: IT202100002471A1

Abstract

The present invention relates to the association of lactoferrin, at least one polysaccharide or salts thereof and at least one sialic acid or salts thereof in order to increase the stability of lactoferrin and consequent bioavailability of the same. The present invention further relates to pharmaceutically compositions comprising said association and the use thereof.

Description

ASSOCIATION OR COMPOSITION COMPRISING STABILISED LACTOFERRIN The present invention relates to the association of lactoferrin,at least one polysaccharide or salts thereof and at least one sialic acid or salts thereof in order to increase the stability of lactoferrin and consequent bioavailability of the same. The present invention further relates to pharmaceutically compositions comprising said association and the use thereof. STATE OF THE ART Recent global pandemic caused by SARS-Cov-2 infection has caused up to date more than 29,000,000 infections and more than 900,000 deaths. At present, no vaccine to prevent the infection is available and various treatment protocols are under investigation. SARS-Cov-2 belongs to the Corinaviridae family. To date 7 coronavirus strains are known to infect humans and no approved therapies or vaccine against them are available. In the past two decades, in addition to SARS-CoV-2, two other β-coronavirus have caused three of the most severe epidemics worldwide: SARS-CoV and MERS-CoV which respectively cause the Severe Acute Respiratory Syndrome (SARS), and the Middle East Respiratory Syndrome (MERS). It is well characterized how SARS-CoV infection is mediated by the high-affinity interactions between the receptor-binding domain (RBD) of the spike (S) glycoprotein and the human-host Angiotensin-converting enzyme 2 (ACE2) receptor. The spike protein is located on the virus envelope and promotes the attachment to the host cell and the fusion between the virus and the cellular membrane. SARS-CoV-2 is characterised by a single-stranded RNA genome and the expression of four essential structural proteins: a spyke glycoprotein, a small envelope protein, a matrix protein and a nucleocapside protein. The spike (S) glycoprotein is composed of two subunits (S1 and S2). Homotrimers of S are responsible for binding with high affinity the ACE2 human receptor which is expressed on the surface of pulmonary, cardiac, renal, intestinal and endothelial human cells. In particular, nasal epithelia show the highest ACE2 expression among all investigated cells of the respiratory tree. Most patients infected by SARS-Cov-2 exhibit mild/moderate symptoms, but about 15% develop acute pneumonia and about 5% progress to acute respiratory distress syndrome, septic shock and/or multiple organ failure, associated with high mortality. Recent publications have disclosed that Lactoferrin inhibits SARS-Cov-2 infection (Carlos Alberto Marques de Carvalho et al “In Vitro Inhibition of SARS-Cov-2 infection by bovine lactoferrin BioRix preprint May 132020, Chang et al “Lactogerrin as potential preventive and adjunct treatment for COVID-19” International Journal of Antimicrobial Agents August 52020, Kell et al “The biology of Lactoferrin, an iron-binding protein that can help defend against viruses and bacteria” Frontiers in immunology May 282020) and have proposed the use of Lactoferrin for the prevention and/or treatment of SARS- Cov-2 infections and, more in general, for the prevention and/or treatment of bacterial or viral infections. Milanetti et al 2020 (Milanetti et al “In-Silico evidence for two receptors based strategy of SARS-CoV-2” Biorix April 2020) have recently that SARS-Cov-2 infection of human airway epithelial cells involves not only high affinity interactions with ACE2 receptor but also low-affinity interactions with sialic acid containing polysaccharides, using a sialic binding region on the spike protein. SUMMARY OF THE INVENTION The authors of the present invention discovered that unlike SARS-CoV but similar to MERS-CoV and influenza A virus, SARS CoV 2 virions, possesses a sialic acid like binding site and possibly interacts with sialic acid containing oligosaccharides. Thus, impairing binding of oligosaccharides could be an important strategy to contrast SARS CoV 2 virus entry into the target cells. Lactoferrin, i.e. bovine lactoferrin (BLf) is an iron-binding glycoprotein folded in two symmetric globular lobes (N- and C-lobes) with antimicrobial and immunomodulatory activities. BLf is physiologically present in external secretions, especially in milk. BLf has been shown to possess antiviral activity against a broad spectrum of RNA and DNA viruses. These viruses typically utilize common molecules on the cell membrane to facilitate their invasion into cells. In the case of influenza virus, the sialic acid binding protein hemagglutinin, located in the viral envelope (analogous to S1 spike protein in SARS CoV 2), plays a significant role in the early steps of viral infection and represents an attractive target for the development of anti-influenza drugs. It has been demonstrated that lactoferrin interaction with influenza hemagglutinin results in the inhibition of the viral entry process. The authors of the present invention have surprisingly discovered that thermal stability of lactoferrin can be strongly increased when the molecule is combined with at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof thereby increasing the bioavalaibility of said molecule in medical formulations. Furthermore, as sialic acids appear to have a key role in the infectious pathways of several viruses, including Sars-Cov-2, the presence of a sialic acid in the formulation can compete with the sialic acid naturally present in the mucin in the aerial ducts, thereby enhancing the entrapment of the virus entering the aerial ducts exerted by the lactoferrin molecule. The invention therefore provides a new association comprising lactoferrin, at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof that is more stable than lactoferrin alone and that, due to the presence of sialic acid entraps the virus entering the aerial ducts disrupting the ability of the virus to infect the cells. In particular, for viruses with an extremely high infectious efficiency, such as Sars-Cov- 2, the association provided in the present invention, when delivered in nosepharyngeal and/or oropharyngeal trait, is a strong weapon in the treatment or in the prevention, or in adjuvating the treatment or the prevention of viral infections, including Sars-Cov-2 infections by entrapping the virus before its penetration and replication into the cell. Therefore, object of the present invention are An association of Lactoferrin, at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof; A composition comprising said association and one or more pharmaceutically/nutraceutically acceptable vehicles and/or diluents and/or excipients and/or additives; A composition or the association comprising Lactoferrin, at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof as defined in the description and in the claims for use in a medical treatment of a viral disease or as an adjuvant in a medical treatment of a viral disease or in the prevention of a viral infection or in a preventive medical treatment against a viral infection comprising the administration of the composition or the association comprising Lactoferrin, at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof as defined in the description and in the claims to a subject in need thereof; A medical device comprising the composition or the association comprising Lactoferrin, at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof as defined in the description and in the claims suitable for administration thereof in the nosepharyngeal and/or oropharyngeal trait; A process for manufacturing the composition as defined in the description and in the claims wherein an association as defined in the description and in the claims is formulated into suitable dosage units with one or more pharmaceutically/nutraceutically acceptable excipients and/or additives. GLOSSARY DETAILED DESCRIPTION OF THE FIGURES Figure 1 circular dichroism spectra of native and unfolded blf (bovine lactoferrin) holoprotein in PBS buffer is shown as continuous line together with the thermally unfolded protein (95 °C for 24 hrs) in the same buffer as dashed line. The thermally unfolded protein CD spectrum is thus used as a reference for the fully denatured blf. The CD spectra recorded during the stability testing are thus considered as a linear combination of the spectra of folded and unfolded protein. Figure 2: Thermal denaturation curves of bovine lactoferrin in PBS buffer as a function of time. Protein concentration was 10 mg/mL in PBS buffer pH 7.4. Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■) 25 °C; (♦) 35 °C; and (●) 45 °C. Continuous lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Figure 3 Thermal denaturation curves of bovine lactoferrin in PBS buffer containing 1% (w/w) sialic acid as a function of time. Protein concentration was 10 mg/mL in PBS buffer pH 7.4. Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■)25 °C; (♦)35 °C; and (●) 45 °C. Continuos lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Figure 4 Thermal denaturation curves of bovine lactoferrin in PBS buffer containing 1% (w/w) carboxymethylglucan as a function of time. Protein concentration was 10 mg/mL in PBS buffer pH 7.4 and carboxymethylglucan. Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■)25 °C; (♦)35 °C; and (●) 45 °C. Continuos lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Figure 5, Thermal denaturation curves of bovine lactoferrin in PBS buffer containing 1% (w/w) carboxymethylglucan and 1 % sialic acid as a function of time. Protein concentration was 10 mg/mL in PBS buffer pH 7.4 containing sialic acid (1%) and carboxymethylglucan (1%). Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■)25 °C; (♦)35 °C; and (●) 45 °C. Continuous lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Values are summarized in Table 1. Figure 6 Thermal denaturation curves of bovine lactoferrin in PBS buffer containing 5% (w/w) carboxymethylglucan and 1 % sialic acid as a function of time. Protein concentration was 10 mg/mL in PBS buffer pH 7.4 containing sialic acid (1%) and carboxymethylglucan (5%). Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■)25 °C; (♦)35 °C; and (●) 45 °C. Continuos lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Values are summarized in Table 1. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an association of Lactoferrin, at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof. The association can additionally comprise at least one zinc salt. In an embodiment of the invention, the association can comprise or consist of Lactoferrin: negatively charged polysaccharide/s or salts thereof: sialic acid/s or salts thereof in a w/w ratio, respectively, from about 1:1:0.1 to about 1:5:1. In an embodiment, the w/w ratio Lactoferrin: negatively charged polysaccharide/s or salts thereof: sialic acid/s or salts thereof is, respectively, from about 1:2:0.1 to about 1:5:1, from about 1:3:0.1 to about 1:5:1; from about 1:4:0.1 to about 1:5:1 or from about 1:5:0.1 to about 1:5:1. In a further embodiment, the association can comprise consist of Lactoferrin: negatively charged polysaccharide/s or salts thereof: sialic acid/s or salts thereof: zinc salt/s in a w/w ratio, respectively, from about 1:1:0.1:0 to about 1:5:1:0.05. In an embodiment, the w/w ratio Lactoferrin: negatively charged polysaccharide/s or salts thereof: sialic acid/s or salts thereof: zinc salt/s is, respectively, from about 1:2:0.1:0 to about 1:5:1:0.05, from about 1:3:0.1:0 to about 1:5:1:0.05; from about 1:4:0.1:0 to about 1:5:1:0.05 or from about 1:5:0.1:0 to about 1:5:1:0.05. The weight is referred to the dry weight of each compound. By way of example, the association can be an association comprising or consisting of the following amounts of compounds suspended in sterile water or in another suitable solvent: Lactoferrin from 0.01 to 1 % (from 0.1 to 10 mg/mL) Negatively charged polysaccharide/s or salts thereof from 0.01 to 5 % (from 0.1 to 50 mg/mL) Sialic acid/s or salts thereof from 0.001 to 1 % (from 0.01 to 10 mg/mL) Zinc salt/s comprised from 0 to 0.0005 % (from 0 to 0.005 mg/mL) According to the description the salts of each compound are intended as pharmaceutically or nutraceutically acceptable salts. Zinc is known to be an essential element that is crucial for growth, development, and the maintenance of immune function. Its known that zinc status is a critical factor that can influence antiviral immunity, particularly as zinc-deficient populations are often most at risk of acquiring viral infections such as HIV or hepatitis C virus and an abundance of evidence has accumulated over the past 50 y to demonstrate the antiviral activity of zinc against a variety of viruses, and via numerous mechanisms. It has been recently reported that most of the risk groups described for COVID-19 are at the same time groups that were associated with zinc deficiency. As zinc is essential to preserve natural tissue barriers such as the respiratory epithelium, preventing pathogen entry, for a balanced function of the immune system and the redox system, zinc deficiency can probably be added to the factors predisposing individuals to infection and detrimental progression of COVID-19. Therefore, the association of the invention can advantageously further comprise Zinc, in the form of a suitable salt, due to the antiviral properties of this ion. According to the invention, suitable zinc salts can be zinc sulphate, Zn(SO4), zinc acetate, zinc chloride or a mixture thereof. According to the invention, lactoferrin can be bovine lactoferrin or recombinantly produced lactoferrin, such as recombinant human lactoferrin. Recombinant human lactoferrin can be produced in any way known to the skilled person, e.g. in genetically transformed bacterial, yeast, animal or plant cells according to protocols known in the art. In a preferred embodiment, dried, lyophilised or spray dried lactoferrin can be used. Due to its easy availability, bovine lactoferrin is preferably used. More than 50 kinds of sialic acid are known, all of which can be obtained from a molecule of neuraminic acid by substituting its amino group or one of its hydroxil groups. In general, the amino group bears either an acetyl or a glycolyl group, but other modifications have been described. Although any sialic acid is suitable for the present invention, in a preferred embodiment the sialic acid is as defined in formula 1 below,

Wherein R₁ is selected from at least one of H, CH₃, COCH₃ R₂ is selected from at least one of H, OH, CH₃, CH₂OH R₃ is selected from at least one of H, OH, CH₃, CH₂OH and R₄ is selected from at least one of H, OH, CH₃, NH COCH₃ as summarised in table 1 below

Above are indicated specific possible sialic acids according to the invention. According to the invention, suitable polysaccharides are anionic polysaccharides of natural origin, such as polysaccharides extracted from yeasts or from plants or from algae, including polysaccaridic fractions of plant extracts, beta glucanes or salts thereof, carboxy-methyl-glucane or salts thereof, alginates or salts thereof. A typical and non-binding example of salts thereof is represented by Na salts thereof. In a preferred embodiment polysaccharides of the association of the invention will comprise or consist of carboxy-methyl-glucane or pharmaceutically or nutraceutically acceptable salts thereof. Due to the protective effect against viral infection exerted by lactoferrin, the association in any of the embodiments provided in the description and in the claims, can be used in a medical treatment of a viral disease or as an adjuvant in a medical treatment of a viral disease or in the prevention of a viral infection. As indicated in the examples below, following the association with at least one polysaccharide and at least one sialic acid as disclosed in the present description, the stability of lactoferrin is strongly increased, consequently, the association of the invention is of particular interest for medical use as the active molecule is more stable when associated as described herein rather than alone. Therefore, among the advantages of the association according to the present invention are the following ones: 1) the solubilisation of lactoferrin and the stabilization thereof, in particular in aqueous solution; 2) the greater thermal stability of lactoferrin in solution constituted an advantage from the industrial point of view for the production of liquid formulations. In fact, this association allows obtaining a prolonged shelf-life of lactoferrin, since the molecule is not subjected to degradation or at least the degradation takes place with delayed mode; 3) at productive level, the solubilization of lactoferrin allows using a lower quantity of this molecule in the production of liquid formulations of pharmaceutical specialties, medical devices, dietary supplies, cosmetic products, functional pieces of food, veterinary products; 4) the greater stability of the present lactoferrin formulation allows a better bioavailability thereof. So far, higher concentrations of this molecule are reached at the tissue level in presence of at least one polysaccharide and at least one sialic acid with respect to the administration of sole lactoferrin, with consequent increase in efficiency; 5) given that polysaccharides and sialic acids make lactoferrin more bioavailable and consequently more effective, it strengthens even the possible synergic effects between lactoferrin and other molecules. Due to the reported role of lactoferrin and sialic acid in Coronaviruses infections, the association of the invention is of particular interest when used in the treatment or prevention, or in adjuvating the treatment or the prevention of viral infections caused by a Coronavirus, rhinovirus, adenovirus, influenza virus. In a preferred embodiment of the inventions, said Coronavirus is Sars-Cov-2, MERS- Cov. The invention therefore also encompasses a method for the treatment or prevention, or for adjuvating the treatment or the prevention of viral infections caused by a Coronavirus wherein therapeutically effective doses of the association as described hereinabove and in the claims or of the composition as described herein below and in the claims are administered at a therapeutically effective dosage to a subject in need thereof. The administration can be repeated several times per die and the association or the composition can be supplied already in the form of single dosages or with a single- dose dispenser. Therapeutically effective amount means an amount that is effective in therapy, or an amount sufficient to provide a desired therapeutic effect. An amount that is effective in therapy is an amount which produces one or more desired biological activities during the treatment or the prevention of a disease. In the present case during the treatment or the prevention of a viral infectious disease as defined herein. According to the invention, the association of the composition can be vehiculated in the nasopharyngeal and/or in the oropharyngeal trait of the subject treated. A further object of the present invention is a composition comprising the association as defined in the description, and in the claims and one or more pharmaceutically/nutraceutically acceptable vehicles and/or diluents and/or excipients and/or additives. According to one embodiment of the invention, the sole therapeutically active agent in the composition of the invention is the association as defined in the description and in the claims. In an embodiment of the present invention, the composition is the form of a solution, a suspension a spray, a syrup, a tablet, a lozenge, a pill, a candy, a jelly, a granulate (such as an orosoluble granulate), a powder, a cream, an emulsion, a gel, a foam. When the composition is in liquid form, lactoferrin can be suitably dissolved in a liquid formulation comprising the other active components and optionally one or more additional excipient or carrier, immediately before use or after opening the vial containing the liquid formulation comprising the other active ingredients. The composition can also be provided already aliquoted in single dosages or in single dosage fractions, or can be provided in a dispenser apt to release single dosage or single dosages fractions, such as, by way of example, a dispenser apt to release a pre- defined amount of a solution in the form of a spray. Among the vehicles and/or acceptable diluents and/or excipients, according to the administration route and the practice of the pharmaceutical/nutritional standards the following are suitable for carrying out the invention although the person skilled in medical composition preparations will know also other suitable vehicles and/or acceptable diluents and/or excipients: - for the liquid formulations, preferably a vehicle comprising: sucrose or sucrose with glycerin and/or mannitol and/or sorbitol, polysorbate 20, PEG, propylene glycol; - for the formulations under suspension and emulsion preferably a vehicle comprising: a natural rubber, agar, sodium alginate, charragenan, pectine, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. - for the solid formulations preferably a vehicle comprising one or more diluents such as: lactose, dextrose, sucrose, cellulose, corn starch; one or more lubricants such as: silica, stearic acid, magnesium or calcium stearate, polyethylene glycols; one or more binding agents (agglutinants) such as: starches, mucilages, gelatine, methycellulose, carboxymethylcellulose, or polyvinylpyrrolidone; one or more disaggregating agents such as: starch, alginic acid, alginates or glycolates; effervescent mixtures; desiccants; sweeteners; one or more humectant agents such as: lecithin, polysorbate, lauryl sulphate; known substances, inactive and not toxic from the pharmaceutical/nutraceutic point of view. The vehiculants, diluents and excipients mentioned above are meant exclusively by way of example, however not constituting an exhaustive list. Provided herein below are examples of the composition according to the invention. The skilled person, based on the present description, will be able to prepare other compositions by adding to the association of the invention suitable formulative compounds known in the field depending on the desired composition form. COMPOSITION EXAMPLES Composition: Nasal spray: 100 g cont.: COMPOSITION #1 Bovine Lactoferrin, 1 g, Carboxymethylglucan sodium salt, 5 g, Sialic acid, sodium salt, 1 g, polysorbate 80, 0.5 g, propylene glycol, 2 g, Sodium phosphate bibasic, 35 mg, Zinc sulphate, 0,5 mg. Benzalkoniumchlorid 10 mg, Camphor, 0.1 mg, Eucaliptol 0.2 mg and purified water up to 100 g. COMPOSITION #2 Bovine Lactoferrin, 600 mg, Charragenan, 200 mg, Carboxymethylglucan sodium salt, 2,8 g, Sialic acid, sodium salt, 600 mg, potassium sorbate, 0.5 g, propylene glycol, 2 g, , Sodium phosphate bibasic, 35 mg, Zinc sulphate, 0,03 mg. Benzalkoniumchlorid 10 mg, Camphor, 0.1 mg, Xilitol 0.2 mg and purified water up to 100 g. COMPOSITION #3 Bovine Lactoferrin, 100 mg, Carboxymethylglucan sodium salt, 500 mg, Sialic acid, sodium salt, 100 mg, potassium sorbate, 0.5 g, propylene glycol, 2 g, , Sodium phosphate bibasic, 35 mg, Zinc sulphate, 0,005 mg. Chlorexidine gluconate 10 mg, Phyllantus emblica extract, 0.2 mg and purified water up to 100 g According to the invention, for the reasons provided above with reference to the association, the composition according any of the embodiments provided in the description or in the claims is useful in a medical treatment of a viral disease or as and adjuvant in a medical treatment of a viral disease or in the prevention of a viral infection. Accordingly, in a preferred embodiment, said viral disease or said viral infection is caused by a Coronavirus as well as other respiratory tract viruses such as rhinovirus, adenovirus, influenza virus etc.. In a further preferred embodiment said Coronavirus is Sars-Cov-2, MERS-Cov as well as other respiratory tract viruses such as rhinovirus, adenovirus, influenza virus etc. A further object of the invention relates to a medical device comprising an association or a composition as defined in the description and in the claims. According to an embodiment of the invention, said medical device can be an inhaler, a nebuliser or an aerosol delivering device, for nasal and/or buccal drug-delivery. In one embodiment, the device can be apt to admixing a suitable amount of dry lactoferrin with a suitable amount of a liquid formulation comprising or consisting of the remaining compounds of the association as defined in the description and in the claims and one or more pharmaceutically effective carrier. Object of the invention is also a process form manufacturing the composition according to any of the embodiments provided in the present description, comprising the step of formulating the association as defined in any of the embodiments provided in the description and in the claims into suitable dosage units with one or more pharmaceutically/nutraceutically acceptable excipients and/or additives. The examples below provide scientific data demonstrating the increased stability of lactoferrin in the association of the invention ad specific and non-limiting examples of the association and of the composition claimed. EXAMPLES Materials and Methods Bovine apolactoferrin and zinc sulphate were obtained from Merck. Carboxymethyl glucan was obtained from and sialic acid was obtained from Merck. Thermal denaturation experiments were carried out by placing solutions containing 10 mg/mL lactoferrin in sterile, sealed, 1 mL vials and placed in a thermostattable oven at 35 or 45 °C. Circular dichroism spectra were measured on a Jasco 650 spectropolarimeter (Jasco Europe srl) equipped with a peltier thermal control unit. Fluorescence spectra were measured on a Shimazu spectrofluorimeter. CD spectra were collected on the samples daily or weekly and were deconvoluted according to the method of Chang [. Chang, C. T.; Wu, C. S.; Yang, J. T. Anal Biochem 1978, 91, 13–31.] in order to estimate the % of secondary structures present in solution. The 100% native protein was estimated to contain 48% alpha helical content, 28% beta structure and 24% assigned to extended coil. Fully denatured protein was considered 100% extend coil, corresponding to the the protein heated at 95 °C of 1 hour and then centrifuged at 10.000 RPM in a eppendorf min centrifuge. The degradation pattern of the product, assuming that it degrades via a first-order reaction, can be described by a simple exponential decay as follows: Y = exp(-kT) [1] Where Y represents the fraction of native protein with respect to the total protein, k is a first order kinetic constant in days^-1, The observed result (Y) from each lot has has been fitted to the single exponential [1]l by the least squares method using the Matlab program (Mathworks Inc. Natick, Ma, US) RESULTS The blf holoprotein native state has been interpreted as the one having a 48% alpha helical content, 28% beta structure and 24% assigned to extended coil. The amount of secondary structure, as obtained from CD spectra (see Fig.1) is in fair agreement with threedimensional structure data [https://www.rcsb.org/structure/2HCA, Mir, R., Prem Kumar, R., Ethayathulla, A.S., Singh, N., Sinha, M., Kaur, P., Sharma, S., Singh, T.P. 2006]. In Fig.1, the spectrum of native blf protein in PBS buffer is shown together with the thermally unfolded protein (95 °C for 24 hrs) in the same buffer. The thermally unfolded protein CD spectrum is thus used as a reference for the fully denatured blf. The CD spectra recorded during the stability testing are thus considered as a linear combination of the spectra of folded and unfolded protein. The denaturation degree Y has been thus established as the fraction of native protein versus the total protein (folded plus unfolded). Thus, the sterile filtered and sealed blf protein was subjected to a regime of heating as a function of time and CD spectra were recorded at regular intervals. The function Y was thus reported as a function of time as described under the methods section. Fig.2 represent the protein in PBS buffer at 25, 35 and 45 °C. Thermal denaturation curves as a function of time of bovine lactoferrin in PBS buffer containing 1% (w/w) sialic acid or carboxymethyl glucan (1% or 5 %) as a function of time are represented in figure 2-6. The data relative to the kinetics of protein denaturation at different temperatures were thus fitted to a single exponential decay and resumed in TABLE I. Values of the first order kinetic constants indicated that (t_1/2 = ln(2)/k) the shelf life (half time for denaturation) of the lactoferrin formulations varied from a minimum of 69 days in plain buffer at 45 °C to a maximum of 368 days in the presence of Carboxymethyl glucan and sialic acid. It must be mentioned that at a temperature of 45 °C or higher (not reported) yellowish color accompanied by ammonium release is observed in the presence of sialic acid after 4-5 weeks. Control experiments on plain sialic acid solutions demonstrated that the effect is due to the co-presence of sialic acid and lactoferrin pointing to partial degradation of sialic acid at T ≥ 45 °C. Under recommended storage temperature of 25±3 °C, a clear increase in lactoferrin secondary structure stability is observed in the presence of at least 1% CMG or 1% sialic acid. Combined presence of sialic acid and CMG each at 1% concentration offers only a modest increase in stability (Fig.5 and 6). As shown in figure 2, protein concentration was 10 mg/mL in PBS buffer pH 7.4. Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■) 25 °C; (♦) 35 °C; and (●) 45 °C. Continuous lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Values are summarized in Table 1 below. As shown in figure 3, protein concentration was 10 mg/mL in PBS buffer pH 7.4. Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■) 25 °C; (♦) 35 °C; and (●) 45 °C. Continuous lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Values are summarized in Table 1 below. As shown in figure 4, protein concentration was 10 mg/mL in PBS buffer pH 7.4 and 1% carboxymethylglucan. Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■) 25 °C; (♦) 35 °C; and (●) 45 °C. Continuous lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Values are summarized in Table 1. As shown in figure 5, protein concentration was 10 mg/mL in PBS buffer pH 7.4 containing sialic acid (1%) and carboxymethylglucan (1%). Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■) 25 °C; (♦) 35 °C; and (●) 45 °C. Continuous lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Values are summarized in Table 1. As shown in figure 6 protein concentration was 10 mg/mL in PBS buffer pH 7.4 containing sialic acid (1%) and carboxymethylglucan (5%). Kinetic data points have been acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. Temperatures were: (■)25 °C; (♦)35 °C; and (●) 45 °C. Continuous lines have been obtained by least squares fitting of the data to the simple exponential decay described in material section. Values are summarized in Table 1. TABLE 1 Results of data fitting relative to Figs.2-6.

Kinetic constant k can be obtained in days^-1 from the relationship k = ln(2) / t_1/2 As clear from the data obtained, the association of the invention strongly increases the stability of lactoferrin at temperatures from 25 to 45°C. In particular, a 6-8 fold increase in the thermal stability of lactoferrin is observed in the presence of at least 1 % carboxymethyl glucan, with a modest increase in stability up to 5% carboxymethyl glucan. Sialic acid at 1% concentration yields a 2-3 fold increase in stability but its concentration cannot be increased due to ammonium release at higher temperature. Mixing sialic acid with carboxymethylglucane provides t_1/2 values that are comparable, or slightly higher, to those observed in the presence of carboxymethylglucane alone. In conclusion, effects of carboxymethyl glucane and sialic acid on lactoferrin thermal stability are synergic though not additive, and increase of carboxymethylglucane concentration above 1% has only modest effects on lactoferrin overall stability.

Claims

CLAIMS 1. An association comprising Lactoferrin, at least one negatively charged polysaccharide or salt thereof and at least one sialic acid or salt thereof and optionally a zinc salt.

2. The association according to claim 1 comprising Lactoferrin: negatively charged polysaccharide/s or salts thereof: sialic acid/s or salts thereof in a w/w ratio, respectively, from about 1:1:0.1 to about 1:5:1.

3. The association according to claim 1 or 2 comprising Lactoferrin: negatively charged polysaccharide/s or salts thereof: sialic acid/s or salts thereof: zinc salt/s in a w/w ratio, respectively, from about 1:1:0.1:0 to about 1:5:1:0.05.

4. The association according to anyone of claims 1 to 3 comprising Lactoferrin from 0.01 to 1 % Negatively charged polysaccharide/s or salts thereof from 0.05 to 5 % Sialic acid/s or salts thereof from 0.001 to 1 % Zinc salt/s comprised from 0 to 0.0005 % up to 100% of distilled water or any other suitable solvent.

5. The association according to any one of claims 1 to 4wherein said zinc salt is zinc sulphate, zinc acetate, zinc chloride or a mixture thereof

6. The association according to claim 5 wherein said zinc salt is Zn(SO₄).

7. The association according to any one of claims 1 to 6 wherein said lactoferrin is Bovine lactoferrin or human recombinant lactoferrin.

8. The association according to any one of claims 1 to 7 wherein said lactoferrin is spray dried lactoferrin.

9. The association according to any one of claims 1 to 6 wherein said sialic acid is of formula

Wherein R₁ is selected from at least one of H, CH₃, COCH₃ R₂ is selected from at least one of H, OH, CH₃, CH₂OH R₃ is selected from at least one of H, OH, CH₃, CH₂OH and R₄ is selected from at least one of H, OH, CH₃, NH COCH₃.

10. The association according to anyone of claims 1 to 9 wherein said polysaccharide is selected from: anionic polysaccharides of natural origin, polysaccharides extracted from yeasts or from plants, polysaccharides extracted from algae, polysaccaridic fractions of plant, yeast or algae extracts, beta glucanes, carboxy-methyl-glucan, alginates.

11. The association according to anyone of claims 1 to 10 for use in the treatment of a viral infection or as adjuvant in a treatment of a viral infection or in a treatment for the prevention of a viral infection.

12. The association for use according to claim 11 wherein said infection is caused by Coronavirus rhinovirus, adenovirus, influenza virus.

13. The association for use according to claim 12 wherein said Coronavirus is Sars- Cov-2 or MERS-Cov.

14 A composition comprising the association as defined in anyone of claims 1 to 10 and one or more pharmaceutically/nutraceutically acceptable vehicle and/or diluent and/or excipient and/or additive.

15. The composition according to claim 14 wherein said composition is in the form of a solution, a suspension, a spray, a syrup, a tablet, a lozenge, a pill, a candy, a jelly, a granulate, a powder, a cream, an emulsion, a gel, a foam.

16. The composition according to claim 15 wherein said excipient is a mucoadhesive excipient.

17. The composition according to claim 16 wherein said mucoadhesive excipient is one or more of, chitosan, hyaluronic acid, resin, gelatin, polyacrylates, cyclodextrins and silicones.

18. The composition according to anyone of claims 14 to 17 for use in the treatment of a viral infection or as adjuvant in a treatment of a viral infection or in a treatment for the prevention of a viral infection.

19. The association for use according to claim 18 wherein said viral disease or said viral infection is caused by a Coronavirus. rhinovirus, adenovirus, influenza virus.

20. The association for use according to claim 19 wherein said Coronavirus is Sars- Cov-2, MERS-Cov.

21. A medical device comprising an association as defined in anyone of claims 1 to 12 or a composition as defined in anyone of claims 14 to 19.

22. The medical device according to claim 21 wherein said medical device is a device apt to nasal and/or buccal drug-delivery.

23. A process form manufacturing the composition according to anyone of claims 14 to 17 wherein the association as defined in anyone of claims 1 to 10 is formulated into suitable dosage units with one or more pharmaceutically/nutraceutically acceptable vehicle and/or diluent and/or excipient and/or additive.