IT202100002471A1 - ASSOCIATION OR COMPOSITION COMPRISING STABILIZED LACTOFERRIN - Google Patents

Description

COMBINATION OR COMPOSITION INCLUDING LACTOFERRIN

STABILIZED

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 the consequent bioavailability? of the same. The present invention also relates to pharmaceutical compositions comprising said association and the use thereof.

STATE OF THE ART

The recent global pandemic caused by SARS-Cov-2 infection has caused to date more? than 29,000,000 infections and more? of 900,000 deaths.

Not at the moment? no vaccine is available to prevent infection and various treatment protocols are being studied.

SARS-Cov-2 belongs to the Coronaviridae family.

To date, 7 strains of coronavirus are known to infect humans, and there are no approved therapies or vaccines available against them.

In the past two decades, in addition to SARS-CoV-2, two other ?-coronaviruses have caused three of the world's largest epidemics? serious in the world: SARS-CoV and MERS-CoV causing Severe Acute Respiratory Syndrome (SARS), and Middle East Respiratory Syndrome (MERS), respectively.

? well characterized how SARS-CoV infection is mediated by high affinity interactions between the receptor binding domain (RBD) of spike (S) glycoprotein and the human host angiotensin converting enzyme 2 (ACE2) receptor. The spike protein? located on the virus envelope and promotes attachment to the host cell and fusion between the virus and the cell membrane.

SARS-CoV-2 ? characterized by a single-stranded RNA genome and the expression of four essential structural proteins: a spike glycoprotein, a small envelope protein, a matrix protein, and a nucleocapsid protein.

The spike glycoprotein (S) ? composed of two subunits? (S1 and S2). Are homotrimers of S responsible for binding with high affinity? to the human ACE2 receptor that ? expressed on the surface of human lung, heart, kidney, intestinal and endothelial cells. In particular, the nasal epithelia show the most? high expression of ACE2 among all cells of the respiratory tree investigated.

Most patients infected with SARS-Cov-2 show mild/moderate symptoms, but approximately 15% develop acute pneumonia and approximately 5% progress to acute respiratory distress syndrome, septic shock and/or multiple organ failure, associated with high mortality.

Recent publications have described 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 13 May 2020, Chang et al ?Lactogerrin as potential preventive and adjunct treatment for COVID-19? International Journal of Antimicrobial Agents 5 August 2020, Kell et al ?The biology of Lactoferrin, an iron-binding protein that can help defend against viruses and bacteria? Frontiers in immunology 28 May 2020) 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 (Milanetti et al ?In-Silico evidence for two receptors based strategy of SARS-CoV-2? Biorix April 2020) recently found that SARS-Cov-2 infection of human airway epithelial cells implies not only high affinity interactions? with the 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 found that unlike SARS-CoV but similar to MERS-CoV and influenza A virus, the virions of SARS CoV 2 possess a similar sialic acid binding site and possibly interact with oligosaccharides containing acid sialic. Thus, impairment of oligosaccharide binding could be an important strategy to counter SARS CoV 2 virus entry into target cells.

Lactoferrin, or bovine lactoferrin (BLf) ? an iron-binding glycoprotein folded into two symmetrical globular lobes (N- and C-lobes) with activity antimicrobial and immunomodulatory. The BLf? physiologically present in external secretions, especially in milk. ? been shown that the BLf has assets? antiviral against a broad spectrum of RNA and DNA viruses. These viruses typically use common molecules on the cell membrane to facilitate their invasion into cells.

In the case of the influenza virus, the sialic acid binding protein hemagglutinin, located in the viral envelope (analogous to the S1 spike protein in SARS CoV 2), plays a significant role in the early stages of viral infection and represents an attractive target for the development of anti-flu drugs. ? The interaction of lactoferrin with influenza hemagglutinin has been shown to inhibit the viral entry process.

The authors of the present invention have surprisingly discovered that the stability? thermal lactoferrin pu? be strongly increased when the molecule ? combined with at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof, thus increasing? the bioavailability? of said molecule in medical formulations. Furthermore, since sialic acids seem to have a key role in the infectious pathways of various 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 air ducts, thus enhancing the the trapping of the virus entering the air ducts by the lactoferrin molecule.

The invention thus provides a novel combination comprising lactoferrin, at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof which is more stable than lactoferrin alone and which, thanks to the presence of sialic acid, traps the virus that enters the air ducts, stopping the ability? of the virus to infect cells.

In particular, for viruses with an extremely high infectious efficiency, such as Sars-Cov-2, the combination provided in the present invention, when conveyed in the nasopharyngeal and/or oropharyngeal tract, is a powerful weapon in the treatment or prevention, or in aiding the treatment or prevention of viral infections, including Sars-Cov-2 infections by trapping the virus before its penetration and replication in the cell. Therefore, the subject of the present invention are

A combination 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 carriers and/or diluents and/or excipients and/or additives;

A composition or combination comprising lactoferrin, at least one negatively charged polysaccharide or salts thereof and at least one sialic acid or salts thereof as defined in the specification and 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 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 specification and claims to an individual in need; A medical device comprising the composition or 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 nasopharyngeal and/or oropharyngeal tract ;

A method of manufacturing the composition as defined in the specification and claims wherein a combination as defined in the specification and claims is formulated in units? dosage suitable 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 are shown as solid line together with thermally unfolded protein (95°C for 24 h) in the same buffer as dashed line. The CD spectrum of the thermally unfolded protein ? hence used as a reference for the fully denatured blf. The CD spectra recorded during the stability test? they are therefore regarded as a linear combination of the folded and unfolded protein spectra.

Figure 2: Thermal denaturation curves of bovine lactoferrin in PBS buffer as a function of time.

The protein concentration was 10 mg/mL in PBS pH 7.4 buffer. Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?) 25 ?C; (?) 35?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section.

Figure 3 Thermal denaturation curves of bovine lactoferrin in PBS buffer containing 1% (w/w) sialic acid as a function of time.

The protein concentration was 10 mg/mL in PBS pH 7.4 buffer. Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?)25 ?C; (?)35 ?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section.

Figure 4 Thermal denaturation curves of bovine lactoferrin in PBS buffer containing 1% (w/w) carboxymethylglucan as a function of time.

The protein concentration was 10 mg/mL in PBS buffer pH 7.4 and carboxymethylglucan. Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?)25 ?C; (?)35 ?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials 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.

The protein concentration was 10 mg/mL in PBS pH 7.4 buffer containing sialic acid (1%) and carboxymethylglucan (1%). Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?)25 ?C; (?)35 ?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section. The 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.

The protein concentration was 10 mg/mL in PBS pH 7.4 buffer containing sialic acid (1%) and carboxymethylglucan (5%). Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?)25 ?C; (?)35 ?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section. The 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 further comprising at least one zinc salt.

In one embodiment of the invention, the association can understand or consist of

Lactoferrin: Negatively charged polysaccharide(s) or salts thereof: Sialic acid(s) or salts thereof in a w/w ratio, respectively, of about 1:1:0.1 to about 1:5:1.

In one embodiment, the w/w ratio of Lactoferrin:negatively charged polysaccharide(s) or salts thereof:sialic acid(s) or salts thereof is, respectively, about 1:2:0.1 to about 1:5 :1, approximately 1:3:0.1 to approximately 1:5:1; approximately 1:4:0.1 to approximately 1:5:1 or approximately 1:5:0.1 to approximately 1:5:1.

In a further embodiment, the association can understand be made up of

Lactoferrin: Negatively charged polysaccharide(s) or salts thereof: Sialic acid(s) or salts thereof: Zinc salt(s) in a w/w ratio of about 1:1:0, 1:0 to about 1, respectively :5:1:0,05.

In one embodiment, the w/w ratio of the negatively charged Lactoferrin:polysaccharide(s) or salts thereof:sialic acid(s) or salts thereof:zinc salt(s) is, respectively, from about 1:2:0, 1:0 to approximately 1:5:1:0.05, approximately 1:3:0.1:0 to approximately 1:5:1:0.05; approximately 1:4:0.1:0 to approximately 1:5:1:0.05 or approximately 1:5:0.1:0 to approximately 1:5:1:0.05.

Weight ? referred to the dry weight of each compound.

By way of example, the association can? be an?association comprising or constituted by the following quantities? of compounds suspended in sterile water or other suitable solvent:

Lactoferrin 0.01 to 1% (0.1 to 10 mg/mL)

Negatively charged polysaccharide(s) or salts thereof 0.01 to 5 % (0.1 to 50 mg/mL)

Sialic acid(s) or salts thereof 0.001 to 1% (0.01 to 10 mg/mL)

Zinc salt(s) between 0 and 0.0005 % (0 to 0.005 mg/mL)

According to the description the salts of each compound are intended as pharmaceutically or nutraceutically acceptable salts.

? known that the zinc ? an essential element crucial for the growth, development, and maintenance of immune function. ? known that the state of the zinc ? a critical factor that can? affect l? immunity? antiviral, in particular since? populations deficient in zinc are often more? at risk of contracting viral infections such as HIV or hepatitis C virus and in the last 50 years yes? accumulated an abundance of evidence to demonstrate the activity? zinc antiviral against a variety of viruses, and by numerous mechanisms. ? It has recently been reported that most of the described risk groups for COVID-19 are at the same time groups associated with zinc deficiency.

because the zinc ? essential to preserve the natural barriers of tissues such as the respiratory epithelium, which prevent the entry of pathogens, for a balanced function of the immune system and the redox system, zinc deficiency can likely to be added to factors that predispose individuals to infection and harmful progression of COVID-19. Therefore, the association of the invention can? advantageously also understand the Zinc, in the form of a suitable salt, because of the properties? antivirals 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 bacterial, yeast, animal or vegetable cells genetically transformed according to protocols known in the art.

In a preferred embodiment, it can dried, freeze-dried or spray-dried lactoferrin can be used. Given its easy availability, bovine lactoferrin is preferably used.

Are they known more? of 50 types of sialic acid, each of which can? be obtained from a neuraminic acid molecule by replacing its amino group or one of its hydroxyl groups. In general, the amino group bears either an acetyl or a glycolyl group, but other modifications have been described.

While any sialic acid is suitable for the present invention, in a preferred embodiment the sialic acid is as defined in formula 1 below,

In which

R1 ? selected from at least one of H, CH3, COCH3

R2 ? selected from at least one of H, OH, CH3, CH2OH

R3 ? selected from at least one of H, OH, CH3, CH2OH e

R3 ? selected from at least one of H, OH, CH3, NH COCH3

as summarized in Table 1 below

Indicated above are specific sialic acids possible according to the invention.

According to the invention, suitable polysaccharides are anionic polysaccharides of natural origin, such as polysaccharides extracted from yeasts or plants or algae, including polysaccharide fractions of plant extracts, beta-glucans or salts thereof, carboxy-methylglucan or salts thereof, alginates or salts thereof. A typical and non-binding example of salts of the same? represented by Na salts thereof. In a preferred embodiment, the polysaccharides of the combination of the invention will comprise or consist of carboxy-methyl-glucan or pharmaceutically or nutraceutically acceptable salts thereof.

Due to the protective effect against viral infection exerted by lactoferrin, the combination in any of the embodiments provided in the description and the claims, may 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 pi? forward, following the association with at least one polysaccharide and at least one sialic acid as described in the present description, the stability? of lactoferrin? strongly increased, as a consequence, the association of the invention ? of particular interest for medical use since? the active molecule? more stable when combined as described herein rather than alone. Therefore, among the advantages of the association according to the present invention there are the following:

1) the solubilization of lactoferrin and its stabilization, in particular in aqueous solution;

2) the greater stability? of lactoferrin in solution has been an advantage from an industrial point of view for the production of liquid formulations. In fact, this association makes it possible to obtain an extension of the shelf-life of lactoferrin, since it is the molecule not ? subject to degradation or at least the degradation takes place with mode? delayed;

3) at the production level, the solubilization of lactoferrin allows you to use a quantity? inferior of this molecule in the production of liquid formulations of specialties? pharmaceuticals, medical devices, dietary supplements, cosmetic products, functional food pieces, veterinary products;

4) the greater stability? of this formulation of lactoferrin allows a better bioavailability? of the same. So far, concentrations are reached more? higher levels of this molecule at the tissue level in the presence of at least one polysaccharide and at least one sialic acid compared to the administration of lactoferrin alone, with a consequent increase in efficiency;

5) given that polysaccharides and sialic acids make lactoferrin more? bioavailable and consequently more? effective, it also strengthens the possible synergistic effects between lactoferrin and other molecules.

Due to the reported role of lactoferrin and sialic acid in Coronavirus infections, the combination of the invention is of particular interest when used in the treatment or prevention, or in aiding the treatment or prevention of viral infections caused by a Coronavirus, a rhinovirus, an adenovirus, an influenza virus.

In a preferred embodiment of inventions, called Coronavirus ? Sars-Cov-2, MERS-Cov.

The invention therefore also comprises a method for the treatment or prevention, or for aiding the treatment or prevention, of viral infections caused by a Coronavirus in which therapeutically effective doses of the combination as described above in the present and in the claims or of the composition as described in this pi? foregoing and in the claims are administered at a therapeutically effective dosage to an individual in need.

The administration can be repeated several times a day and the association or composition can be provided already? in the form of single doses or with a single dose dispenser.

quantity therapeutically effective means a quantity? That ? effective in therapy, or a quantity? sufficient to provide a desired therapeutic effect. A quantity? That ? effective in therapy? a quantity? which produces one or more activity? biological pathways desired during the treatment or prevention of disease. Herein during the treatment or prevention of a viral infectious disease as defined herein. According to the invention, the association of the composition can be conveyed in the nasopharyngeal and/or oropharyngeal tract of the treated individual.

A further object of the present invention ? a composition comprising the association as defined in the specification and claims and one or more? pharmaceutically/nutraceutically acceptable carriers and/or diluents and/or excipients and/or additives. According to one embodiment of the invention, the only therapeutically active agent in the composition of the invention is ? the association as defined in the description and in the claims. In one embodiment of the present invention, the 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 (such as a buccal granule), a powder, a cream, an emulsion, a gel , a foam.

When the composition? in liquid form, lactoferrin pu? be suitably dissolved in a liquid formulation comprising the other active components and optionally one or more? additional excipients or carriers, 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 doses or in single dose fractions, or pu? be supplied in a dispenser suitable for releasing a single dose or single dose fractions such as, by way of example, a dispenser suitable for releasing a quantity pre-defined solution in the form of a spray.

Of the carriers and/or diluents and/or excipients which are acceptable, depending on the route of administration and practice of pharmaceutical/nutritional standards, the following are suitable for carrying out the invention although the person skilled in the preparation of medical compositions will know also other suitable acceptable vehicles and/or diluents and/or excipients:

- for liquid formulations, preferably a carrier comprising: sucrose or sucrose with glycerin and/or mannitol and/or sorbitol, polysorbate 20, PEG, propylene glycol;

- for suspension and emulsion formulations preferably a carrier comprising: a natural gum, agar, sodium alginate, carrageenan, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.

- for 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 (caking agents) such as: starches, mucilages, gelatin, methylcellulose, carboxymethylcellulose, or polyvinylpyrrolidone; one or more disintegrating agents such as: starch, alginic acid, alginates or glycolates; effervescent blends; desiccants; sweeteners; one or more humectant agents such as: lecithin, polysorbate, lauryl sulfate; known, inactive and non-toxic substances from a pharmaceutical/nutraceutical point of view.

The carriers, diluents and excipients mentioned above are intended as examples only, however they are not an exhaustive list.

Examples of the composition according to the invention are given below.

The expert person, on the basis of this description, will be capable of preparing other compositions by adding to the combination of the invention suitable formulation compounds known in the art depending on the desired composition form. COMPOSITION EXAMPLES

Composition: Nasal spray: 100 g cont.:

COMPOSITION #1

Bovine Lactoferrin, 1g, Carboxymethylglucan Sodium, 5g, Sialic Acid, Sodium, 1g, Polysorbate 80, 0.5g, Propylene Glycol, 2g, Dibasic Sodium Phosphate, 35mg, Zinc Sulfate, 0.5mg . Benzalkonium chloride 10mg, Camphor, 0.1mg, Eucalyptol 0.2mg and purified water up to 100g.

COMPOSITION #2

Bovine Lactoferrin, 600mg, Carrageenan, 200mg, Carboxymethylglucan Sodium, 2.8g, Sialic Acid, Sodium, 600mg, Potassium Sorbate, 0.5g, Propylene Glycol, 2g, Dibasic Sodium Phosphate, 35mg, Zinc sulphate, 0.03 mg. Benzalkonium chloride 10mg, Camphor, 0.1mg, Xylitol 0.2mg and purified water up to 100g.

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, Dibasic sodium phosphate, 35 mg, Zinc sulphate, 0.005 mg. Chlorhexidine gluconate 10mg, Phyllantus emblica extract, 0.2mg and purified water up to 100g

According to the invention, for the reasons given above with respect to the association, the composition according to any of the embodiments provided in the description or in the claims is useful 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.

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 called Coronavirus? Sars-Cov-2, MERS-Cov as well as other respiratory tract viruses such as rhinoviruses, adenoviruses, influenza viruses, etc.

A further object of the invention relates to a medical device comprising an association or composition as defined in the description and in the claims.

According to an embodiment of the invention, said medical device can be an inhaler, nebulizer or aerosol delivery device, for nasal and/or buccal drug delivery.

In one embodiment, the device can be able to mix a quantity? adequate dry lactoferrin with a quantity? suitable preparation of a liquid formulation comprising or consisting of the remaining compounds of the association as defined in the specification and claims and one or more? pharmaceutically effective carriers.

Object of the invention ? Also a method of producing 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 in units? dosage suitable with one or more? pharmaceutically/nutraceutically acceptable excipients and/or additives.

The following examples provide scientific data demonstrating the increase in stability? of lactoferrin in the combination of the invention and specific and non-limiting examples of the claimed combination and composition.

EXAMPLES

Materials and methods

Bovine apolactoferrin and zinc sulfate were obtained from Merck. carboxymethylglucan? was obtained from and sialic acid ? been obtained from Merck. Thermal denaturation experiments were conducted by placing solutions containing 10 mg/mL lactoferrin into sterile, sealed 1 mL vials and placed in a heat-settable oven at 35 or 45°C. The circular dichroism spectra were measured on a Jasco 650 spectropolarimeter (Jasco Europe srl) equipped with a unit? Peltier thermal control. The fluorescence spectra were measured on a Shimazu spectrofluorometer. The CD spectra were acquired on the samples daily or weekly and were deconvolved according to Chang's method [. Chang, C.T.; Wu, C.S.; Yang, J. T. Anal Biochem 1978, 91, 13?31.] to estimate the % of secondary structures present in the solution. The 100% native protein is estimated to contain 48% alpha-helical content, 28% beta-structure, and 24% allotted extended coil. The completely denatured protein ? was considered 100% extended spiral, corresponding to the protein heated at 95 ?C for 1 hour and then centrifuged at 10000 RPM in an eppendorf minicentrifuge.

The degradation pattern of the product, assuming that it degrades with a first-order reaction, can be described by a simple exponential decay as follows: Y = esp(-kT) [1]

Where Y represents the fraction of native protein to total protein, k ? a first order kinetic constant in days<-1>,

The observed result (Y) from each batch ? has been adapted to the single exponential [1] with the least squares method using the Matlab program

RESULTS

The native state of the blf holoprotein ? interpreted as having an alpha-helix content of 48%, beta structure at 28%, and 24% assigned to extended spiral. The quantity? of secondary structure, as obtained from CD spectra (see Figure 1) ? in fair agreement with the three-dimensional structure data [https://www.rcsb.org/structure/2HCA,

In Figure 1, the spectrum of native blf protein in PBS buffer ? shown together with the thermally unfolded protein (95 ?C for 24 h) in the same buffer. The CD spectrum of the thermally unfolded protein is then used as a reference for the fully denatured blf. The CD spectra recorded during the stability test? they are therefore regarded as a linear combination of the folded and unfolded protein spectra. The degree of denaturation Y ? was then determined as the fraction of native protein versus total protein (folded plus unfolded).

Therefore, the sterile filtered and sealed blf protein ? was subjected to a heating regime as a function of time and the CD spectra were recorded at regular intervals. The Y function? was then reported as a function of time as described pi? continue in the methods section. Figure 2 represents the protein in PBS buffer at 25, 35 and 45°C.

Thermal denaturation versus time curves of bovine lactoferrin in PBS buffer containing 1% (w/w) sialic acid or carboxymethylglucan (1% or 5%) versus time are shown in Figures 2-6 .

The data relating to the kinetics of protein denaturation at different temperatures were then fitted to a single exponential decay and summarized in TABLE I. The values of the first order kinetic constants indicated that (t1/2 = ln(2)/k) the shelf life (half-life for denaturation) of lactoferrin formulations ranged from a minimum of 69 days in unspiked buffer at 45°C to a maximum of 368 days in the presence of carboxymethylglucan and sialic acid. It should be noted that at a temperature of 45 ?C or higher (not reported) a yellowish color is observed accompanied by ammonium release in the presence of sialic acid after 4-5 weeks. Control experiments on non-added sialic acid solutions have shown that the effect is ? due to the co-presence of sialic acid and lactoferrin indicating partial degradation of sialic acid at T ? 45?C. At the recommended storage temperature of 25?3?C, a marked increase in stability is observed. of the secondary structure of lactoferrin in the presence of at least 1% CMG or 1% sialic acid. The combined presence of sialic acid and CMG each at a 1% concentration offers only a modest increase in stability. (Fig.5 and 6).

As shown in Figure 2, the protein concentration was 10 mg/mL in PBS buffer pH 7.4. Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?) 25 ?C; (?) 35?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section. The values are summarized in Table 1 below.

As shown in Figure 3, the protein concentration was 10 mg/mL in PBS buffer pH 7.4. Kinetic data points were acquired via UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?) 25 ?C; (?) 35?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section. The values are summarized in Table 1 below.

As shown in Figure 4 , the protein concentration was 10 mg/mL in PBS buffer pH 7.4 and 1% carboxymethylglucan. Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?) 25 ?C; (?) 35?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section. The values are summarized in Table 1.

As shown in Figure 5 , the protein concentration was 10 mg/mL in PBS buffer pH 7.4 containing sialic acid (1%) and carboxymethylglucan (1%). Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?) 25 ?C; (?) 35?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section. The values are summarized in Table 1.

As shown in Figure 6 the protein concentration was 10 mg/mL in PBS buffer pH 7.4 containing sialic acid (1%) and carboxymethylglucan (5%). Kinetic data points were acquired by UV-CD spectroscopy thus considering the progressive loss of secondary structure. The temperatures were: (?)25 ?C; (?)35 ?C; and (?) 45 ?C. The solid lines were obtained by least squares fitting of the data to the simple exponential decay described in the materials section. The values are summarized in Table 1.

TABLE 1

Data fitting results compared to Figs.2-6.

The kinetic constant k can? be obtained in days<-1 > from the relation k = ln(2) / t1/2

As evident from the data obtained, the association of the invention strongly increases the stability? of lactoferrin at temperatures from 25 to 45? C. In particular, is there a 6-8-fold increase in stability? temperature of lactoferrin in the presence of at least l? 1% of carboxymethylglucan, with a modest increase in the stability? up to 5% carboxymethylglucan. Sialic acid at a concentration of 1% produces a 2-3 fold increase in stability? but his concentration can not? be increased due to the release of ammonium at temperatures pi? elevated. Mixing sialic acid with carboxymethylglucan gives t1/2 values that are comparable to, or slightly higher than, those observed in the presence of carboxymethylglucan alone. In conclusion, the effects of carboxymethylglucan and sialic acid on the stability thermal stability of lactoferrin are synergistic although not additive, and increasing the concentration of carboxymethyl glucan above 1% has only modest effects on stability? total lactoferrin.

Claims (23)

CLAIMS A combination 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. 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, of about 1:1:0.1 to about 1:5:1. 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 of about 1:1:0, 1:0 to about 1, respectively :5:1:0,05. 4. Association according to any one of claims from 1 to 3 comprising Lactoferrin from 0.01 to 1% Negatively charged polysaccharide/s or salts thereof 0.05 to 5 % Sialic acid(s) or salts thereof 0,001 to 1 % Zinc salt(s) between 0 and 0.0005% up to 100% distilled water or any other suitable solvent. 5. Association according to any one of claims 1 to 4 wherein said zinc salt is zinc sulphate, zinc acetate, zinc chloride or a mixture thereof. 6. The association of claim 5 wherein said zinc salt is Zn(SO4). 7. Association according to any one of claims 1 to 6 wherein said lactoferrin ? bovine lactoferrin or recombinant human lactoferrin. 8. Association according to any one of claims 1 to 7 wherein said lactoferrin ? spray-dried lactoferrin. 9. Association according to any one of claims 1 to 6 wherein said sialic ? of formula In which R1 ? selected from at least one of H, CH3, COCH3 R2 ? selected from at least one of H, OH, CH3, CH2OH R3 ? selected from at least one of H, OH, CH3, CH2OH e R3 ? selected from at least one of H, OH, CH3, NH COCH3. 10. Association according to any one of claims 1 to 9 wherein said polysaccharide ? chosen from: anionic polysaccharides of natural origin, polysaccharides extracted from yeasts or plants, polysaccharides extracted from algae, polysaccharide fractions of extracts from plants, yeasts or algae, beta glucans, carboxy-methyl-glucan, alginates. The combination according to any one of claims 1 to 10 for use in the treatment of a viral infection or as an adjuvant in a treatment of a viral infection or in a treatment for the prevention of a viral infection. 12. The combination for use according to claim 11 wherein said infection is caused by Coronavirus, rhinovirus, adenovirus, influenza virus. 13. Association for use according to claim 12 wherein said Coronavirus ? Sars-Cov-2 or MERS-Cov. 14. Composition comprising the association as defined in any one of claims 1 to 10 and one or more? pharmaceutically/nutraceutically acceptable carriers and/or diluents and/or excipients and/or additives. 15. A composition according to claim 14 wherein said composition is in the form of a solution, suspension, spray, syrup, tablet, lozenge, pill, candy, jelly, granulate, powder, cream, emulsion, gel, foam. 16. A composition according to claim 15 wherein said excipient is a mucoadhesive excipient. 17. A composition according to claim 16 wherein said mucoadhesive excipient is one or more of, chitosan, hyaluronic acid, resin, gelatin, polyacrylates, cyclodextrins and silicones. The composition according to any one of claims 14 to 17 for use in the treatment of a viral infection or as an adjuvant in a treatment of a viral infection or in a treatment for the prevention of a viral infection. 19. The combination for use according to claim 18 wherein said viral disease or viral infection is caused by a Coronavirus, a rhinovirus, an adenovirus, an influenza virus. 20. Association for use according to claim 19 wherein said Coronavirus ? Sars-Cov-2, MERS-Cov. 21. Medical device comprising a combination as defined in any of claims 1 to 12 or a composition as defined in any of claims 14 to 19. 22. A medical device according to claim 21 wherein said medical device is a device suitable for the nasal and/or buccal delivery of drugs. 23. Process for producing the composition according to any one of claims 14 to 17 wherein the association as defined in any one of claims 1 to 10 is formulated in units? dosage suitable with one or more? pharmaceutically/nutraceutically acceptable carriers and/or diluents and/or excipients and/or additives.