EP4304552A1

EP4304552A1 - Novel antiviral compositions and their use in therapy and in the treatment of viral infections

Info

Publication number: EP4304552A1
Application number: EP22706666.9A
Authority: EP
Inventors: Paolo Colombo; Eride QUARTA; Gaia Colombo; Sabrina BANELLA
Original assignee: Plumestars Srl
Current assignee: Plumestars Srl
Priority date: 2021-02-01
Filing date: 2022-01-31
Publication date: 2024-01-17
Also published as: IT202100002003A1; WO2022162635A1

Abstract

The present invention refers to novel pharmaceutical compositions in the form of powder, which comprise antiviral drugs alone or in combination, as well as the use of said compositions in therapy, in particular for the treatment and/or prevention of respiratory tract infections.

Description

"Novel antiviral compositions and their nse in therapy and in the treatment of viral infections"

Summary

Technical field

The pandemic caused by the novel SARS-CoV-2 coronavirus led to the disease called COVID-19 by OMS, which has determined a huge study on finding not only a vaccine treatment for prevention, but also for the need of having available a fast and safe therapy medical aid for the subjects which get infected. To this end, a research was made for known drugs which could show an activity in containing the diffusion and manifestation of the symptoms related to a viral infection such as SARS-CoV-2, in order to compose them in a medical preparation in the form of a nasal powder.

To date specific antiviral treatments against coronavirus consisting of newly- proposed drugs are starting to be approved. However, a great number of known pharmacologically active substances has been empirically used to fight the vims replication, in particular some licensed as antimalarials, antivirals, antiparasities in combination with antibiotics for concurrent infections caused by bacteria. These drugs have been studied in limited clinical trials and with dubious success, both due to the small number of patients involved and because the recruited patients were in the pulmonary phase of the disease with hypoxia (hyperinflammatory stage). In this stage, the viral pathogenicity was a minor aspect of the general physiopathology, while the inflammatory responses of the host were the dominant pathology.

The antiviral drugs are generally administered by systemic route in the treatment of infections caused by various viruses. Due to the small dimensions, the viruses are often highly infectious, therefore the most effective medical approach for viral diseases remains the vaccination. Even with the availability of the vaccine, the possibility of being subjected to the infection in the future requires the simultaneous availability of a pharmacological treatment to quickly treat and/or limit the development of the infection.

After months of full blown infection, the evolution of COVID-19 disease is known. The entrance of the virus and the virus replication occur essentially in the upper respiratory tract. Today we know that vast majority of the COVID-19 patients have an asymptomatic infection or a mild disease with fever, loss of taste and smell, and respiratory and gastrointestinal symptoms, which are addressed with antipyretic or anti-inflammatory support therapies. The severe disease, which occurs in a minority of subjects 5-7 days after the onset of the symptoms, is a huge immune response, due to the setting up of the viral pneumonia. An analysis of the dynamics of the viral replication revealed that there is, during the infective stage, a window of antiviral opportunity where suitable countermeasures can be employed to avoid the disease from worsening as the virus appears in the lungs.

To date few antiviral drugs, which selectively act on specific viruses, have been developed. For example, neuraminidase inhibitors are actually licensed for the treatment and prophylaxis of seasonal, pandemic or zoonotic influenza, with a debate on their efficacy and safeness and, therefore, also on the suitability of employing these drugs for future influenza pandemics. Public healthcare points out that they are not an alternative to vaccination and have to be always taken on medical prescription and supervision, as soon as the symptoms of infection appear.

For example, the two classes of antiviral drugs for the prophylaxis and treatment of influenza are neuraminidase inhibitors (oseltamivir and zanamivir) and M2 viral protein inhibitors (amantadine and rimantadine). The prophylactic use of antivirals is not recommended as a routine during seasonal influenza pandemic, due to the risks of creating resistance. Notwithstanding, recent studies on influenza have suggested that antiviral drugs administered immediately at the onset of the symptoms can reduce infection and infectiousness to other persons.

Chloroquine (CQ) and hydroxychloroquine (HCQ) are drugs widely used for the treatment of malaria. HCQ has shown good in vitro results and a clear antiviral action against coronavirus in primate cells, also at low concentrations. Being drugs with potential antiviral activity and being already widely used in antimalarial therapy, their pharmacokinetic and toxicological profile is known. No data to date showed that the in vitro activity of CQ and HCQ against SARS-CoV-2 resulted in validated clinical efficacy after systemic administration. The studies carried out on the clinical efficacy of the CQ and HCQ administration in the COVID- 19 disease have often shown contradictory results, also because they have been carried out on hospitalized patients with a severe respiratory syndrome. Moreover, it has been noticed that these studies have been performed only for the systemic administration of the drugs (via oral route or by injection), given that the virus enters the human body through the upper respiratory tract, i.e. mouth and nose. The systemic administration determines a low concentration of drug at the infection site in the upper respiratory tract which, in the case of increase of the dose, raises the risk of amplifying the side-effects.

Giuliani et a., Drug Delivery, vol. 25(1), 2018, 376-387, describe the nasal administration of ribavirin agglomerates for its transport to the brain (not for a local action on the airways), said agglomerates having a diameter from 350 to 400 pm.

US2001/0053359 describes compositions for the nasal administration, containing the ICAM-I protein (Intercellular Adhesion Molecule 1) which is a glycoprotein of about 90 kDa, also known as CD54, as antiviral. ICAM-I is typically present on endothelial cells and immune system cells and it is also used by rhinoviruses as a receptor. In said compositions the concentration of ICAM-I is from 0.1 to 50%, preferably from 1 to 20% and chitosan is also present, which is known to be an absorption enhancer, as also described in Giuliani et al. mentioned above.

US 2017/0246262 Al, Latec N, describes a liquid preparation containing antiviral substances, which is deposited as spray drops on various mucosas including the nasal one. When the drops dry off, they form a protective film which filters the particles or microorganisms being inhaled and also has an antiviral activity. The solution may also contain the dissolved nutraceutical substance lactoferrin, which is reputed to have antiviral properties by US 2017/0246262 Al.

There is therefore the continuous need of providing novel therapeutic means targeted at a particular region of the body which solve the problems of actual antiviral therapies (activity, toxicity), in particular for the treatment of the upper respiratoiy tract diseases. Objects of the invention

It is a first object of the invention to provide novel inhalation pharmaceutical compositions of substances with antiviral activity, alone or in combination with other substances, in form of an aerosolizable and inhalable powder capable of settling and accumulating the drug composing them in the cells of the epithelium of the upper respiratory tract.

It is another object of the invention to provide said compositions in a passive nasal inhaler favoring the deposition of the active substances in the different regions of the upper respiratory tract, at a dose lower than the oral or parenteral dose.

It is a further object of the invention to provide said compositions to be used in therapy, in particular for the prevention, treatment and limitation of the upper respiratoiy tract viral infections, for example, but without limitation, of the coronavirus infections, as in the COVID-19 disease.

Brief description of the Figures

Figure 1 shows the permeation profiles through nasal mucosa of rabbit of hydroxychloroquine, starting from a saturated solution (triangle) and from powdered hydroxychloroquine (circle) in the form of microparticles.

Figure 2 shows the amount of hydroxychloroquine (HCQ) accumulated in the nasal mucosa of rabbit at the end of the permeation experiment (4 hours) of Figure 1, starting from the solution of HCQ and from the spray dried powder of HCQ MPs-SD.

Figure 3 show's the aerodynamic behavior and the in vitro deposition of an exemplary composition of the invention (Example 1) after being aerosolized at 60 L/min with an inhalation device for dry’ powder of passive type RS 01.

Figure 4 shows the SEM image of the microparticles of powder of Example

1 , which consists of 99% chloroquine and 1% sodium stearate.

Figure 5 show's the SEM image of the microparticles of powder of Example

2, which consists of chloroquine diphosphate and zinc chloride.

Figure 6 show's the SEM image of the microparticles of powder of Example

3, which consist of chloroquine diphosphate and zinc gluconate and are covered w'ith lactoferrin.

Figure 7 shows the air flows produced by healthy volunteers during nasal inspiration through the device for the administration/inhalation of the powder of Example 3, by using the passive device Turbospin, PH&T, Milan, Italy, for the inhalation of dry powder.

Figure 8 shows the Covinhale device (known as Turbospin, PH&T, Milan), as an example of a device suitable for the administration of the composition of the invention.

Description of the invention

According to a first aspect, subject-matter of the invention is a loco-regional pharmaceutical composition, in the form of microparticle powder, which comprise at least one antiviral agent of low molecular weight, characterized in that at least 90% of said microparticles have volume diameter from 1 to 20 microns, in that said antiviral is present at a concentration of 60-100 wt% with respect to the total weight of the composition and in that said composition is to be administered via a device for dry powder inhalation.

The expression "loco-regional" is well known to one skilled in the field and defines the administration targeted to a specific region of the body. In the present invention the expression "loco-regional pharmaceutical composition" means a composition adapted to the administration to the respiratory tract, preferably at least the upper respiratory tract such as nose, throat, and trachea and possibly also bronchi and lungs. The administration of the invention is essentially a topical administration to accumulate the drug on the mucosas of the upper respiratory tract, while keeping the systemic concentration significantly reduced.

According to the present invention, the "antiviral agent" is the active ingredient of the composition of the invention and the expression "low molecular weight antiviral agent" means herein that said at least one active ingredient is preferably a so called "small molecule", a concept well known to one skilled in the field, i.e. it is not a compound of high molecular weight such as for example a polymer, such as a protein. The antiviral agent constituting the active ingredient of the composition of the invention is therefore characterized in that it is not a polymer, for example it is not a peptide or a nucleotide.

The term "aerosol" and derived terms are clear to one skilled in the field and refers herein to the dispersion of a solid (the microparticles as herein defined) in the air.

According to a preferred embodiment, the administration is carried out via nasal route, preferably by an inspiratory act, herein also called "by inhalation" where by ’’inhalation" it is meant that the administration occurs by an inspiratory act, opposite to what happens through an administration by "insufflation". This difference is of particular relevance since it allows a targeted and deep administration of the composition, which can thus reach also the lowest part of the upper respiratory tract, as it will be explained in detail below, also with reference to the appended Figures.

The nasal administration by "inhalation" according to the invention can be made through the device for the inhalation of dry powder ("dry' powder inhaler"), which means herein any device routinely used for the administration of powders via buccal route, for example those employed for the treatment of asthma. The nasal administration of a powder, according to the invention, occurs with an inspiratory act done through a nose nostril, by using a dry powder inhaler which has a nasal "mouthpiece" adapted to be inserted into the cavity of the nasal nostril. It has been found that the air flows exerted by volunteers, during a nasal inspiration test of the powder of Example 3 with the device fitted to the nose nostril, allowed a quantitative extraction of the powder contained in the capsule. It has been found that of the 40 mg of powder charged in the capsule, 20-27 mg have been inhaled in two inspiratory acts performed in the two nostrils by the volunteers. They reported the appearance of a bitter taste, thus confirming that the powder containing chloroquine, typically bitter, of the composition of Example 3 also settled in the throat.

According to a preferred embodiment, subject-matter of the invention is a pharmaceutical powder composition consisting of microparticles as herein defined, which comprises at least one antiviral agent as defined above and at least one agent selected from an enhancing agent of the antiviral activity and a bioadhesive agent.

According to a preferred embodiment, subject-matter of the invention is a pharmaceutical powder composition of microparticles as herein defined, which comprises at least one antiviral agent as defined above and at least one enhancing agent of the antiviral activity and/or at least one bioadhesive agent. According to a preferred embodiment, the pharmaceutical powder composition of microparticles as herein defined, comprises at least one antiviral agent as defined above, at least one enhancer of the antiviral activity and at least one bioadhesive agent.

According to an embodiment, subject-matter of the invention is a pharmaceutical powder composition consi sting of microparticles as herein defined, which comprises at least one antiviral agent as defined above and at least one agent selected as enhancer of the antiviral activity, said composition not comprising any bioadhesive agent.

According to a preferred embodiment, subject-matter of the invention is a pharmaceutical powder composition consisting of microparticles (herein also only "particles") as herein defined, which comprises an antiviral agent as defined above and at least one bioadhesive agent, said composition not comprising any enhancing agent of the antiviral activity.

The pharmaceutical microparticle powder composition of the invention is also herein simply denoted as the "composition of the invention" or the "composition”.

The expression "microparticle powder composition" is well known in the art and refers to a mixture of substances in the form of particles with micrometric dimensions. More specifically, the composition has at least 90% of the particles with a dimension, as volume diameter, lower than 20 microns, preferably from 1 to 20 microns.

According to a preferred embodiment, the microparticles of the invention have a median volume diameter from 0.1 to 15 microns, for example from 1 to 10 microns, more preferably from 1 to 7 microns.

The terms median diameter, volume diameter, average diameter and aerodynamic diameter denote the dimensions of the powder particles as a function of the quantitative determination procedures and are concepts well known to one skilled in the art of inhalation powders. The average diameter of the microparticles of the invention has been measured with a laser diffractometer.

According to a most preferred embodiment, the microparticles of the invention have a median aerodynamic diameter from 2 to 12 microns (determined with an impactor as the New Generation Impactor, measured in an air flow of 60 L/min at room temperature), for example from 4 to 7 microns; this particle dimension allows to achieve a fine dispersion of the particles emitted by the device following an inspiratory act through the nose, thus forming a stable powder aerosol, which can easily enter the upper respiratory tract through the nose and settle thereby sequentially covering the respiratory- mucosas at least of nose, throat, pharynx, in part trachea, and possibly also primary bronchi.

A powder as defined above, i.e. containing microparticles with a median aerodynamic diameter from 2 to 12 microns, measured in an air flow⁷ of 60 L/min at room temperature, has to be considered as a pulmonary "aerosolizable powder", the type of powder which is indeed administered via buccal route with a dry powder inhaler (also called pulmonary device). The aerodynamic behavior of an inhalation powder describes its ability to form a stable aerosol from which the dispersed particles settle with a rate function of their dimension, density and form. The coarsest particles (>10 pm) of an inhaled aerosol will settle in the upper parts of the respiratory tract. The aerodynamic dimension of the powder particles emitted by the inhaler depends on the air flow used for their emission.

The inspiration through the nose generates fluxes of 15-25 L/min (as evidenced in Figure 7 showing the air flows of the volunteers), lower than those generated by a mouth inhalation (>50 L/min). As a consequence an inhalation powder, inspired at low flux by the nose, will be emitted under the form of microparticle aggregates, with an aerodynamic dimension mostly greater than 10 pm and settled in the upper respiratory tract (nose, throat, pharynx and trachea).

The composition of the invention can be used in therapy for the prevention, treatment and containment of the diseases of the upper respiratory tract, for example the viral diseases of the respiratory tract such as, but without limitation, the infections caused by coronavirus for example, but without limitation, the COVID- 19 disease.

The at least one antiviral agent according to the invention is a drug adapted for the prevention, treatment and/or containment of viral diseases of the respiratory tract, for example known drugs which during past infections have shown activity against coronaviruses, or novel antiviral drugs. For example, the following may be used as antiviral agents: the antimalarials as chloroquine and hydroxychloroquine, the anti-parasite as ivermectin, the antivirals as remdesivir, ribavirin, lopinavir and other drugs proposed for other viral infections such as oseltamivir, zanamivir, molnupiravir, favipiravir or M2 viral protein inhibitors as amantadine and rimantadine, and the mixtures of antiviral agents included their pharmaceutically acceptable salts. These substances have the advantage of being already known for their activity, toxicity and side-effects.

According to a preferred embodiment, the at least one antiviral agent is selected from chloroquine, hydroxychloroquine, amantadine, remdesivir, favipiravir and mixture thereof including their pharmaceutically acceptable salts.

According to a preferred embodiment, the at least one antiviral agent is chloroquine or a pharmaceutically acceptable salt thereof, for example the diphosphate salt.

According to a preferred embodiment, the at least one antiviral agent is hydroxychloroquine or a pharmaceutically acceptable salt thereof, for example the sulfate salt.

According to a preferred embodiment, the at least one antiviral agent is favipiravir or a pharmaceutically acceptable salt thereof.

According to a preferred embodiment, the at least one antiviral agent is molnupiravir or a pharmaceutically acceptable salt thereof.

The expression "enhancer of the viral activity" means a substance which can strengthen and increase the antiviral activity of the antiviral agent in the composition of the invention.

According to an embodiment, the enhancer of the antiviral activity is one or more other antiviral agents.

According to an embodiment, the enhancer of the antiviral activity can be an agent acting against the viral proteins which enhances the antiviral activity of the antiviral agent itself and can be, for example, selected from a pharmaceutically acceptable sale of zinc such as zinc chloride, zinc gluconate and zinc acetate, preferably zinc gluconate and zinc chloride. Mixtures of enhancers can be also comprised in the composition of the invention. According to a preferred embodiment, in the composition the molar ratio of the at least one antiviral agent to the moles of the zinc salt enhancer ranges from 3.25 to 1.0, more preferably from 3.25 to 2.0.

The term "bioadhesive" identifies the ability of a compound to prolong the contact of the composition with the epithelia of the upper respiratory' tract and denotes herein a polymeric or oligomeric biocompatible substance, such as for example a water-soluble polymer soluble of medium-high viscosity, so that the 7.5% solution in water at 25°C has a viscosity from 400 to 1000 mPa s. Suitable bioadhesive agents are for example the salts of hyaluronic acids, cyclodextrins, sodium chondroitin sulfate, alginic acid, collagen, poloxamers and soluble derivatives of cellulose. According to a preferred embodiment, said bioadhesive agent is sodium hyaluronate. According to a preferred embodiment of the invention, the sodium hyaluronate has advantageously an average molecular weight from 700 to 1,300 kDa.

According to another preferred embodiment said bioadhesive agent is the lactoferrin protein. The inventors have indeed found, unexpectedly, that lactoferrin is capable of providing a strong bioadhesive effect and it is particularly useful in the composition of the invention.

Subject-matter of the invention, according to another of the aspects thereof, is the use of lactoferrin as bioadhesive agent, in particular as bioadhesive agent in a composition for the administration of substances to the human being or animals, for example a pharmaceutical, nutraceutical or cosmetic composition.

It has been also observed that when the bioadhesive agent is the lactoferrin protein, the particles of the composition take a corrugated form favorable to their aerodynamic behavior and representative of the fact that the protein encapsulates the rest of the composition. The microparticles of the composition of the invention comprising lactoferrin as bioadhesive agent are therefore also better aerosolizable, since they are more easily carried by the inhalation air flow.

According to a preferred embodiment, the pharmaceutical microparticle powder composition of the invention comprises at least one antiviral agent as herein defined, a pharmaceutically acceptable zinc salt, preferably selected from zinc chloride, zinc gluconate, zinc acetate and mixtures thereof, and a bioadhesive, preferably lactoferrin and/or sodium hyaluronate. Preferably, when present, the bioadhesive is present in the composition in a percentage from 10 to 20% of the total weight of the composition.

According to a preferred embodiment, the composition does not comprise absorption promoters, for example it does not comprise chitosan.

All the components of the composition are commercially available or can be prepared according to known methods and techniques.

Pharmaceutically acceptable excipients, known in the art, can be added to the composition of the invention.

According to an embodiment, the composition of the invention comprises from 65 to 100 wt% of at least on antiviral agent as defined herein, preferably from 70 to 100 wt%, from 80 to 100 wt%, from 70 to 99 vrt%, from 80 to 99 wt%, from 90 to 100 wt%, from 95 to 100 wt%, from 99 to 100 wt%, from 90 to 99 wt%, from 90 to 99.50 wt%, from 95 to 99 wt% or from 95 to 99.95 wt%, with respect to the total weight of the composition.

Contrary/ to the composition of the invention, the compositions known for the nasal administration have a low concentration of active ingredient. The high concentration of active ingredient is particularly important in a composition for the nasal route administration according to the invention, in order to limit the amount of powder to be inhaled and improve therefore the compliance.

According to a preferred embodiment, the composition consists of:

- from 60.00 to 100% of at least one antiviral agent, preferably from 65.00 to 99.95%;

- from zero to 20.00% of at least one enhancer of the antiviral activity- selected for example from the pharmaceutically acceptable salts of zinc, present from 5.00 to 20.00%, more preferably from 10.00 to 20.00%;

- from zero to 20.0% of a bioadhesive, for example from 10.00 to 18.00%;

- from zero to 5% of pharmaceutically acceptable excipients; said % being expressed as weight of each component with respect to the total weight of the composition.

Some excipients which improve the organoleptic and technological characteristics of the composition can be added to the composition of the invention. In another preferred embodiment said excipient is a sweetener derived from sugar. such as mannitol, xylitol or sorbitol. According to a preferred embodiment, said excipient is mannitol, which can be present in an amount lower than 5 wt% with respect to the total weight of the composition.

The composition can also contain other excipients used to favor the physical characteristics of the nasal powder and its ability of being aerosolized, such as for example sodium stearate, leucine and isoleucine. Moreover, the composition can contain corrective excipients of the organoleptic properties and preservatives such as chlorhexidine, possessing also antiviral properties.

According to a preferred embodiment, the composition comprises sodium stearate, preferably in an amount of 0.1-2 wl%, more preferably of 0.5 -1.5 wt%, for example 1 wt%, with respect to the total weight of the composition.

According to a preferred embodiment, the composition comprises also further optional excipients such as perfumes and/or other preservatives.

Said preferred compositions comprise: chloroquine or a pharmaceutically acceptable salt thereof, with or without sodium stearate; hydroxychloroquine or a pharmaceutically acceptable salt thereof, and sodium stearate; chloroquine or a pharmaceutically acceptable salt thereof, and a zinc salt selected from zinc chloride, zinc acetate, zinc gluconate and mixtures thereof; chloroquine or a pharmaceutically acceptable salt thereof, and a zinc salt and a bioadhesive agent, preferably ferritin; chloroquine or a pharmaceutically acceptable salt thereof and a bioadhesive agent, preferably ferritin; hydroxychloroquine or a pharmaceutically acceptable salt thereof and a bioadhesive agent, preferably ferritin; hydroxychloroquine or a pharmaceutically acceptable salt thereof, and a zinc salt selected from zinc chloride, zinc acetate, zinc gluconate and mixtures thereof; favipiravir or a pharmaceutically acceptable salt thereof and sodium stearate and optionally a zinc salt and optionally a bioadhesive; molnupiravir or a pharmaceutically acceptable salt thereof and sodium stearate and optionally a zinc salt and optionally a bioadhesive.

Subject-matter of the invention, according to another of the aspects thereof, is the composition as defined above for its use through nasal administration by means of a dry powder inhaler, active or passive, preferably a passive type device.

Subject-matter of the invention, according to another of the aspects thereof, is the composition as defined above for its use in therapy, preferably in the treatment and/or prevention of viral diseases of the respiratory tract, such as for example the coronavirus infections, for example but without limitation by SARS-CoV-2 (COVID-19 disease).

Subject-matter of the invention, according to another of the aspects thereof, is the use of a dry powder inhaler, active or passive, for the nasal administration of the composition of the invention, preferably a passive type device which is operated by the inspiration made by the patient through the nose.

Subject-matter of the invention, according to another of the aspects thereof, is a method for the prevention, treatment and/or containment of the viral diseases of the respiratory tract, such as for example the coronavirus infections, for example but without limitation by SARS-CoV-2 (COVID-19 disease), which for its characteristics favors the administration to a subject in need thereof of a composition of the invention as defined above, preferably through a dry powder inhaler, advantageously a passive type device operated by the nasal inspiration by the patient.

The composition of the invention has to be administered via nasal route to a mammalian subject, preferably the human being or a pet or a cattle, for example, cows, sheep, horses and the like.

According to a preferred embodiment, the composition of the invention is packaged in pre-dosed rigid capsules containing a single dose of the composition, for example packaged in blisters, which can be marketed as such or together with a type of the device to be used for the nasal inhalation.

Each unit dose can preferably comprise from 20 to 65 mg of powder comprising:

- from 10 to 40 mg, preferably from 20 to 30 mg of at least one antiviral agent as defined above; and optionally further comprising:

- from 0 to 5.0 mg, for example from 0.5 to 5.0 rag, preferably from 1.0 to 3.0 mg, for example about 2 mg, of at least one enhancer of the antiviral activity, preferably a pharmaceutically acceptable salt of zinc for example selected from zinc chloride, zinc acetate, zinc gluconate and mixtures thereof; and/or

- from 0 to 15 mg, for example from 1.0 to 15 mg, preferably from 3.0 to 7.0 mg, for example about 5 mg, of at least one bioadhesive, preferably lactoferrin and/or sodium hyaluronate, optionally together with one or more pharmaceutically acceptable excipients.

It is understood, from the composition above, that said composition can comprise one or more antiviral agents alone or in combination with one or more enhancers and/or one or more bioadhesives.

As stated, in addition to the mentioned substances, pharmaceutically acceptable excipients can be present such as preservatives, sweeteners, aromas and/or perfumes, in an amount generally lower than 5 wt% with respect to the total weight of the composition.

Also where not explicitly stated, all the components present in the composition of the invention must be pharmaceutically acceptable.

The present inventors have understood that the therapeutic challenge for respiratory infections, for example but without limitation, COVID-19, is how to increase the concentration of antiviral drugs in the upper respiratory tract, exactly where the vims has infected the epithelial cells of nose and throat. A drug absorbed in such cells provides locally high loco-regional concentrations, keeping at the same time low blood levels. Since the viruses infect the organism through the upper respiratory tract, it has been thought to follow' the virus through the same way. Therefore, the composition in the form of a powder of the invention has been devised to be used by inspiration through a device for inhalation of a dr}' powder, as a novel therapeutic approach and produced for the viral infections, such as for example the coronavirus infections for example, but without limitation, COVID- 19.

In general the administration by inhalation requires the control of the deposition of the dmg on the infected epithelium of the respiratory tract. In the early stage of the infection, the localization of the virus is essentially in the upper respiratory tract, whereas in a later stage the virions reproduced in the cells of such respiratory tract can be distributed in the lung. Inspiration operations and suitable dimensional characteristics of the composition, appropriate for the place where the virus is present, are needed to control the nasal or pulmonary deposition.

Unlike the conventional powders administered via nasal route, which have a limit for the particles lower than 10 microns to provide a local action, i.e. they must not have dimensions lower than 10 microns, the micronized powder composition of the invention has been designed for the nasal inhalation so that it is settled and remains for long time not only in the nose but also in the throat and in the lower respiratory tract, for a loco-regional antiviral activity. Should the particles penetrate the bronchi, their effect would not be absolutely negative, unlike with normal nasal powders.

The composition of the invention, indeed, activates in the infectious respiratory epithelia an antiviral action against the virus penetrated in the cells. The composition plays also a protective action with regard to other people, since the viral load of the sputum and nasal mucus of the infected subject to whom the composition has been administered is significantly reduced.

A subject-matter of the invention is to administer antiviral powders via nasal inhalation with an inhaler which allows the powder to enter the upper respiratory' tracts by means of an inspiratory act through the nose for a widespread deposition on the epithelial surface.

An important element of the invention has been the use of the powders in the form of microparticles, for example prepared with a spray drying technique of a composition in aqueous solution, capable of settling in different positions of the respiratory tract and occupying a wide surface depending on the dimensional distribution of the powder and the device used.

As stated, it is indeed known that the compositions for the nasal administration to solve local problems are not micronized powders as micronization could allow the powder to pass through the nose and down through the upper respiratory' tract, which de facto is an undesired event for the solution of the problems exclusively at the nasal level. Therefore, an original aspect of the invention has been to devise a composition for inhaling an antiviral agent through the nose, said agent capable of being deposited at the loco-regional level, i.e. on the nasal mucosa, in the throat and the upper respiratory tract depending on the powder particle dimension and the inhalation technique used, that is the inspiratory air flows; the smaller the particle size, the greater the possibility for said particles to reach the lower respiratory tract.

The invention consists therefore in antiviral drug compositions in the form of powders to be administered via nasal inhalation in the early stage of the viral disease, such as the coronavirus diseases for example, but without limitation, COVID-19.

The composition of the invention can be administered with a device for the inhalation of dry powder, preferably of passive type, and preferably with the simultaneous deep nasal inspiration.

The microparticles of the composition in the form of powder, which settle on the epithelium of the respiratory tract, dissolve in situ and the active substances are absorbed by the cells, giving rise to antiviral concentrations therein that prevent the reproduction of the virus. The deposition of the microparticles on the epithelium of the upper respiratory’ tract produces, locally in the cells, a high concentration of the drug, which cannot be achieved by a systemic administration. Moreover, as stated, the absorption in the respiratory tract results in a high local concentration, while the blood drug level remains significantly lower and therefore safer than oral or injection administration.

It has been indeed observed that the drug dose to be administered nasally in order to have a local therapeutic effect is from 1/10 to 1/20 of the dose normally administered via oral route or by other systemic administration route. It is understood that the dose reduction, the low blood level of the drug and the simultaneous high drug concentration in the respiratory' tract together constitute a result due to a significant technical advancement involving the powder and the device.

In the pulmonary inhalation devices, the composition can be introduced pre- dosed in a container, such as a perforable rigid capsule. The device, as stated, is preferably of passive type, meaning that the powder is extracted from the capsule thanks to a respiratory act made by the user with the nose through the nasal adapter of the device. The nasal adapter, to be inserted in the nostril, is a cylindric device portion connected to the capsule. By closing with one finger the other nostril and by deeply inhaling, a sufficient air flow is produced of between 10 and 30 L/min, capable of extracting the powder from the capsule and directing the aerosol containing the powder into the nose. According to an alternative embodiment, the inhaler can also be of active type, meaning that the air flow produced in a mechanical way extracts the powder from the capsule and releases it as an aerosol. In this case the deposition will be mainly in the nasal cavities.

The person skilled in the art will easily understand that the powder composition of the invention is actually, due to the dimensions of its particles, a breathable powder as those that can be administered by inhalation through the mouth. In fact, the dry powder inhalers are used for the pulmonary administration through the mouth, for example of anti-asthmatic drugs.

Even though the nasal inhalation through a dry powder inhaler is a key- feature of the invention for the above reasons, if desired or necessary, the composition of the invention may be also taken by buccal inhalation, by using the same buccal device we have demonstrated to be suitable for the nasal administration. Such device never proposed before for the nasal administration, called Covihnale and known as Turbospin (PH&T, Milan), is schematized in Figure 8.

The composition of the invention can be prepared by mixing the above components, preferably by preparing a solution of the at least one antiviral agent to which, when present, the zinc salt enhancer, the bioadhesive and the any other components may be added in water, in which, mannitol and the further optional components of the invention have been dissolved under stirring, when present. Said solution is made up to volume with water to achieve a solid residue from 0.5% w/v to 10.0% w/v. According to the preferred method, the so-obtained solution is transformed into a micronized powder with the spray drying technique, well known to the one skilled in the art.

Therefore, according to the preferred embodiment, the composition of the invention is realized through a process comprising the following steps: i. dissolving in water under stirring the at least one antiviral agent and, optionally, at least one agent selected from an enhancer of the antiviral activity and one bioadhesive, and the further optional components; after dissolution, add a water volume so as to achieve, if dried, a solid residue from 0,5 to 10,0% w/v; ii. drying the so-obtained solution, preferably with a spray dryer to obtain a dry powder of microparticles.

According to another alternative preparation form, the solution of the composition can be dried by lyophilisation, thus obtaining a powder mass which can be ground, for example through a 400 mesh grid.

A detailed description of the process of the invention is set forth in the following Experimental Section.

The composition is particularly useful in the treatment and/or containment of the diffusion of upper respiratory tract infections such as the coronavirus infections, for example by SARS-CoV-2, not only in the respiratory tract but also in the external environment.

As stated, when the microparticle powder composition of the invention is inhaled in the nasal cavities with an inhaler described above, it settles on the surface of the respiratory epithelium of nose, throat, trachea and up to the bronchi. The microparticles deposited on the epitheliums infected by the virus dissolve in the mucus on which they have been deposited and the drug molecules are absorbed by the underlying cells.

In an ex vivo experiment, in which an antiviral composition has been deposited on the nasal mucosa of rabbit, we found that the nasal mucosa quickly absorbed the antiviral agent, in an amount that locally produced a high active concentration against the virus of the order of millimoles, whereas the antiviral activity of hydroxychloroquine is of the order of micromoles. It is therefore understood that the composition of the invention in combination with the selected administration method and route, allows depositing the antiviral agent in very' high concentrations in the region of interest, i.e. the respiratory' tract.

It has been also noted that the absorption of hydroxychloroquine, by the cells of the nasal epithelium of rabbit (Figure 1), was much more intense when the composition deposited on the mucosa was in the form of microparticles, compared to a saturated solution of the antiviral agent.

For the treatment of the infection and/or for the prevention of the pathological symptoms of the upper respiratory tract, such as cough, sneezes, inflammation, abundant nasal secretions or sore throat, the composition can be administered one or, preferably, several times per day, at regular intervals, until the disappearance of the symptomatology. This posology can be made more effective from the bioadhesive properties of the composition in the form of micronized powder which promote the retention of the antiviral on the cells of the respiratory epithelium until full local absorption. The high loco-regional concentration of the drug is the very relevant advantage of the nasal powder compared to the known technique of administration by pills or injection. The amount of active substance taken by mouth, at least 10 times higher than the nasal dose, by spreading throughout the body, produces a much lower concentration in the respiratory' tract, where needed.

Figure 2 shows the amount of hydroxychloroquine found within the nasal mucosa of rabbit at the end of the permeation experiment in Figure 1. When the microparticles dissolve, they accumulate a high amount of hydroxychloroquine in the nasal mucosa of rabbit where 0.35 ± 0.10 mg are present, an amount at least three times higher than that found by using the solution (0.11 ± 0.04 mg).

As stated, when the dose of the composition is administered to a subject, following inspiration an aerosol is produced in the nose which passes through the nose, enters the throat, goes down the trachea and eventually to the bronchi and lungs. During this path in the respiratory’ apparatus, the aerosol, depending on the particle size, deposits fractions of the composition to cover the anatomical surfaces with which it conies into contact.

This process, causing the deposition of the anti viral drug on the mucosas or epitheliums potentially infected by the virus, requires a mechanical action to extract the powder from the capsule and form the aerosol . Such action is provided or caused by the inhaler used, in particularly by its manufacturing characteristics. We found that by using two types of commercial dry/ powder devices defined active or passive, it is possible to schedule the deposition of the dose of powder on the target structure to fight the viral replication. The use of either device, combined with a deep inhalation, results in a useful deposition to treat the ongoing infection and to limit the spread of the infection by the infected subject- in detail, the active device is an insufflator using a rubber bulb connected to the nasal adapter containing the capsule. After piercing the capsule by a pin, the nasal adapter is inserted in the nostril of the nose and, by pressing the rubber bulb, an air flow is created which extracts the powder from the capsule and generates the aerosol entering the nose. This insufflation is carrier out several times, until the capsule powder is completely released, alternating the two nasal nostrils. During the insufflation in one nostril, the other is closed with the external pressure of a finger. The user may be passively undergo the emission of the powder but, preferably, also simultaneously inhale. In the two cases, the deposition of the powder occurs mainly in the nose and to a lesser extent in the throat. This administration technique is more suitable for a containment action of the infection.

With the preferred passive device according to the invention the procedure is similar, but the air flow producing the powder aerosol is provided by a deep inspirator}' act through the nose of the user. The device, in this case, does not have a rubber bulb but only the nose adapter where the capsule pierced in a similar way as the active device, is inserted. In this mode as well, the inhalation is carried out several times until the complete extraction of the powder, by alternating the two nose nostrils and closing the opposite nostril during the inspiration. This administration technique is more suitable for the deep treatment of the infection and in turn contributes to the containment of the infection by limiting the presence of virions in the mucus of nose or throat.

We have therefore found that the nasal inhalation with the two types of devices leads to a different deposition of the powder which, in the case of the active device or insufflator, occurs mainly in the nose. By using the passive device, the deposition can reach not only the nose but the throat, pharynx, trachea and also the bronchi .

This different mode of deposition, which depend on the device used and also on the particle size of the composition, and complies with the therapeutic action to be performed, is an important technical advantage.

Since under applicative conditions the powder of the composition settles on a wet mucosa surface, conditions for a prolonged action of the product on the surfaces of the respiratory tracts arise. We found that the deposition of the product in the solid state is strategic to the effect, since the dissolution over time of the solid microparticles produces a concentration on the epithelial cells which remains saturated until the whole solid is dissolved. Indeed, as can be seen from Figure 2, the comparison between the solid state deposition of the composition with the deposition of the same composition as saturated solution, produces a very high concentration in the potentially infected cells and guarantees a very effective antiviral activity for a long time.

Therefore, the composition of the invention is capable of providing respiratory mucosas with microparticles of the antiviral agent which contain antiviral agents within them, which enhance themselves by acting on the different mechanisms of action against virus replication, for example of the SARS-CoV-2. The antiviral agents could not enter the infected cells if they were not kept in contact with these cells for a sufficient time after being deposited in the solid state, thus creating a fast and prolonged diffusive flux of drug. We have seen that this occurs thanks to the possible presence of bioadhesive substances, generally of polymeric type, preferably of natural origin, in the microparticles.

This is a solution to increase the permanence of the composition on the surface of the cells where the virus penetrated, which, unexpectedly, was identified by comparing the ability of the powder composition to penetrate and accumulate in the cells where the virus is present. The discovery' that the solid-state composition deposited on the nasal mucosa of rabbit produces a cellular accumulation at least three times more than the same dose of the composition applied in the liquid state was unexpected, since it had never been observed for this kind of active substances.

Moreover, we found that the two different types of nasal devices can cause depositions of the active powders which are suitable to fight viral infections, for example but without limitation the COVID-19 disease, at different levels in the respiratory tract. These devices are perfectly interchangeable depending on the required function in terras of the desired deposition site. Indeed, the composition they can emit is the same for each of the two devices, if this is marketed pre-dosed in a rigid capsule. Such capsule is perfectly suitable to be inserted and used in both types of nasal devices, notwithstanding their different emission mechanism of the powder contained in the capsule.

Combined with the active or, preferably, passive device, the composition allows the treatment and protection of the nose wall, the throat and the trachea, not only against the vims replication, but during the infectious stage it protects the external environment from the virion dispersion. As a result of the powerful local antiviral action at the nasal and oral mucosa surfaces, no active virions are emitted with the drops from the mouth or nose.

The deposition of a powder on the respiratory tract following an inhalation was simulated and measured in vitro with a device called Next Generation Impactor (NGI), Copley Scientific, Nottingham, UK. The simulation allows quantifying the aerosolized and inhaled dose fraction which could deposit in the nose, throat, bronchi and deep lung, depending on the dimensions of the particles composing the aerosol.

Figure 3 shows the in vitro behavior of the powder of Example 1 inhaled and deposited on the stages which simulate the anatomic sequence from the nose to the lung. The powder, leaving the device as an aerosol, has an aerodynamic dimensional distribution of the particles which causes, in the respiratory tree, a deposition in quantitatively decreasing fraction from the largest to the smallest particles. This deposition sequence reproduces what could happen in vivo from the nose to the throat, trachea, bronchi to the lungs. These are the structures which could be potentially infected by the vims when it is inhaled from the external environment.

A detailed study of the respirability by means of the Next Generation Impactor has been carried out by supplying three capsules of type 3 charged with 26.5 mg of the composition of Example 1, corresponding to 26.2 mg of chloroquine phosphate. The capsules have been inserted in a passive pulmonary inhaler RS01 (Plastiape, Osnago, Italy) representing a standard of which there are many literature references. After piercing the capsule, a 60 L/min air flow has been activated, simulating the pulmonary inhalation capacity of a subject. The simulated inhalation act resulted in the emission and deposition of different size fractions of the powder particles in the various separating stages of the instrument which can be compared to the different anatomic sites of the respiratory' tract. Figure 3 illustrates the simulated physiological state following the aerosol inhalation under the described conditions, referred to as fraction of the powder mass charged into the capsule deposited on each stage of the NGI impactor. It can be observed that, for the investigated composition, about 10% of the dose remained in the device, almost 30% was able to be deposited on the stage representing the nose and throat, about 20% between the throat and trachea, about 30% in the bronchi and the remaining amount would reach the lung. The composition charged in the capsule, which was deposited in the specified stages, corresponded to the aerodynamic dimensions of the progressively smaller particles that can be related to the different structures of the respiratory tract corresponding to such dimensions.

The fractions deposited in the NGI allowed to measure the median aerodynamic diameter of this powder, which was about 5.5-6.4 microns.

Subject-matter of the invention, according to another of the aspects thereof, is a kit comprising at least one composition of the invention, preferably in the form of unit dose, and at least one dry powder inhaler for the nasal administration of the said composition. Preferably the device is of passive type of the type shown in Figure 8 and allows the nasal administration of the composition through a deep inspiration. Preferably, in the kit of the invention the composition is packaged in unit doses as defined above. More preferably, the kit of the invention also comprises a package leaflet.

According to a preferred embodiment, the kit of the invention comprises a dry powder inhaler, preferably a passive device and at least three, preferably at least 12, more preferably from 12 to 30 unit doses, or even more.

The invention will be now illustrated by the following examples, which are not intended limiting the protection scope of the invention.

Experimental section

Example 1

It has been produced a composition of antiviral agent with sodium stearate with a ratio of 99: 1 w/w, comprising:

Procedure for the preparation of 5 g of a composition according to the invention i) Preparation of the solution of antiviral agent and sodium stearate a) 4.95 g of chloroquine diphosphate, or hydroxychloroquine sulfate, have been dissolved in 350 ml of purified water at 30°C under stirring. The dissolution occurred quickly in few minutes and a clear solution was achieved. pH of the solution was 7.0. b) 0.05 g of sodium stearate has been dissolved in 150 ml of ethanol 90° at 30°C. The full dissolution occurred after about 5 minutes while keeping the mechanical stirring at 300 rpm. c) The solution of sodium stearate has been added to the aqueous solution of the drug, while keeping the magnetic stirring at 300 rpm at 30°C until the solution was clear with 6.5 pH. ii) Spray drying

The final solution has been spray dried by a Spray Dryer (Buchi B-290) by setting the following process parameters: inlet air temperature 125°C, air flow of the nozzle 600L/h, speed of liquid feeding 3ml/min, suction 35 ra³/h, nozzle 0.7 mm and outlet air temperature 75°-80°C.

The yield of the process was about 70%. A picture of the particles produced according to Example 1 is depicted in Figure 4.

Example 2

It has been also prepared a solution to be dried at 1% w/v of chloroquine diphosphate and zinc chloride by keeping the 3.25: 1 molar ratio according to the invention. 986 mg of chloroquine have been dissolved in 50 ml of water, and when completely dissolved, 80 mg of zinc chloride have been added (MW: 136.28 g/moles) and it was made up to a volume of 100 ml. The total solid content was 1.066 g in 100 ml of water. Such solution has been spray dried by using the following parameters: inlet air temperature 150°C; nozzle 0.7 mm; nozzle air flow 600 L/h; liquid feeding 3 ml/min (11%); suction 100% (35 m '/'h). The yield of the process was about 80%. A picture of the particles produced according to Example 2 is depicted in Figure 5.

Example 3

A composition of chloroquine enhanced with zinc salt and made bioadhesive by covering the particles with the lactoferrin protein according to the invention is produced.

The lactoferrin is dissolved in a 1% w/v solution containing chloroquine diphosphate and zinc gluconate, in a 3.25: 1 molar ratio. The following have been weighted:

I. 986 mg of chloroquine diphosphate (MW: 515.86 g/moles) 0.0019 moles

II. 270 mg of zinc gluconate (MW : 455.68 g/moles) 0.000587 moles

III. 200 mg of lactoferrin (MW⁷: 80 kDa)

986 mg of chloroquine have been dissolved in 50 ml of water, and when completely dissolved, zinc chloride has been added and finally the lactoferrin, by stirring with a magnetic bar.

When the dissolution is completed, it was made up to a volume of 150 ml. The total solid content was 1.456 g in 150 ml of water. Such solution has been spray dried by using the following parameters: inlet air temperature 150°C; nozzle 0.7 mm; nozzle air flow 600 L/h; liquid feeding 3 ml/min (11%); suction 100% (35 m⁵/h). The yield of the process was about 80%. All the powder was in the container and the cyclone remained clean. The residual water content was 1.3% w/w (Figure 6).

The so-obtained composition has the form of powder consisting of microparticles which, measured with a laser particle analyzer Spraytech, Malvern Instruments, UK, showed a median volume diameter D_V(50) from 3.0 to 6.0 microns, a percentile D_V(10) from 1.5 to 2.3 microns and a percentile D_V(90) from 7.0 to 20 microns.

Example 4

Production and characterization of a spray dried powder based on Favipiravir and Sodium Stearate

A nasal powder has been prepared by the spray drying technique containing Favipiravir and sodium stearate in a 99: 1 ratio starting from a 1% w/v concentrated solution (PCT/EP2008/0591 10; WO 2010/003465 A2).

The preparation of the formulation is set forth in the following:

* 1.980 g of favipiravir have been dissolved in 140 ml of ultrapure water at 30°C under 300 rpm stirring. For the mechanical stirring a magnetic bar of 60 mm length and 5 min diameter has been used. The dissolution occurred quickly in few minutes and a clear solution was achieved . The pH of the solution was 7.0;

* 0.02 g of sodium stearate have been dissolved in 60 ml of ethanol 90° at 30°C. The full dissolution occurred after about 5 minutes by keeping the mechanical stirring at 300 rptn. pH of the solution was 6.0.

The solution of sodium stearate has been dropped into the aqueous solution of chloroquine while keeping the stirring at 300 rpm at 30°C by the same magnetic bar described previously. A clear solution with a pH of 6.5 was obtained.

The 200 ml of the final solution (water/ ethanol 70:30) have been dried by the Spray Dryer (Buchi B-290) by setting the parameters:

Inlet temperature 150°C;

Air flow speed 600 L/h;

Feeding speed 3 ml/min (11%);

Suction 100% (35 ra3/h),

Nozzle 0.7 mm

The outlet temperature was 83°C. The yield of the process was 65%. All the powder was in the container, the cyclone remained clean and the powder was fine and brownish. The residual water content in the formulations was 3.5% w/w.

The respirability of the powders has been tested by FSI, using the Fast Screening Impactor (FSI). The determination of the respirability values has been carried out by gravimetry. An HPMC capsule size 3 has been charged with 25 mg of favipiravir powder and then inserted in an average resistance RS01® inhaler and aerosolized at 60 L/min. The following parameters have been measured: amount of emitted powder (ED): 21.5± 0.3 mg, amount and respective fraction of fine particles below 5 pm (FPD): 5.5± 0.2 mg with respect to the amount of powder charged in the inhaler. Then, the emitted fraction (86%) and the fraction of fine particles (25.6%) have been calculated.

The dimensional distribution of the obtained powder is shown in the following Table:

Claims

1. A loco-regional pharmaceutical composition in the form of microparticle powder comprising at least one low molecular weight antiviral agent, characterized in that at least 90% of said microparticles have volume diameter from 1 to 20 microns, in that said antiviral is present at a concentration of 60-100 wt% with respect to the total weight of the composition and in that it is administered via nasal route by means of a device for the inhalation of dry powder.

2. The composition according to claim 1, characterized in that said antiviral is present at a concentration of 65.00-99.95 wt% with respect to the total weight of the composition.

3. The composition according to claim 1 or 2, characterized in that it comprises at least one enhancing agent of the antiviral action, selected from the pharmaceutically acceptable salts of zinc, preferably selected from zinc chloride, zinc gluconate and zinc acetate.

4. The composition according to any one of claims 1 to 3, characterized in that the microparticles of the composition have median aerodynamic diameter measured with an air flow of 60 L/min of from 2 to 12 microns.

5. The composition according to any one of claims 1 to 4, characterized in that said low molecular weight antiviral agent is selected from chloroquine and hydroxychloroquine, ivermectin, remdesivir, ribavirin, lopinavir and oseltamivir, zanamivir, molnupiravir, favipiravir, M2 viral protein inhibitors, amantadine, rimantadine, and mixtures thereof.

6. The composition according to any one of claims 1 to 5, characterized in that it also comprises a bioadhesive component, preferably lactoferrin.

7. The composition according to any one of claims 1 to 6, characterized in that the composition consists of:

- from zero to 20.00% of at least one enhancer of the antiviral activity, selected from zinc chloride, zinc gluconate and zinc acetate;

- from zero to 20.00% of at least one bioadhesive component;

- from zero to 5% of pharmaceutically acceptable excipients; said % being expressed as weight of each component with respect to the total weight of the composition,

8. The composition according to any one of claims 1 to 7, characterized in that it is in the form of unit doses as a rigid capsule, each comprising:

- from 10 to 40 mg, preferably from 20 to 30 mg, of at least one antiviral agent, and optionally further comprising:

- from 0 to 5.0 mg, preferably from 1.0 to 3.0 mg, of at least one enhancer of the antiviral activity, selected from zinc chloride, zinc gluconate, zinc acetate and mixtures thereof; and/or

- from 0 to 15 mg, preferably from 3.0 to 7.0 mg, of at least one bioadhesive component, preferably lactoferrin and/or sodium hyaluronate, together with one or more pharmaceutically acceptable excipients.

9. The composition according to any one of claims 1 to 8, for use in therapy via administration by nasal inhalation.

10. The composition for the use according to claim 9, in the treatment and/or prevention of viral respiratory tract diseases, preferably viral infections, more preferably by coronavirus.

11. A kit comprising at least one composition according to any one of claims 1 to 8 and at least one device for the inhalation of dry powder.

12. Use of the lactoferrin protein as a bi oadhesi ve agent.