EP3946430A1

EP3946430A1 - Topical oral and/or systemic oral formulations containing a mixture based on lactoferrin, more efficacious in iron uptake, for the prevention and treatment of several pathologies

Info

Publication number: EP3946430A1
Application number: EP20721106.1A
Authority: EP
Inventors: Piera Valenti; Rosalba Paesano
Original assignee: Uragme Srl
Current assignee: Uragme Srl
Priority date: 2019-03-27
Filing date: 2020-03-26
Publication date: 2022-02-09
Also published as: WO2020194225A1; IT201900004599A1

Abstract

Mixture containing lactoferrin, sodium citrate and sodium bicarbonate and whose molar ratio intended as moles of lactoferrin/ (moles of sodium citrate+ moles of sodium bicarbonate) is between 10"4 and 10"3 for use in the treatment of infections colonising the epithelia in the oral mucous membranes for the purpose of: • decreasing the number of pathogenic microorganisms, including those associated with tooth pigmentation such as black stains, • inhibiting the formation of biofilm, • inhibiting the microbial ability to adhere and invade, • helping injury repair, • inhibiting or decreasing the inflammatory process caused by infections optionally associated with iron deficiency, anaemia, inflammation anaemia or diabetes. The mixture is administered only topically in the form of an oral topical formulation in the absence of iron homeostasis disorders or the oral topical formulation is administered in combination with a systemic oral formulation including lactoferrin, sodium citrate and sodium bicarbonate in the same molar ratio range as above, when these infections and the associated inflammatory process in the oral mucous membranes are associated with iron deficiency, anaemia or inflammation anaemia, or when the inflammatory process is associated with diabetes.

Description

TOPICAL ORAL AND/OR SYSTEMIC ORAL FORMULATIONS CONTAINING A MIXTURE BASED ON LACTOFERRIN, MORE EFFICACIOUS IN IRON UPTAKE, FOR THE PREVENTION AND TREATMENT OF SEVERAL PATHOLOGIES

Description

FIELD OF INVENTION

The present invention concerns an innovative composition to significantly increase the efficacy of Lactoferrin of either recombinant or any natural origin, in chelating iron, significantly increasing its antimicrobial, anti-biofilm and, consequently, anti- adhesive, anti-invasive, anti-inflammatory action as well as mucosal lesion repair.

STATE OF THE ART

The human mucous membranes, even if protected by the commensal microbiota, are exposed to the attack of several pathogenic microorganisms. For this reason, in addition to the commensal microbiota which prevents the colonization of pathogens, the mucous membranes are wetted by secretions which, being made up of proteins and peptides of natural immunity, ensure an effective and complete protection against infections by pathogenic microorganisms. Among the natural immunity proteins, lactoferrin is of particular importance, as it is the only one with a strong antimicrobial activity and a remarkable multifunctionality (Rosa et al 2017). Lactoferrin, a highly cationic bilobate glycoprotein (lobe N and lobe C) belonging to the transferrin family with a MW of about 80 kDa, is able to chelate two ferric ions per molecule. This primary function allows it to remove iron from microorganisms that need it for multiplication. Like all iron chelating proteins, lactoferrin captures iron through four canonical amino acids that make up the chelating site. This site is present in each lobe and is constituted in lobe N by Asp58, Tyr93, Tyr193 and His254 and in lobe C by Asp396, Tyr434, Tyr527 and His596. The stability of this bond varies according to temperature, pH and integrity of the glycoprotein (Rosa et al 2018) which can be digested by proteolytic enzymes into fragments without the amino acid sequences essential for capturing the ferric ion. Iron, on the other hand, is so important for almost all living cells that microorganisms, in order to acquire it, have developed multiple transport systems ranging from the synthesis of siderophores, small MW molecules of about 600 kDa able to chelate one ferric ion per molecule (Petrik et al 2017), to that of enzymes with iron-reductase activity that reduce ferric ions to easily acquire ferrous ions and to that of capturing iron directly from lactoferrin (Beddek and Schryvers 2010). The microorganisms, thus, engage in a real battle of chelating agents against lactoferrin, on the result of which the host's infections and diseases depend. Obviously, the result of this battle depends on the different affinity for the ferric ion possessed by the various microbial acquisition systems compared to that of lactoferrin. Although lactoferrin, among the proteins belonging to the transferrin family, is the glycoprotein with the highest affinity for the ferric ion (Kd of about 10-20 M), under particular conditions, iron may be released or no longer captured. Particularly interesting is the fact that, unlike commensal microorganisms, pathogenic microorganisms have, in order to multiply, a strong dependence on the ferric ion. On the other hand, the ability of lactoferrin to capture iron has an inhibiting effect on bacterial multiplication and is therefore classified as a natural protein with antimicrobial activity (Valenti and Antonini 2005). In addition, the antimicrobial action of lactoferrin is particularly important in diseases associated with iron accumulation in tissues and secretions as well as the presence of chromogenic bacteria in the oral cavity, the primary cause of unsightly pigmentation of teeth including black stains. In fact, some chromogenic bacteria synthesize metabolic products such as hydrogen sulphide, which is of considerable interest because, reacting with the iron available in the saliva under pathological conditions (iron homeostasis disorders), it forms black precipitates consisting of ferric sulphide. Moreover, if microorganisms are inhibited in multiplication they do not reach a cell density such as to activate the quorum sensing sequences that lead to the formation and development of biofilm (a combination of bacteria and the exopolysaccharide synthesized by themselves), a particular microbial lifestyle very difficult to eradicate with the antimicrobial drugs known so far. In addition, lactoferrin, being a multifunctional protein, as mentioned above, also carries out simultaneously an activity inhibiting microbial adhesion and internalization in host cells as well as an anti-inflammatory activity. Obviously, microbial growth inhibition leads to biofilm inhibition as well as a significant decrease in microbial adhesion and invasiveness (Rosa et al 2017). Furthermore, if the number of intracellular pathogenic bacteria decreases, the production of pro -inflammatory cytokines by infected epithelial cells and thus the inflammatory process also decreases, although it cannot be excluded that lactoferrin's anti-inflammatory activity depends, for the same number of intracellular bacteria, on its entry into the nucleus where it binds to pro- inflammatory cytokine-encoding sequences (Puddu et al 2011; Paesano et al 2012; Rosa et al 2017). However, it should be stressed that lactoferrin can only perform this function if the cells are infected (Berlutti et al 2006; Frioni et al 2014). Finally, the action of lactoferrin to repair lesions of the oral cavity is closely related to its anti-inflammatory activity as the continuation of inflammation increases the severity and persistence of the lesions.

SUMMARY OF THE INVENTION

The Applicant has now found that it is possible to enhance lactoferrin's iron- chelating activity through the addition of sodium citrate and sodium bicarbonate in a molar ratio expressed as lactoferrin moles / (sodium citrate moles + sodium bicarbonate moles) between 10^-4 and 10^-3.

In fact, this combination administered topically and/or orally systemically is capable of performing a more powerful antimicrobial activity, by removing iron more effectively and by chelating it more permanently than lactoferrin alone or in the absence of sodium citrate and sodium bicarbonate.

The object of this invention is therefore a mixture containing lactoferrin, sodium citrate, sodium bicarbonate in the above mentioned range of molar ratio to be used in the treatment and prevention of infections colonizing epithelia at the level of the oral mucous membranes in order to:

• decrease the number of pathogenic microorganisms including those associated with tooth pigmentations such as the so called black stains,

• inhibit the formation of biofilm, the microbial ability to adhere and invade,

• promote the repair of injuries,

• inhibit or decrease the inflammatory process caused by infections optionally associated with iron deficiency, anaemia or inflammation anaemia or diabetes.

The mixture is administered only topically in the form of a topical oral formulation in the absence of iron homeostasis disorders, or the topical oral formulation is administered in combination with a systemic oral formulation comprising lactoferrin, sodium citrate and sodium bicarbonate where the lactoferrin, sodium citrate and sodium bicarbonate are present in the same molar ratio range as above, when said oral mucosal infections and their inflammatory process is associated with iron deficiency, anaemia or inflammatory anaemia or when the inflammatory process of said oral mucous membranes is associated with diabetes.

DESCRIPTION OF FIGURES

Figure 1 represents the ferric ion uptake site of the lactoferrin containing the citrate in the N-lobe.

Figure 2 shows the ferric ion uptake site containing the bicarbonate ion in the N-lobe. Also in lobe C, the ferric ion uptake site can be made more efficient in metal uptake by the presence of citrate and bicarbonate in the same way as in lobe N.

Legenda of the figures:

His=histidine Tyr=tyrosine Asp=asparagine Arg=arginine

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this invention, lactoferrin means lactoferrin of any origin (human, bovine, equine, murine, caprine, camel, buffalo and donkey) and recombinant lactoferrin.

The improvement of the iron-chelating activity seems to take place according to the following reaction pattern: [1]

2 sodium citrate + Fe³⁺ ferric dicitrate (monocitrate-Fe³⁺ -sodium monocitrate) ferric dicitrate + sodium bicarbonate - 1 bicarbonate Fe³⁺ + 2 sodium monocitrate bicarbonate Fe³⁺ + lactoferrine lactoferrine-Fe³⁺- sodium bicarbonate

[1]

This reaction, which highlights the ability of sodium bicarbonate to displace the citrate in the ferric ion uptake, however, does not exclude the possibility that ferric monocitrate may form a complex with lactoferrin and then be displaced by the bicarbonate that stabilizes the lactoferrin-Fe³⁺ bond according to the following reaction scheme [2] :

2 sodium citrate + Fe³⁺ ferric dicitrate (monocitrate-F^’e³⁺ - sodium monocitrate)

ferric monocitrate + lactoferrine lactoferrine-Fe³⁺- sodium citrate lactoferrine-Fe³⁺ -citrate + sodium bicarbonate lactoferrine-Fe³⁺- bicarbonate + sodium monocitrate [2]

In other words, sodium citrate is used to solubilize and chelate the ferric ion through the formation of ferric dicitrate which, in turn, exchanges iron with bicarbonate according to the following reaction: [3]

ferric dicitrate+ sodium bicarbonate bicarbonate Fe³⁺ + 2 sodium monocitrate

[3]

or forms with lactoferrin a complex (lactoferrin-Fe³⁺-citrate) from which it is then displaced by bicarbonate (lactoferrin-Fe³⁺-bicarbonate) returning to sodium monocitrate, which, in turn, reactivates the uptake cycle of the available ferric ions. Ferric citrate and ferric bicarbonate are ideal compounds to transport the ferric ion to the lactoferrin uptake sites, according to the scheme shown in Figures 1 and 2, respectively.

Lactoferrin, highly conserved in the various species, is a glycoprotein comprising 685 to 691 amino acids depending on the species, with an isoelectric point of about 9 and a molecular weight of about 80 kDa with 3 or 5 glycosylation sites (human or bovine, respectively). The bond affinity with iron is about 10^-20 M, which increases significantly in the mixture described above and is greater than the transferrines that are responsible for the transport of iron and therefore release it more easily (Rosa et al 2017). Native lactoferrin is produced in large quantities by extraction from bovine milk, while recombinant human or bovine lactoferrin is produced by recombinant DNA. Bovine lactoferrin is preferably used.

The term 'mixture' refers to a composition having as active ingredients lactoferrin, preferably of bovine origin, associated with sodium citrate and sodium bicarbonate in which the molar ratio related to moles of lactoferrin /(moles of sodium citrate + moles of sodium bicarbonate) is between 10^-4 and 10^-3.

The mixture may be used in liquid, semi-solid, solid or granular form with sodium citrate and sodium bicarbonate and with the addition of excipients or other biologically acceptable compounds.

The mixture thus composed of lactoferrin, citrate and sodium bicarbonate chelates the ferric ion more effectively, as shown in Table 1, and is applied in the prevention and treatment of infections and lesions of the oral mucous membranes. According to the invention the molar ratio related to moles of lactoferrin/(moles of sodium bicarbonate + moles of sodium citrate) in the mixture is preferably comprised between 3 × 10^-4 and 7 ×10^-4).

In the mucous membranes of the oral cavity the mixture is administered topically in the form of oral topical preparations when the microbial infection is associated with inflammation of the oral mucous membranes and in particular with halitosis, gingivitis, periodontitis, tooth pigmentations including black stains and oral mucosal lesions (Calvani et al 2018) and is not associated with iron deficiency or iron deficiency anaemia or inflammation anaemia. This topical application is instead combined with systemic oral administration, when inflammation of the oral mucous membranes caused by microbial infections, such as halitosis, gingivitis, periodontitis, tooth pigmentations including black stains is associated with iron deficiency, iron deficiency anaemia, inflammation anaemia or diabetes.

For this reason, the combined use of the topical oral formulation of the mixture with that of the systemic oral formulation represents the best treatment not only of microbial infections, but also of iron deficiency, iron deficiency anaemia and inflammation anaemia possibly associated with pathological inflammation or diabetes.

The mixture comprising lactoferrin of any natural or recombinant origin, sodium citrate and sodium bicarbonate in the above mentioned molar ratio ranges, administered for topical and/or oral use, carries out simultaneously:

i) antimicrobial activity also against chromogenic bacteria as well as iron- dependent and protective antiviral activity against infections

ii) iron-dependent anti-biofilm activity

iii) activity inhibiting the production of iron-dependent reactive oxygen species

iv) activity inhibiting inflammatory processes induced by iron-dependent reactive oxygen species

v) anti-adhesive activity dependent on iron-dependent microbial multiplication inhibition

vi) anti-invasive activity dependent on the inhibition of iron-dependent microbial multiplication vii) anti-inflammatory activity against the expression and synthesis of pro- inflammatory cytokines such as IL-1b, IL-6, IL-8, TNF-a and NF-kB dependent on the significant decrease in the number of iron-dependent intracellular microorganisms

viii) anti-inflammatory activity dependent on intracellular microbial killer ix) activity modulating the expression of epcidine and ferroportin

x) activity inhibiting iron homeostasis disorders (iron excess in tissues and secretions and iron deficiency in the circulation)

xi) activity inhibiting the increased susceptibility of the host to infections induced by iron accumulation in tissues and secretions

xii) prevention and treatment of iron deficiency, iron deficiency anaemia and inflammation anaemia

xiii) repair of mucosal lesions linked or not linked to the perduring of iron- dependent or independent inflammation

xiv) prevention and treatment of diabetes dependent on the anti-inflammatory activity of lactoferrin.

Moreover, since lactoferrin present in the mixture, thanks to sodium citrate and sodium bicarbonate, chelates iron more effectively, in addition to the numerous activities described above, the higher concentration of the form of iron-saturated lactoferrin makes it more resistant to heat and digestion of proteolytic enzymes present in all human secretions including saliva. Furthermore, even if the proteolytic enzymes digest lactoferrin at least partially, enzymatic digestion would separately produce the two lobes (lobe N 1-280 and lobe C 345-692) which, possessing an iron-binding site per lobe, would perform the same functions as intact lactoferrin provided that the two lobes are added together with sodium citrate and sodium bicarbonate. The two lobes as well as the intact protein can chelate not only iron but also copper, zinc, cobalt, nickel and manganese which are also necessary for bacterial development and therefore involved in the antimicrobial activity of lactoferrin which, even in the uptake of the ions mentioned above, is enhanced by sodium citrate and sodium bicarbonate.

Preferred forms for topical administration in the oral cavity comprise freeze-dried anhydrous powders of lactoferrin, sodium citrate and sodium bicarbonate for direct use (e.g. freeze-dried sprinkled powders, orosoluble tablets, granules and capsules), solutions ready to use or to be dissolved in water at the time of use (e.g. rinse or wash solutions), gels, toothpastes, toothpastes with added hydroxyapatite, chewing gum, tablets to be dissolved in the mouth, spray. The preferred topical formulation contains lactoferrin in orosoluble freeze-dried form which is administered directly to the oral mucosa along with sodium citrate and sodium bicarbonate in the molar ratio between 10^-4 and 10^-3.

In a second aspect, the invention concerns a combined preparation for simultaneous, sequential or separate administration, which includes a first topically administered oral formulation, which contains the mixture with bovine lactoferrin, sodium citrate and sodium bicarbonate in the above molar ratios and a second systemically administered oral preparation, which contains bovine lactoferrin, sodium citrate and sodium bicarbonate in the above molar ratios. Benefits obtained from the preparation according to the invention include the advantage of simultaneously resolving states of infection and inflammation that occur together with iron deficiency, anaemia and inflammation anaemia or diabetes. Currently, known products and compositions not formulated as combined preparations for topical and oral use are not capable of achieving such an effect simultaneously.

The two compositions can be defined by means of the same required and optional characteristics indicated with regard to the forms for topical and/or oral administration described above in the context of mixture use.

In order to achieve an appreciable effect on both acute or chronic infection and destructive pathological inflammation, wound repair, iron deficiency, anaemia and inflammation anaemia or diabetes, it is highly advantageous to follow the dosage regimen described below.

If the first (topical) composition is in a solid single-dose form (e.g. soluble tablets or granules in the oral cavity), the mixture to be administered two or more times a day until the symptoms disappear contains a total amount of bovine lactoferrin preferably ranging from 50 to 400 mg plus sodium citrate and sodium bicarbonate in a mole range preferably between 0,01 and 1. Although repeated administration several times a day would be the best and most effective option, you may choose to administer the mixture at a higher concentration once a day only.

For topical administration in the oral cavity, lozenges, capsules and tablets and orosoluble granules containing 50 mg to 200 mg of lactoferrin, each with added sodium citrate and sodium bicarbonate, may be used in a range of moles between

0.01 and 1 to be administered two or several times a day until the symptoms disappear.

If the first composition is in liquid or semi-solid multi-dose form (e.g. rinse solution), the mixture contains bovine lactoferrin in the range of 0.1-10% w/w and sodium citrate with sodium bicarbonate in the 0.01-1 mole range. It is advantageous to remain in the range of 50-400 mg of bovine lactoferrin per day until the symptoms disappear as indicated above for solid compositions.

If the second (systemic oral) composition is in solid and single dose form (e.g. tablets, capsules or granules), preferably the mixture contains a total amount of bovine lactoferrin from 50 mg to 200 mg per tablet or capsule or granulate with sodium citrate and sodium bicarbonate in the 0,01-1 mole range to be administered two or several times a day until the symptoms disappear or more preferably for a total of 100 to 200 mg lactoferrin two or several times a day, at least for one month.

Both topical and oral administration should be performed away from meals.

By way of example, the in vitro efficacy of the mixture in the treatment of halitosis, gingivitis, periodontitis, viral gingivo-stomatitis, tooth pigmentations including black stains is subsequently described using cultures of gingival fibroblasts infected with supragingival or subgingival plaque, aerobic or anaerobic Gram-negative bacteria or demonstrated in teeth extracted from patients with black stains.

The administration of the mixture reduces more effectively than lactoferrin alone the high expression and synthesis of pro-inflammatory cytokines produced by infected epithelial cells and inhibits the production of superoxides, which, in turn, carry out a pro-inflammatory action.

Similarly, the anti-inflammatory activity of the mixture administered topically and/or orally systemically can be used to prevent and treat insulin-resistant diabetes associated with inflammation (Mohamed and Schaalan 2018). As regards in vivo tests, the effectiveness of the mixture administered topically and orally systemically in the event of iron deficiency, anaemia and inflammation anaemia in the treatment of halitosis, gingivitis, periodontal disease, viral gingivitis, pigmentations including black stains and lesions is assessed not only by local objective examination but also by clinical parameters, such as the concentration of pro-inflammatory cytokines and epcidine in blood or urine, and haematological parameters, with particular attention to haemoglobin values, red blood cell count, total serum iron and serum ferritin before and after treatment. The greater inflammation reduction in vivo due to the action of the mixture compared to lactoferrin alone allows this new formulation to modulate iron homeostasis by inhibiting epcidine synthesis and restoring ferroportin synthesis, preventing the formation of superoxides in the tissues, while inhibiting microbial replication and inducing the restoration of haematological values. The new formulation, administered both topically and orally systemically, also modulates resistance to insulin which gives rise to type 2 diabetes.

Furthermore, again as an example, the effectiveness of the mixture in the treatment of fungal infections by Candida Albicans is demonstrated using in vitro cultures of fibroblasts as a model for oral fungal infections. In this type of infection the addition of the mixture powerfully inhibits the expression and synthesis of interleukin-6 by cultured cells. Anti-inflammatory activity of the mixture in vivo allows restoration of iron homeostasis. In these fungal infections, in case of severe iron deficiency, anaemia or inflammation anaemia, topical administration must be combined with systemic oral administration in the treatment of gingival mucosal infections caused by fungi while the effectiveness of the mixture is assessed not only by objective examination but also by clinical parameters, such as the concentration of pro -inflammatory cytokines, epcidine in blood and urine as well as blood parameters.

Using the mixture in the treatment of infectious and inflammatory diseases in human oral mucous membranes and its ability to increase haematological parameters are just some of the possible therapeutic applications of the activity of simultaneously inhibiting the expression and synthesis of pro -inflammatory cytokines and the production of superoxides. The examples shown here show that the therapeutic activity of the mixture in infections and inflammatory processes affecting the mucous membranes can be used against infectious and inflammatory diseases with particular attention to human mucous membranes, such as oral mucous membranes, with particular reference to selected disorders including halitosis, gingivitis, periodontitis, tooth pigmentation, lesions, bacterial, fungal and viral oral infections, iron deficiency, anaemia and inflammation anaemia and other acute and relapsing disorders. Treatment with the mixture can be considered extremely advantageous compared to treatments with lactoferrin alone or with other drugs currently in use, being also characterized by the absolute lack of toxicity and the fact that it can treat several diseases simultaneously ranging from infections to pathological inflammation, iron homeostasis disorders and diabetes.

In particular, the mixture, by inhibiting the interleukin-6 synthesis, consequently prevents the synthesis of epcidine, modulated by it, restoring the action of ferroportin, which is otherwise inhibited by epcidine. In any case, a direct action of lactoferrin, mainly bovine lactoferrin, cannot be excluded in the up-regulation of ferroportin.

Finally, in the event of periodontal disease treatment, the mixture replaces an expensive and invasive therapy such as surgical removal of subgingival plaque, which only resolves the problem for short periods, after which repeated infectious and inflammatory recurrences appear until the loss of the tooth attachment and the partial loss of bone. In addition, in the treatment of mucosal lesions, the mixture heals wounds in an extremely short time compared to classical therapies or lactoferrin alone. Even in the treatment of mandibular or maxillary bone degradation, the mixture appears more effective than lactoferrin alone by significantly increasing osteoblasts and decreasing osteoclasts.

Other features and advantages of the present invention will be best illustrated by the description of the following preferred forms of realization, including but not limited to the following.

Following is the procedure demonstrating the higher effectiveness of the mixture in capturing ferric ions compared to bovine lactoferrin in the absence of sodium citrate and sodium bicarbonate. The in vitro ability of lactoferrin from any source to chelate iron is demonstrated by a series of optical density (OD) readings at 468 nm based on an extinction coefficient of 0.54 for a 1% solution of 100% iron-saturated lactoferrin, referred to as holo-lactoferrin.

Specifically, 10 mg of bovine lactoferrin (bLf) are dissolved in 10 ml of a physiological solution and the first reading is taken at 468 nm. The value obtained indicates the amount of iron contained in the commercial preparation of lactoferrin or its iron saturation level by the following proportion:

holo-lactoferrin: 0,54 coefficient of holo-lactoferrin extinction = X (sample saturation level: sample OD at 468 nm

Using the same procedure, 10 mg of the same commercial lactoferrin sample are dissolved in 10 ml of a 0,2M sodium citrate solution and the first reading is performed at 468 nm. The value obtained indicates both the quantity of iron contained in the commercial preparation of lactoferrin or its iron saturation level through the previous ratio and any interference by sodium citrate checked against the OD value recorded after dissolution of lactoferrin in saline solution.

Using the same procedure 10 mg of the same commercial lactoferrin sample are dissolved in 10 ml of a 0,1 M sodium bicarbonate solution and the first reading is performed at 468 nm. The value obtained indicates both the quantity of iron contained in the commercial preparation of lactoferrin or its iron-saturation level through the previous ratio and the possible interference by sodium bicarbonate checked against the OD value recorded after dissolution of lactoferrin in saline solution.

Using the same procedure 10 mg of the same commercial lactoferrin sample are dissolved in 10 ml of a solution containing 0,2M sodium citrate and 0,1M sodium bicarbonate and the first reading is performed at 468 nm. The value obtained indicates both the quantity of iron contained in the commercial preparation of lactoferrin or its iron-saturation level by the previous ratio and the possible interference of sodium bicarbonate and sodium citrate checked against the OD value recorded after dissolution of lactoferrin in saline solution. After the first reading of each of the four samples, 2,5 ml of ferric citrate obtained by mixing 0,1M ferric chloride with 0,2M sodium citrate are added to each sample and mixed before reading at 468 nm. This procedure is repeated until a sample reaches the complete iron saturation indicated by the OD value of 0,540 at 468 nm. Once this value has been reached, a further 25 ml of the same ferric citrate solution is added to the above solution as a single addition in order to check that balance has been reached.

The results obtained are shown in Table 1. You can see that, regardless of the mixture components, the values of the first reading are similar and indicate an iron saturation of commercial lactoferrin of 9.4%. On the other hand, the values obtained after the addition of 2.5 ml ferric citrate are different from those obtained when lactoferrin is added to sodium citrate (0.2M) and sodium bicarbonate (0.1M), indicating the greater effectiveness of the mixture in chelating iron. Lactoferrin dissolved in saline or sodium citrate only or sodium bicarbonate only has a low effectiveness in chelating iron and similarly under all three described conditions. Table 1. Ability to chelate the ferric ion by a commercial preparation of bovine lactoferrin (bLf) saturated at 9.44% (as shown by first reading) in different solutions.

Therefore, as is very evident from Table 1, bovine lactoferrin is able to chelate the ferric ion until it saturates the uptake sites to 100% only in the presence of sodium bicarbonate and citrate, while in saline or in the presence of sodium bicarbonate or citrate only lactoferrin is never 100% but at most 29% saturated indicating the poor ability to chelate iron in the above conditions.

The sodium bicarbonate and citrate solution enhances the ability to chelate iron also by human, bovine, equine, murine, murine, goat, camel, buffalo and donkey lactoferrin and recombinant human and bovine lactoferrin.

The solutions of 0.2 M concentration for sodium citrate and 0.1 M concentration for sodium bicarbonate are optimal although, despite different effectiveness, sodium citrate and sodium bicarbonate can be added in a range of moles between 0.01 and 1 including all possible combinations. All concentrations of sodium citrate and sodium bicarbonate above are not cytotoxic and do not affect antimicrobial activity in the absence of lactoferrin.

After this unequivocal demonstration of the increased capacity of lactoferrin, when combined with sodium citrate and sodium bicarbonate at the above mentioned concentrations, to chelate the iron which is at the basis of the multiple functions of lactoferrin, this mixture's effectiveness was compared with bovine lactoferrin alone in infections and inflammations of the oral mucous membranes and in particular against halitosis, gingivitis, periodontitis, black stains, oral mucosal lesions including post-surgical lesions, diabetes, iron deficiency, anaemia and inflammation anaemia and other acute and relapsing disorders.

QUANTITY BY WEIGHT OF THE DIFFERENT MOLES OF BOTH SODIUM CITRATE AND SODIUM BICARBONATE

The different quantities by weight of both sodium citrate and sodium bicarbonate referred to the different moles, shown in the text, have been calculated according to the following formula:

where X indicates the grams required for the citrate or bicarbonate and the Molecular Weight refers to the molecular weight of sodium citrate dihydrate (294,1) or sodium bicarbonate (84,0).

Table 2 shows the different mg required for the lactoferrin mixture containing different moles of sodium citrate dihydrate and sodium bicarbonate.

Example 1. Mixture is efficacious against halitosis

A previous trial carried out on adult subjects treated twice a day after oral hygiene with orosoluble tablets containing 50 mg of bovine lactoferrin showed good resolution of halitosis after 10 days of treatment.

The new trial carried out on adult subjects treated twice a day after oral hygiene with orosoluble tablets containing 50 mg of bovine lactoferrin combined with 1 mole sodium citrate and 1 mole sodium bicarbonate showed a complete resolution of halitosis already after 5 days of treatment, clearly demonstrating the increased effectiveness of the mixture.

Example 2. Mixture is efficacious against bacterial and fungal gingivitis

In vivo experiments were performed by taking 10 ml of saliva first (time 0) and then after 7 days of treatment (twice a day after thorough oral hygiene) with 50 mg of bovine lactoferrin or with 50 mg of bovine lactoferrin with added 1 mole citrate and 1 mole sodium bicarbonate in patients with gingivitis. After one week of treatment, the decrease of bacteria in the saliva was greater in patients treated with the mixture than in those treated with bovine lactoferrin (10⁴ compared to 10⁷/ml of saliva). Similarly, after one week of treatment, the decrease of saliva fungi was greater in patients treated with the mixture than in those treated with bovine lactoferrin (10² compared to 10⁴/ml of saliva). In addition, the gums appeared less inflamed and the bleeding was no longer evident, especially in patients treated with the mixture. In case of iron deficiency, anaemia or inflammation anaemia, the topical therapy with the mixture was also combined with systemic oral therapy always with the mixture. The blood values increased back to normal after 15 days of treatment and the IL-6 serum concentration decreased back to normal (from 10 to 2pg/ml). Oral administration of lactoferrin alone (50 mg) required 30 days of therapy to reach the values obtained from the mixture.

Example 3. Mixture is efficacious against periodontal disease

It is known that pro-inflammatory cytokines that significantly increase in periodontal disease can be tested in the crevicular fluid of patients with periodontal disease. Treatment for one month with only 50 mg of bovine lactoferrin contained in orosoluble tablets to be consumed twice a day after thorough oral hygiene significantly decreased the concentration of pro -inflammatory cytokines as well as the depth of pockets, gingival index and bleeding. The treatment with orosoluble tablets with the mixture containing 50 mg of bovine lactoferrin combined with 1 mole sodium citrate and 1 mole sodium bicarbonate to be consumed twice a day after thorough oral hygiene, already after 7 days, induced a significant decrease in IL-6, pocket depth, gingival index, bleeding and surprisingly plaque index (0.1 compared to 0.5 of lactoferrin alone). In addition, superoxide production was greatly inhibited by the mixture.

In case of iron deficiency, anaemia or inflammation anaemia, systemic oral therapy was also associated with the topical therapy with the mixture. The blood values increased and returned to normal after 15 days of treatment and the IL-6 serum concentration decreased and returned to normal (from 10 to 2pg/ml). Oral administration of lactoferrin alone (50 mg) required 30 days of therapy to reach the values obtained from the mixture.

Example 4. Mixture is efficacious against black stains

Among the unaesthetic pigmentations on the tooth surface, particularly important, in black stains, is the accumulation of iron in the tissues and secretions which, together with chromogenic bacteria, is the primary cause of this aesthetic problem. In fact, hydrogen sulphide, synthesized by some bacteria in the oral cavity, reacts with the iron available in the saliva under pathological conditions (iron homeostasis disorders) forming black precipitates hence the name black stains.

The treatment with 50 mg of lactoferrin twice a day for 6 months eliminates this aesthetic problem, while the treatment, twice a day for 30-40 days, with the mixture containing 50 mg of bovine lactoferrin combined with 1 mole sodium citrate and 1 mole sodium bicarbonate eliminates black stains already after 30-40 days of treatment. Obviously, this is a further demonstration that the mixture chelates the ferric ion more effectively and consequently eliminates the substrate for the formation of black stains in addition to the fact that it has a greater antibacterial power than lactoferrin alone. No relapses have been observed with lactoferrin alone or with the mixture. In case of iron deficiency, anaemia or inflammation anaemia, the topical therapy with the mixture is also combined with oral therapy with the mixture. Example 5. Mixture is efficacious against mucosal lesions

Oral cavity lesions, whether due to orthodontic braces or surgical treatments including those performed to remove osteonecrosis, are characterized by prolonged inflammatory phases that lead to wounds not healing and also increase the risk of infection. Lactoferrin, as already mentioned, is a multifunctional glycoprotein capable of both antimicrobial and anti-inflammatory activity.

In this type of experiment, bovine lactoferrin or a mixture containing 50 mg of bovine lactoferrin combined with 1 M sodium citrate and 1 M sodium bicarbonate are used to soak sterile gauze pads to be applied to the lesion.

In particular, 100 mg of sterile bovine lactoferrin are dissolved in 2 ml of sterile saline and absorbed on a sterile gauze pad measuring 50 mm × 50 mm. The application shall be carried out in accordance with the size of the lesion. On the other hand, the mixture (100 mg of bovine lactoferrin dissolved in 2 ml containing a solution of 1M sodium citrate and 1M sodium bicarbonate) applied to the lesion leads to healing of the mildest lesions within 24-48h compared to 7 days of treatment with lactoferrin alone while the most severe lesions within 7-10 days compared to one month with lactoferrin dissolved in saline solution. In case of iron deficiency, anaemia or inflammation anaemia, the topical therapy with the mixture is also combined with oral therapy always with the mixture in orosoluble tablets or granules. The oral administration of lactoferrin alone requires 30 days of therapy to reach the values obtained from the mixture in 15 days. Example 6. Mixture is efficacious in the treatment of iron deficiency and anaemia

In the treatment of iron deficiency or anaemia, subjects must take capsules containing 100 mg of lactoferrin two or three times a day away from meals and for at least one month or until the disease is resolved.

Table 3 compares the blood parameters (mean values) obtained in 87 patients enrolled with iron deficiency or anaemia, of whom the study ended with 41 patients treated with capsules containing 100 mg of lactoferrin and 41 patients treated with capsules containing 100 mg of lactoferrin combined with sodium citrate (1 mole) or sodium bicarbonate (1 mole) twice a day away from meals for 30 days.

Table 3. Observational study on 82 patients suffering from iron deficiency and anaemia, of whom 41 treated twice daily for one month with capsules containing 100 mg of lactoferrin and 41 treated twice daily with capsules containing 100 mg of lactoferrin combined with sodium citrate (1 mole) or sodium bicarbonate (1 mole)

Example 7. Mixture is efficacious in the treatment of inflammation anaemia

In the treatment of inflammation anaemia, subjects must take capsules containing 100 mg of lactoferrin three or four times a day away from meals and for at least one month or until the disease is resolved.

Table 4 compares the blood parameters (mean values), the IL-6 serum concentration of 45 patients enrolled with inflammation anaemia of whom the study was completed with 20 patients treated with capsules containing 100 mg of lactoferrin and 23 patients treated with capsules containing 100 mg of lactoferrin combined with sodium citrate (1 mole) or sodium bicarbonate (1mole) three times a day away from meals for 30 days.

Table 4: Blood parameters in patients with inflammation anaemia treated with lactoferrin or the mixture

Bibliography

Berlutti F, Schippa S, Morea C, Sarli S, Perfetto B, Donnarumma G, Valenti P. Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive Escherichia coli strains. Biochem Cell Biol. 2006 Jun;84(3):351-7

Beddek, A.J.; Schryvers, A.B. The lactoferrin receptor complex in Gram negative bacteria. Biometals 2010, 23,377-386.

Calvani F, Cutone A, Lepanto MS, Rosa L, Valentini V, Valenti P. Efficacy of bovine lactoferrin in the post-surgical treatment of patients suffering from bisphosphonate-related osteonecrosis of the laws: an open-label study.Biometals. 2018 Jun;31(3):445-455. Frioni, A.; Conte, M.P.; Cutone, A.; Longhi, C.; Musci, G.; di Patti, M.C.; Natalizi, T.; Marazzato, M.; Lepanto, M.S.; Puddu, P.; et al. Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases. Biometals 2014, 27, 843-856.

Mohamed WA, Schaalan MF. Antidiabetic efficacy of lactoferrin in type 2 diabetic pediatrics: controlling impact on PPAR-g. SIRT-1. and TLR4 downstream signaling pathway.DiabetolMetabSvndr. 2018 Dec4;10:89.

Paesano R, Natalizi T. Berlutti F. Valenti P. Body iron delocalization: the serious drawback in iron disorders in both developing and developed countries. Pathog Glob Health. (2012)

Petrik, M.; Zhai, C.; Haas, H.; Decristoforo, C. Siderophores for molecular imaging applications. Clin. Transl. Imaging 2017, 5, 15-27.

Puddu, P.; Latorre, D.; Carollo,M.; Catizone, A.; Ricci, G.; Valenti, P.; Gessani, S. Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells. PLoS ONE 2011, 6, e22504.

Rosa L, Cutone A, Lepanto MS, Paesano R, Valenti P. Lactoferrin: A Natural Glycoprotein Involved in Iron and Inflammatory Homeostasis.lnt J

Mol Sci. 2017 Sep 15;18(9).

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Valenti P, Antonini G. Lactoferrin: an important host defence against microbial and viral attack. Cell Mol Life Sci. 2005 Nov;62(22):2576-87.

Claims

1. Mixture containing lactoferrin of either recombinant or any natural origin, sodium citrate, sodium bicarbonate in a molar ratio, meant as moles of lactoferrin/(moles of sodium citrate + moles of sodium bicarbonate), comprised between 10^-4 and 10^-3, preferably between 2 × 10^-4 and 7 × 10^-4 for use in the treatment or prevention of infections that colonize epithelia at the level of oral mucosa in order to:

• decrease the number of pathogenic microorganisms,

• inhibit the formation of biofdms, the microbial capacity to adhere and invade,

• promote the repair of injuries,

• inhibit or decrease the inflammatory process caused by infections, optionally associated with iron deficiency, anaemia, inflammation anaemia or diabetes;

said mixture being administered only topically in the form of a topical oral formulation in the absence of iron homeostasis disorders, or the aforementioned topical oral formulation is administered in combination with a systemic oral formulation comprising lactoferrin, sodium citrate and sodium bicarbonate in the same range as the same molar ratio range reported above, when these infections and the related inflammatory process occur at the level of the oral mucosa and are associated with iron deficiency, anaemia or inflammation anaemia or when the inflammatory process is associated with diabetes.

2. The mixture for use according to claim 1, wherein the following effects are simultaneously obtained:

- significant inhibition of microbial multiplication,

- inhibition of biofilm formation,

- inhibition of microbial adhesiveness,

- inhibition of microbial invasiveness,

- inhibition of the synthesis of pro -inflammatory cytokines,

- prevention and treatment of diabetes,

- restoration of iron homeostasis e

- restoration of the physiological values of haemoglobin, of the number of red blood cells, of total serum iron, of serum-ferritin and of the percentage of haematocrit.

3. The mixture for use according to claim 1 or 2, wherein the acute or chronic infections are selected from those associated with adhered or intracellular, optional or compulsory microorganisms in platonic or biofilm form.

4. The mixture for use according to claim 3, wherein the acute or chronic infections associated with inflammation are selected from one or more of: halitosis, gingivitis, periodontitis, black stains and lesions of the oral mucosa.

5. The mixture for use according to any one of the claims 1-4, wherein the treatment of orally systemically obtained iron deficiency or anaemia or inflammation anaemia involves the restoration of the physiological distribution of iron between the tissues and the secretions and the circle.

6. A combined preparation comprising a first oral formulation suitable for topical administration in the oral cavity comprising lactoferrin, sodium citrate and sodium bicarbonate, wherein the molar ratio, meant as moles of lactoferrin/ (moles of sodium citrate + moles sodium bicarbonate) is comprised between 10^-4 and 10^-3 and preferably between 2 × 10^-4 and 7 × 10^-4, and a second systemic oral formulation containing lactoferrin, sodium citrate and sodium bicarbonate as the active ingredient in which the molar ratio meant as moles of lactoferrin/(moles of sodium citrate + moles of sodium bicarbonate) is comprised between 10^-4 and 10^-3, preferably between 2 and 7 × 10^-4.

7. The mixture for use according to any one of claims 1-5, or the combined preparation according to claim 6, wherein lactoferrin has an iron saturation degree from 0% to 100%, preferably from 0% (limit included) to 100% (limit not included), more preferably from 0% to 20% (limit included).

8. The mixture for use according to any one of claims 1 to 5, or the combined preparation according to any one of claims 6 or 7, wherein the saturation degree is obtained with one or more of Fe(III), Zn, Cu and Mn and wherein the lactoferrin is selected from one or more of the following: intact lactoferrin, the lobe N, the lobe C and the sequences that contain the iron binding site.

9. The mixture for use according to any one of claims 1-5, or a combined preparation according to any one of claims 6-8, wherein the topical oral formulation is in the form of a powder, of ready-to-use or extemporaneous solutions to be dissolved in water at the time of use, for example solutions for rinses or washes, or is in the form of gels, of toothpaste eventually added with hydroxyapatite, or in the form of chewing gums, tablets to be dissolved in the mouth, sprays, capsules or buccal soluble tablets.

10. The mixture for use according to claim 9, wherein from 50 to 400 mg of lactoferrin are topically added with sodium citrate and sodium bicarbonate in a range of between 0.01-1 mole from two to four times a day until the resolution of the pathology.

11. The mixture for use according to claim 9, wherein multiple doses of aqueous solutions containing lactoferrin are topically administered in concentrations between 0.1 and 10% weight/volume added with sodium citrate and sodium bicarbonate, each in a range between 0.01 and 1 mole.

12. The mixture for use according to any one of claims 1-5 and 11, wherein with the oral systemic formulation lactoferrin is administered in amounts ranging from 50 to 400 mg and sodium citrate and sodium bicarbonate each in amounts ranging from 0.01 and 1 mole from two to four times a day until the resolution of the pathology.