FR3064486A1 - PREVENTION OF SYNCYTIAL RESPIRATORY VIRUS INFECTION IN UPPER RESPIRATORY TRACT - Google Patents

Abstract

The present invention relates to the use of an anti-RSV immunoglobulin immunoglobulin (Ig) composition for the prophylactic treatment of RSV infection in a subject by application of the mucosal composition. upper respiratory tract.

Description

Holder (s): FRENCH LABORATORY OF FRACTIONATION AND BIOTECHNOLOGIES.

Extension request (s)

Agent (s): CABINET BECKER ET ASSOCIES.

PREVENTION OF SYNCYTIAL RESPIRATORY VIRUS INFECTION IN THE UPPER RESPIRATORY TRACT.

The present invention relates to the use of an immunoglobulin (Ig) composition hyper-enriched in anti-RSV immunoglobulins in the prophylactic treatment of an infection with the RSV virus in a subject, by application of the composition. in the mucous membranes of the upper respiratory tract.

FR 3 064 486 - A1

The present invention relates to the use of an immunoglobulin (Ig) composition hyper-enriched in anti-RSV immunoglobulins in the prophylactic treatment of an infection with the RSV virus in a subject, by application of the composition to the tissues targets, namely at the level of the mucous membranes of the upper respiratory tract, more particularly at the level of the mucous membranes of the nasal cavities and / or of the nasopharynx.

TECHNOLOGICAL BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is a common infectious agent of the respiratory tract. RSV usually causes a mild, asymptomatic or mild symptoms (cold) infection in immunocompetent people, but which can be responsible for several infectious episodes per year on the same individual, which can be the cause of absenteeism from school or work. job.

In addition, this virus is an important factor of infections in hospitals in immunocompromised patients, awaiting or leaving a transplant, in infants or young children or in the elderly. RSV is the most common cause of bronchiolitis and pneumonia in infants and young children, and is largely responsible for respiratory infections in institutions for the aged.

Currently, there is no effective vaccine available to prevent RSV infection. Although anti-viral drugs are available to treat RSV infections, their effectiveness is questionable, especially in children.

There is therefore a need for a preventive treatment capable of neutralizing the respiratory syncytial virus (RSV) as soon as it comes into contact with the body and thus preventing infection with RSV.

SUMMARY OF THE INVENTION

The present invention relates to an immunoglobulin (Ig) composition hyper-enriched in anti-respiratory syncytial virus (RSV) immunoglobulins, for its use in the prophylactic treatment of an RSV virus infection in a subject, by application of the composition in the mucous membranes of the upper respiratory tract, more particularly in the mucous membranes of the nasal cavities and / or the nasopharynx.

Said Ig composition hyper-enriched in anti-RSV immunoglobulins further comprises a muco-adhesive, preferably selected from the group consisting of: polyacrylic acid or its derivatives, carbomer, polycarbophyle, polymethyl methacrylate or its derivatives, cellulose or its derivatives, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, chitosan or its derivatives and its pharmaceutically acceptable salts, chitosan glutamate, alginates, microparticles of alginates conjugated with lectins, pregelatinized starches, pectins, hyaluronic acid, chondroitinine sulfate, chondroitinine sulfate, chondroitin sulfate dextrans and its derivatives such as amino dextrans or DEAEdextran, poly hydrogel nanoparticles (methacrylic acid grafted with poly (ethylene glycol)), thiol polymers, natural oligosaccharide gums, xanthan gums, carbopol® 974P, sea curve 240, scleroglucans, polycarbophils .

Said Ig composition hyper-enriched in anti-RSV immunoglobulins can also comprise a bacteriostatic compound, in particular said bacteriostatic compound can be selected from the group consisting of: benzalkonium chloride, benzyl alcohol, chlorobutanol, parabens, phenylethyl alcohol, acid benzoic, EDTA, 2-pyrrolidone, and their derivatives.

Preferably, the Ig composition hyper-enriched in anti-RSV immunoglobulins useful according to the present invention comprises a concentration of anti-RSV immunoglobulins of between 10 and 250 g / L.

According to a particular embodiment, said Ig composition hyper-enriched in anti-RSV immunoglobulins is intended to be administered in the form of particles or droplets having a size between 2 and 100 μm, more preferably between 5 and 50 μm.

According to a particular embodiment, said Ig composition hyper-enriched in anti-RSV immunoglobulins is intended to be administered or applied in the form of nasal drops, suspension, powder, emulsion, gel, ointment, cream, aerosol.

According to a particular embodiment, said Ig composition hyper-enriched in anti-RSV immunoglobulins is intended to be administered or applied using a device, said device being selected from the group consisting of: a dispersion system powder, drip, atomization or spray system, nasal spray, nebulizer.

According to a particular embodiment, said Ig composition hyper-enriched in anti-RSV immunoglobulins is intended to be administered or applied to the nostril of the subject, preferably with an administration volume of between 50 and 250 μl per nostril.

According to a particular embodiment, the Ig composition hyper-enriched in anti-RSV immunoglobulins is intended to be administered or applied once a day and through one nostril.

Advantageously, the application of the Ig composition hyper-enriched in anti-RSV immunoglobulins is adapted so that the anti-RSV immunoglobulins bind to RSV in the mucous membranes of the upper respiratory tract, preferably in the mucous membranes of the nasal cavities. and / or the nasopharynx.

According to a particular embodiment, said Ig composition hyper-enriched in anti-RSV immunoglobulins is used in the prophylactic treatment of an infection with the RSV virus in a subject belonging to at least one of the following groups: children, infants, immunocompromised people, including premature infants and children or the elderly with lung, heart or immune disease, people being treated for a transplant, especially a lung transplant, people with chronic obstructive pulmonary disease (COPD).

LEGEND OF FIGURES

Figure 1. Analysis of viral replication in mice, 1 day after infection with the RSV virus. Study by in vivo imaging (dorsal view) of viral replication 1 day after infection with RSV virus in mice having received preventively, by the nasal route: a composition comprising non-specific Ig (negative control) or a composition enriched in anti Ig -RSV at a dose of lmg / kg or a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg.

A) First group of mice tested (2 mice per condition)

B) Second group of mice tested (2 mice per condition)

C) Average of the results obtained for the groups of mice A and B grouped in a histogram for each condition. Antibody irrelevant = Negative control; LFB 1 = composition enriched in anti-RSV Ig at a dose of 1 mg / kg; LFB 0.1 = a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg.

Figure 2. Analysis of viral replication in mice, 4 days after infection with the virus

RSV. In vivo imaging study (dorsal view) of viral replication 4 days after infection with RSV virus in mice having received preventively, via the nasal route: a composition comprising non-specific Ig (negative control) or a composition enriched in anti Ig -RSV at a dose of lmg / kg or a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg.

A) First group of mice tested (2 mice per condition)

B) Second group of mice tested (2 mice per condition)

C) Average of the results obtained for the groups of mice A and B grouped in a histogram for each condition. Antibody irrelevant = Negative control; LFB 1 = composition enriched in anti-RSV Ig at a dose of 1 mg / kg; LFB 0.1 = a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg.

Figure 3. Analysis of viral replication in mice, 1 to 4 days after infection with the RSV virus.

A) Study by measurement of the radiance, in lysates of nasal fossae of mice, of viral replication after an infection with the RSV virus in mice having received preventively, by the nasal route: a composition comprising non-specific Ig (negative control) or a composition enriched in anti-RSV Ig at a dose of lmg / kg or a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg. The radiance measurements were carried out on the lysates of the nasal passages of mice sacrificed 1 day or 4 days after their infection. This study included 6 mice / day of sacrifice / condition. Antibody irrelevant = Negative control; LFB 1 = composition enriched in anti-RSV Ig at a dose of 1 mg / kg; LFB 0.1 = a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg.

B) Study by measurement of the radiance, in lysates of mouse lungs, of viral replication after an infection with the RSV virus in mice having received preventively, by the nasal route: a composition comprising non-specific Ig (negative control) or a composition enriched in anti-RSV Ig at a dose of lmg / kg or a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg. The radiance measurements were carried out on the lysates of the lungs of mice sacrificed 1 day or 4 days after their infection. This study included 6 mice / day of sacrifice / condition. Antibody irrelevant = Negative control; LFB 1 = composition enriched in anti-RSV Ig at a dose of 1 mg / kg; LFB 0.1 = a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg.

DETAILED DESCRIPTION OF THE INVENTION

Preventive treatment

H is described here a method for the prophylactic treatment of an infection with the RSV virus in a subject, said method comprising the topical and targeted application of an immunoglobulin (Ig) composition hyper-enriched with anti-respiratory syncytial virus immunoglobulins (RSV).

The subject can be any mammal, preferably a human being, whatever their age or sex. It may in particular be a subject at risk with respect to an infection with the RSV virus, for example a child, an infant, or an immunocompromised person, in particular in premature infants and children or the elderly suffering from '' a lung, heart or immune disease. Preferably, the subject, if infected, is asymptomatic, that is, he has not yet developed symptoms associated with the infection.

In particular, the subject may belong to at least one of the following groups: children, infants, immunocompromised persons, in particular premature infants and children or elderly persons suffering from pulmonary, cardiac or immune disease, persons treated for a transplant, in particular a lung transplant, people with chronic obstructive pulmonary disease (COPD).

The term "prophylactic treatment" refers to preventing or stopping the development of the infection, before it reaches the lower respiratory tract, causing inflammation of the lungs. It thus prevents the appearance of symptoms, namely congestion or runny nose, but especially lung inflammation, especially in premature infants and children with lung, heart or immune disease.

Ig composition hyper-enriched in anti-RSV Ig

The immunoglobulin composition useful according to the invention is an immunoglobulin composition hyper-enriched in anti-RSV immunoglobulins which exhibits a neutralizing action with respect to RSV and has therapeutic efficacy for the patient. More particularly, the composition is hyper-enriched in immunoglobulins directed specifically against the RSV virus. The term "specific" means that the immunoglobulins recognize and bind to an immunogen of said virus, substantially without cross-reaction with another virus. Throughout the description, the terms "Ig", "immunoglobulins" and "antibodies" may be used interchangeably.

The immunoglobulin composition includes antibodies which bind to the same viral immunogen (this is called a monovalent population of antibodies), or to several different immunogens from RSV (we can then speak of a polyvalent population of antibodies). Essentially, these are polyclonal antibodies.

Said immunoglobulin composition essentially contains polyvalent human immunoglobulins which may be immunoglobulins A (IgA), immunoglobulins E (IgE), immunoglobulins M (IgM) or immunoglobulins G (IgG). Advantageously, the immunoglobulin composition essentially contains IgG and / or IgM and / or IgA.

In a particular embodiment, the immunoglobulin composition essentially comprises IgG, whatever their subclass (IgGl, IgG2, IgG3 and IgG4).

Preferably, the composition is suitable for local administration and preferably contains essentially IgG and / or IgA, the IgA allowing the recruitment of cells such as mucosal cells having receptor alfa.

By “Ig composition hyper-enriched in anti-RSV immunoglobulins” is meant an immunoglobulin composition comprising at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%; at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% by weight of immunoglobulins neutralizing against RSV. In a particular embodiment of the invention, the immunoglobulin concentrate hyper-enriched in anti-RSV immunoglobulins comprises at least 10% by weight of polyvalent immunoglobulins capable of recognizing and / or neutralizing the RSV virus or one of its parts. In another particular embodiment of the invention, the immunoglobulin concentrate hyper-enriched in anti-RSV immunoglobulins comprises at least 30% by weight of polyvalent immunoglobulins capable of recognizing and / or neutralizing the RSV virus or the one of its parts. In another alternative and specific embodiment of the invention, the immunoglobulin concentrate hyper-enriched in anti-RSV immunoglobulins comprises at least 60% by weight of polyvalent immunoglobulins capable of recognizing and / or neutralizing the RSV virus or one of its parts. The immunoglobulins directed specifically against RSV can bind to any of the proteins of the virus, preferably a surface protein, playing a role in the mechanisms of infection, in particular during recognition, of the binding of the particle. viral to its host cell, the fusion of the viral particle to its host cell and the spread of the virus from host cells to host cell. Mention may in particular be made of the SH protein, the G protein or the F protein of RSV.

The composition is generally concentrated. In particular, the composition can comprise a concentration of anti-RSV immunoglobulins of between 10 and 250 g / l, preferably between 10 and 100 g / l.

Methods for preparing the Ig composition hyper-enriched in anti-RSV Ig

Several methods of preparing the Ig composition hyper-enriched in anti-RSV Ig are possible. Preferably, a method of preparing immunoglobulins is used from plasma pools, advantageously human plasma. Such a method involves operations of concentration of the plasma preparations, typically by a factor of 100 to 1000.

According to a particular embodiment, immunoglobulin preparations derived from plasma or from blood plasma fractions are subjected to affinity chromatography using a viral protein as affinity ligand, to obtain an immunoglobulin preparation hyper-enriched in immunoglobulins capable of recognizing, and advantageously neutralizing, said RSV. This method uses affinity chromatography supports onto which one or more RSV viral proteins, or antigenic fragments thereof, are grafted. Among the viral proteins useful as ligands, mention may be made, for example, of the envelope proteins: the SH protein, the G protein (highly glycosylated transmembrane glycoprotein responsible for binding to the host cell), the F protein (transmembrane glycoprotein responsible for fusion to the host membrane, the entry of the virus into the cell and the formation of syncitia).

Chromatography uses a matrix comprising a support based on polymer particles, and is preferably in the form of a gel or a resin. These polymer particles are preferably spherical or oblong in shape, they may in particular be balls. The polymer can be natural or unnatural, organic or inorganic, crosslinked or uncrosslinked. The polymer is preferably an organic polymer, preferably crosslinked. In a preferred embodiment, the polymer is cellulose, and the particles are preferably porous cellulose beads. Other possible types of polymers include agarose, dextran, polyacrylates, polystyrene, polyacrylamide, polymethacrylamide, copolymers of styrene and divinylbenzene, or mixtures of these polymers. The particles can provide a chromatography medium that can be used to fill a column, for example.

On the support is grafted a RSV viral protein, or an antigenic fragment thereof, either directly or via a spacer, which covalently binds the ligand to the particles of the chromatographic support. In a preferred embodiment of the invention, the protein grafted on the support is a RSV surface protein, for example RSV L protein or RSV G protein, or an antigenic fragment thereof, either directly , or via a spacer, which covalently binds the ligand to the particles of the chromatographic support.

In a particular embodiment of the invention, the grafted support has several different proteins and / or several different antigenic fragments belonging to RSV, in order to obtain an immunoglobulin concentrate having different antigenic targets. A particle can carry several spacers. The bond between the ligand and the spacer can be, for example, an amide bond.

The matrix in the form of a gel is prepared by conventional addition of a buffer to the polymer particles carrying the ligands, as is known to those skilled in the art, so as to obtain a matrix in the form of a gel, suitable for chromatography. of affinity. The matrix as defined here is useful in affinity chromatography binding immunoglobulins. The matrix is particularly useful in mixed bed affinity chromatography. Such a matrix advantageously comprises several different antigens specific to RSV and is therefore capable of binding immunoglobulins directed against several different epitopes of RSV. The anti-RSV immunoglobulins present in the plasma or the plasma fraction bind to the matrix, and the adsorbed product is eluted and recovered, enriched in anti-RSV immunoglobulins. Advantageously, the conditions for implementing affinity chromatography are adapted by a person skilled in the art to guarantee the reuse of the matrix while maintaining non-degrading elution conditions for the product of interest.

The plasma or the starting plasma fraction subjected to the enrichment process consists of plasma originating from blood plasmas or pools of blood plasmas of mammalian subjects (human or non-human) or a fraction of this plasma, either all or part -part of the plasma, having undergone one or more purification steps. In a particular embodiment, the plasma or the starting plasma fraction subjected to the enrichment process advantageously consist of plasma originating from pools of blood plasmas of normal human subjects or a fraction of this plasma, either any part or sub- part of the plasma, having undergone one or more purification steps. The plasma fractions that can be used thus include the cryoprecipitate plasma supernatant, the plasma cryoprecipitate (resuspended), the fractions I to V obtained by ethanolic fractionation (according to the Cohn or Kistler & Nitschmann method), the supernatant and the precipitate obtained. after precipitation with caprylic acid and / or caprylate, the chromatography eluates and the non-adsorbed fractions of the chromatography columns, and the filtrates.

In a particularly advantageous manner, the plasma or the starting plasma fraction subjected to the enrichment process come from pools of blood plasmas of normal human subjects, without prior selection of the donors, in particular, without selection of the donors on the basis of their possible rate plasma in anti-RSV immunoglobulins. The plasma or the starting plasma fraction subjected to the enrichment process thus advantageously comprises an anti-RSV immunoglobulin level similar or equal to the anti-RSV immunoglobulin level of a plasma originating from the pool of at least 1000 human donors. randomly selected normal. The plasma or the starting plasma fraction subjected to the enrichment process is advantageously not enriched in anti-RSV immunoglobulins of interest compared to the anti-RSV immunoglobulin level of a plasma originating from the pool of at least 1000 donors chosen from randomly.

In a further embodiment, the plasma or the starting plasma fraction subjected to the enrichment process consist of plasma originating from blood plasmas or pools of blood plasmas from non-human mammalian subjects, male and / or female, advantageously from goat , sheep, goat, bison, buffalo, camel, llama, mouse, rat, cow, bull, pig, sow, rabbit, horse, mare. The non-human mammal is advantageously transgenic and has been previously exposed to the RSV virus in order to produce human anti-RSV immunoglobulins in its plasma.

The plasma or the starting plasma fraction subjected to the enrichment process contains polyvalent human immunoglobulins which may be immunoglobulins A (IgA), immunoglobulins E (IgE), immunoglobulins M (IgM) or immunoglobulins G (IgG). Advantageously, the plasma or the starting plasma fraction essentially contains IgG whatever their subclass (IgGl, IgG2, IgG3 and IgG4) and / or IgA.

The method can typically be an independent method, dedicated to the purification of anti-RSV immunoglobulins, and is then advantageously carried out using plasma or a plasma fraction.

In this embodiment, the method advantageously comprises the following steps: supply of a plasma sample or of a plasma fraction, passage of the plasma sample or of a plasma fraction on an immunoaffinity chromatography on the matrix described here, recovery of the fraction adsorbed on the matrix corresponding to the immunoglobulin composition hyper-enriched in anti-RSV immunoglobulins.

Advantageously, the method may further comprise, after the capture step on anti-RSV affinity chromatography, a dedicated step of depletion of certain immunoglobulins, in particular immunoglobulins E (IgE) and / or immunoglobulins (IgM) , which may be involved in the mechanisms of deleterious side effects in patients.

The method advantageously further comprises a subsequent step consisting in subjecting the immunoglobulin composition hyper-enriched in anti-RSV immunoglobulins to at least one step of inactivation and / or viral elimination.

In yet another particular embodiment, the method of obtaining anti-RSV immunoglobulins is coupled to a method of polyvalent immunoglobulins G in order to obtain, from a same pool of starting plasma, both a concentrate of polyvalent immunoglobulins G and a concentrate of immunoglobulins hyper-enriched in anti-RSV.

The process then typically includes a prior step of obtaining the plasma fraction, by ethanolic fractionation and / or caprylic fractionation and / or chromatographic separation.

In this embodiment, the method advantageously comprises the following steps:

supply of a sample of a prepurified plasma fraction obtained by ethanolic fractionation and / or caprylic fractionation and / or chromatographic separation passage of the prepurified plasma fraction on an immunoaffinity chromatography on the matrix described here, recovery of the fraction adsorbed on the matrix corresponding to the immunoglobulin composition hyper-enriched in anti-RSV immunoglobulins.

By “chromatographic separation” is meant any chromatography step, whether it is ion exchange chromatography (anion exchanger and / or cation exchanger), mixed mode, affinity (using chemical type ligands, antibodies, antibody fragments, aptamers). By chromatographic separation is meant both a single chromatography column, or a set of chromatography columns, using or not the same type of support, in series (multi-column mode for example) or in parallel.

More specifically, the plasma fraction can be obtained by an ethanolic fractionation originally developed by Cohn et al (Cohn et al, 1946. J. Am. Chem. Soc. 68, 459; Oncley et al, 1949, J. Am Chem. Soc. 71, 541), or else by a chromatographic separation, as described for example in EP 0 703 922 and WO 99/64462, or also by a caprylic fractionation as described by Steinbuch et al 1969, Arch Biochem Biophys. 134 (2): 279-84). Particularly preferred are the methods developed by the Applicant in patent applications WO 94/29334 and WO 02/092632, and very particularly that described in WO 02/092632. In this case, a blood plasma, or a fraction of blood plasma enriched in Ig, is subjected to caprylic fractionation (prepurification by precipitation of non-immunoglobulin contaminants), and a single chromatography on an anion exchange resin support carried out at pH alkaline. A viral inactivation treatment can be carried out, preferably carried out by solvent-detergent, as described by Horowitz in US Pat. No. 4,764,369, optionally supplemented by a viral elimination step by nanofiltration on a 75N, 35N pore size filter. , 20N and / or 15N.

The Ig fraction thus collected is already concentrated, but can then undergo additional concentration stages by ultrafiltration, and sterilizing filtration.

This concentrate is then subjected to an immunoaffinity chromatographic step on the matrix as described above.

Advantageously, the method can also comprise, preferably after the capture step on anti-virus affinity chromatography, a dedicated step of depletion of certain immunoglobulins, in particular immunoglobulins A (IgA), and / or immunoglobulins E (IgE) and / or immunoglobulin M (IgM), which may be involved in mechanisms of deleterious side effects in patients. The immunoglobulins to be depleted are advantageously selected in particular according to the route of administration chosen for the immunoglobulin concentrate hyper-enriched in anti-virus immunoglobulins and / or according to the content of the plasma sample or the starting plasma fraction.

The method may further include the subsequent steps of adding one or more pharmaceutically acceptable stabilizers; and optionally freeze or freeze-dry the concentrate thus obtained.

Formulation

Advantageously, an immunoglobulin composition as described above is in the form of a pharmaceutical composition comprising the anti-RSV immunoglobulins, in combination with at least one pharmaceutically acceptable excipient. The term “pharmaceutically acceptable excipient” means excipients which are not harmful or toxic vis-à-vis the mucous membrane of the upper respiratory tract, in particular vis-à-vis the nasal epithelium and nasal mucociliary clearance.

The formulation of the pharmaceutical composition useful according to the invention is suitable for application to the mucous membranes of the upper respiratory tract. In particular, the mucous membranes of the upper respiratory tract consist of the mucous membranes of the nasal cavities and / or the nasopharynx. Thus, the pharmaceutical composition can be prepared according to the usual methods, using appropriate excipients, preferably suitable for application to the mucous membranes of the upper respiratory tract. For example, sodium chloride is an interesting excipient for aqueous nasal compositions.

In addition, other additives can be used in the formulation, such as preservatives, viscosity agents, agents influencing the osmolarity, complexing agents (such as for example sodium edetate), surfactants. active ingredients and agents that influence the pH and tone of the nasal formulation.

The pharmaceutical composition further comprises one or more muco-adhesive (s). In particular, within the meaning of the present invention, the term “muco-adhesive” means a compound or a mixture of compounds capable of adhering to the mucosa, that is to say to the tissue covering of the body cavities in communication with the outside by natural orifices. The mucoadhesive advantageously makes it possible to increase the clearance time of the composition once it has been applied to the mucous membranes of the upper respiratory tract. In particular, the muco-adhesive may be selected from: polyacrylic acid or its derivatives, carbomer, polycarbophyl, polymethyl methacrylate or its derivatives, cellulose or its derivatives, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, chitosan or its derivatives and its pharmaceutically salts acceptable, chitosan glutamate, alginates, lectin conjugated alginate microparticles, pregelatinized starches, pectins, hyaluronic acid, chondroitin sulfate, polyvinylpyrrolidone, gelatin, dextrans and its derivatives such as amino dextrans or DEAEdextran nanoparticles, nanoparticles grafted with poly (ethylene glycol)), thiol polymers, natural oligosaccharide gums, xanthan gums, carbopol® 974P, sea curve 240, scleroglucans, polycarbophils or mixtures of the mucoadhesives listed above.

In a particular embodiment of the invention, the muco-adhesive is of the polymer type, advantageously selected from: cellulose or its derivatives, polyacrylic acid or its derivatives, dextrans and its derivatives such as amino dextrans or DEAE-dextran, hydrogel nanoparticles poly (methacrylic acid grafted with poly (ethylene glycol)), alginate microparticles conjugated to lectins, thiol polymers, natural oligosaccharide gums, xanthan gums, carbopol® 974P, sea curve 240, scleroglucans, hydroxypropylcellulose, pectins, polycarbophils.

In another particular embodiment, the muco-adhesive is a polymer of biological origin, in particular cellulose and its derivatives.

In a particular embodiment of the invention, the muco-adhesive may also have other advantageous properties for the formulation of the composition, this may in particular produce a stabilizing effect for the composition.

In particular, the pharmaceutical composition has a pH, an osmolarity, one or more muco-adhesive agents suitable for the anti-RSV Ig included in the composition to act on the mucous membranes of the upper respiratory tract, preferably on the mucous membranes of one or more nasal cavities and / or nasopharynx.

According to a particular embodiment of the invention, the composition according to the invention further comprises a bacteriostatic compound. By bacteriostatic compound is meant any compound capable of interfering with the growth of microorganisms, in particular bacteria, in particular capable of suspending or inhibiting the growth of microorganisms, in particular bacteria.

In particular, said bacteriostatic compound can be selected from the group consisting of: benzalkonium chloride, benzyl alcohol, chlorobutanol, parabens, phenylethyl alcohol, benzoic acid, EDTA, 2-pyrrolidone, and their derivatives.

Administration and Dosage

Said immunoglobulin (Ig) composition hyper-enriched in anti-RSV immunoglobulins as described above is applied to the upper respiratory tract, more particularly to the mucous membranes of the upper respiratory tract. In particular, said composition is applied to the mucous membranes of the nasal cavities and / or the nasopharynx.

The term “application to the mucous membranes of the upper respiratory tract” means any application, release and / or administration of the composition to the mucous surfaces of the upper respiratory tract, these surfaces constituting a territory susceptible to infection by the RSV virus. and are therefore of interest in the prophylaxis of RSV infection. In particular, said composition is applied topically and targeted to the mucous membranes of the upper respiratory tract. By "upper respiratory tract" or "upper airway" means any duct or extra thoracic cavity in contact with air such as the nose, nasal cavities, nasopharynx. In particular, the application allows the composition to reach and / or penetrate the mucous membranes of the nasal cavities and / or the nasopharynx. The mucous membranes of the nasal cavities and the nasopharynx are characterized by a total area of approximately 140 cm ² . The widely vascularized nasal mucosa allows rapid absorption of the composition used according to the invention.

Advantageously, the application of the composition is adapted so that the anti-RSV immunoglobulins bind to RSV at the level of the mucous membranes of the upper respiratory tract, preferably at the level of the mucous membranes of the nasal cavities and / or of the nasopharynx.

In a particular embodiment, said composition is intended for administration by the nasal route, in particular for nasal inhalation.

Said immunoglobulin (Ig) composition hyper-enriched in anti-RSV immunoglobulins can thus be administered by nasal or intranasal route in the form of nasal drops, suspension, powder, emulsion, gel, ointment, cream, aerosol.

Said immunoglobulin (Ig) composition hyper-enriched in anti-RSV immunoglobulins can be applied or administered using a dispenser or a device, for example: a unit dose vial, an atomizer, a nebulizer, a pump, a nasal tampon, a pre-filled syringe, a nasal sponge or a capsule, or any other method of administration by the nasal route, known in the pharmaceutical literature. In a particular embodiment of the invention, the device further comprises a filter limiting bacterial contamination. Preferably the administration device allows the administration of said composition in the form of an aerosol. Preferably, the administration device is a nebulizer allowing the spraying of said composition in the form of an aerosol. A nebulizer is defined here as a device capable of aerosolizing a liquid material in a dispersed liquid phase.

According to a particular embodiment, the composition of the invention is applied or administered using a device selected from the group consisting of: a powder dispersion system, a drip, an atomization system or spray, nasal spray, nebulizer.

Preferably, said composition is in the form of particles or droplets having a size and a morphology adapted to target the mucous membranes of the upper respiratory tract, and preferably the mucous membranes of the nasal cavities and / or of the nasopharynx. Preferably, the size of the particles (solids) or the droplets (liquids) is between 2 and 100 μm, more preferably between 5 and 50 μm.

The size of the particles can be measured by any technique known to a person skilled in the art, in particular laser diffraction techniques. The size of the droplets can be measured by any technique known to a person skilled in the art, in particular techniques using laser diffraction and techniques based on the Doppler effect. In particular, a person skilled in the art will be able to carry out his measurements using a device implementing laser diffraction and in particular the Visisize D30, Visisize P15, Visisize N60 and Visisize S200 device models (Oxford Lasers Inc).

In a particular embodiment of the invention, said composition is in the form of an aerosol comprising particles or droplets having a size between 2 and 100 μm, more preferably between 5 and 50 μm. An aerosol is defined here as the dispersion of solid particles or very fine liquid droplets in a gas. In particular, the aerosol comprises a continuous gas phase and, dispersed therein, a discontinuous or dispersed phase of particles or droplets.

The pharmaceutical composition according to the invention can be in the form of unit doses (the device allows only one application) or multidoses (the device allows several applications).

In a particular embodiment of the invention, the pharmaceutical composition is advantageously in the form of unit doses.

According to a particular embodiment, the pharmaceutical composition according to the invention is applied or administered in the nostril of the subject. Preferably, said pharmaceutical composition applied to the nostril of the subject has a volume of administration of between 50 and 250 μl per nostril.

By "administration volume" is meant the volume of said composition administered per dose or intake.

In a particular embodiment of the invention, said dose has a volume of administration suitable for the anti-RSV Ig included in the composition to remain localized in the mucous membranes of the upper respiratory tract, preferably in the mucous membranes of one or more nasal cavities and / or nasopharynx.

In a particular embodiment of the invention, the composition is administered using a nasal spray delivering a volume of administration of approximately 150 μl per use.

The dosage can be adjusted appropriately to achieve the desired levels of immunoglobulins locally, following application to the mucous membranes of the upper respiratory tract. For example, in a particular embodiment of the invention, said Ig composition hyper-enriched in anti-RSV immunoglobulins is applied to the mucous membranes of the upper respiratory tract once a day and through one nostril.

The following examples illustrate the invention without limiting its scope.

EXAMPLES

EXAMPLE 1 Preparation of a composition enriched in anti-RSV Ig, from plasma fractions

In this example, the sample used is a plasma fraction obtained from the immunoglobulin purification process as described in patent application WO02092632. The purification process includes the following steps:

Recovery of a fraction I + II + III, obtained from blood plasma treated with ethanol Precipitation with caprylic acid Solvent-detergent treatment (Triton / TnBP)

Anion exchange chromatography (TMAE) at basic pH

The eluate from anion exchange chromatography at basic pH is then subjected to immunoaffinity chromatography, as described below.

1. Anti-RSV (F protein) affinity chromatography

The RSV-F 11049-V08B protein (Sino Biological Inc), a synthetic protein derived from a hRSV DNA sequence (RSS-2) from Met 1 to Thr 529, containing 518 amino acids after cleavage of the propeptide and whose predicted molecular mass is 58kDa. In SDS-PAGE analysis and reduction conditions, the apparent molecular weights are 45-55 kDa and 18 kDa. The RSV-L 11049-V08B ligand used for grafting is in post-fusion conformation.

2mL of NHS-activated SepharoseTM 4 Last Llow gel are placed in a chromatography column with a diameter of 1.1cm and the buffers used are those recommended by the gel supplier. After washing (ImM HCL at 4 ° C.) and equilibration in coupling buffer, 1.98 mg of protein L to be grafted are added to the gel and the whole is stirred. The reactive groups are blocked on the gel by adding 6 mL of 0.5M ethanolamine buffer, 0.5M NaCl, pH 8.3 (6mL), and the gel is stirred for 12 h. The coupled gel is washed with an alternation of 3 volumes of basic then acid buffers: 0.1M TrisHC1 pH 8.4 and 0.1M acetate, 0.5M NaCl pH 4, this cycle being repeated three times. The gel is then stored in lOmM citrate, 0.5M NaCl, O.lg / L sodium azide pH 6.6 buffer.

The coupling was evaluated at 98%, corresponding to a ligand density of 0.89 mg / ml of grafted gel.

The sample is balanced in the injection buffer by adding a concentrated solution of NaCl buffer and trisodium citrate qs 0.05M NaCl and 0.01M Trisodium citrate. The product is then passed over the column for 3.3 min (contact time). After washing, the elution is carried out in buffer O, 1M glycine, 30% propylene glycol at pH 2.57. The eluate is immediately neutralized with IM Tris-HCl pH 9 buffer to bring the pH up to about 6. The gel is then regenerated in order to unhook the uneluted Ig with a 6M guanidine pH 6 buffer. The fraction recovered is also neutralized by adding IM Tris-HCl pH9 to bring the pH back to 6.

2. Anti-RSV (protein G) affinity chromatography

Alternatively, or in addition, the RSV-G protein strain A (ref 11070-V08H2, SB), synthetic protein derived from a DNA sequence of hRSV (strain rsbl734) from Asn 66 to Arg 297, containing 242 amino acids after cleavage of the propeptide and whose predicted molecular mass is 26.3kDa. In SDS-PAGE analysis and reducing conditions, the apparent molecular mass is understood due to a significant glycosylation between 60 kDa and 90 kDa.

strain B (ref 13029-VO8H, SB), synthetic protein derived from a DNA sequence of hRSV (strain 036633-1) from His 67 to Ala 299, containing 244 amino acids after cleavage of the propeptide and the mass of which predicted molecular weight is 27kDa. In SDS-PAGE analysis and reducing conditions, the apparent molecular mass is understood due to a significant glycosylation between 80 kDa and 90 kDa.

The coupling is carried out according to the same protocol as that used in section 1.

Three columns are produced:

GA gel, corresponding to a density of Protein G strain A of 1 mg / ml of grafted gel, gel GB, corresponding to a density of Protein G strain B of 1 mg / ml of grafted gel, gel G-AB, corresponding to a mixture of 50% GA gel and 50% GB gel.

The design of the experiment also made it possible to observe that the anti-RSV IgGs purified on variant A of protein G also interacted with variant B of protein G and conversely (anti-varB on variantA) suggesting that there is cross-reactivity between polyclonal anti-RSV antibodies for the two variants of protein G.

A high proportion of anti-RSV immunoglobulins bind to the hRSV-G protein affinity chromatography support, whatever the strain, which represents in this test at least 6.7 g of anti-RSV immunoglobulins per ml of gel . The non-adsorbed fraction and the washing fraction are depleted in anti-RSV immunoglobulins. The fraction of immunoglobulins eluted from the support at acidic pH and in the presence of Propylene glycol shows a minimum enrichment of 109 times in anti-RSV immunoglobulins.

The test shows that the affinity chromatography using a protein G ligand of strains A or B grafted via NHS chemistry allows the purification of anti-RSV immunoglobulins with a yield of at least 26.2% under the conditions tested, corresponding to a minimum enrichment factor of 109 times.

The composition obtained after affinity chromatography on protein G ligand therefore comprises at least 30% by weight of immunoglobulins directed specifically against at least one epitope of protein G.

EXAMPLE 2 Study of the Effectiveness of a Preventive Treatment by Nasal Administration of the Composition Enriched with Anti-RSV Ig

The composition enriched in anti-RSV Ig as prepared in Example 1 above and dialyzed using a buffer solution containing mannitol (0.15 M) and glycine (0.1 M) to pH = 4 has been tested as a prophylactic treatment against RSV virus, by nasal administration.

Material and methods

The study is carried out over 4 days (D0, D1, D2 and D4), with female BALB / c mice 6 weeks old and made sensitive to the RSV virus.

^1st stage: administration of the prophylactic treatment.

An initial blood sample is taken at the start of the study from all the mice tested.

Prophylactic treatment is administered at D0 - 1 hour.

More particularly, an intranasal administration is carried out according to 3 distinct conditions:

Composition administered Amount administered Administered volume Composition of Nonspecific Antibodies (negative control) 10 mg / kg 10 pL

Composition enriched in anti-RSV Ig 1 mg / kg Composition enriched in anti-RSV Ig 0.1 mg / kg

The study was carried out on 2 groups of 18 mice (6 mice / condition / group).

2 ^nd step: Infection with the RSV virus.

The RSV virus infection was carried out on OJ day, by intra-nasal injection of a solution comprising the RSV virus associated with luciferase (administration of 10 pL).

^3rd step: Analysis of viral replication in mice

Viral replication in mice is then analyzed on day D1 (in a first group of 10 mice, N = 18) and then on day D4 (in a second group of mice, N = 18).

Viral replication was analyzed by an in vivo imaging system. Then the mice were sacrificed, the nostrils and lungs were removed in order to conduct a bioluminescence test (based on the detection of luciferase, complexed with the RSV virus).

Results

Analysis of viral replication by in vivo imaging:

The luminescence analysis in the first group of mice provides the following results. DAY D1 after infection (Figure IA):

Mouse (Figure IA) Radiance in the nose Radiance in lungs 1A1 ++ 0 1 TO 2 ++ 0 1A3 0 0 1A4 0 0 1A5 0 0 1A6 0 0

Figured Equivalence in radiance (maximum of the peak) 0 No signal detected + 0.2 ++ 0.6 +++ 0.8 ++++ 1

DAY D4 after infection (Figure 2A):

Mouse (Figure 2A) Radiance in the nose Radiance in lungs

2A1 ++++ 0 2A2 ++++ 0 2A3 0 0 2A4 0 0 2A5 0 0 2A6 0 0

Figured Equivalence in radiance (maximum of the peak) 0 No signal detected + 0.2 ++ 0.6 +++ 0.8 ++++ 1

The luminescence analysis in the second group of mice provides the following results. DAY D1 after infection (Figure IB):

Mouse (Figure IB) Radiance in the nose Radiance in lungs 1B1 ++++ 0 1B2 +++ 0 1B3 0 0 1B4 0 0 1B5 0 0 1B6 0 0

Figured Equivalence in radiance (maximum of the peak) 0 No signal detected + 0.2 ++ 0.6 +++ 0.8 ++++ 1

DAY D4 after infection (Figure 2B):

Mouse (Figure 2B) Radiance in the nose Radiance in lungs 2B1 ++++ 0 2B2 ++ + 2B3 0 0 2B4 0 0 2B5 0 0 2B6 0 0

Figured Equivalence in radiance (maximum of the peak) 0 No signal detected + 0.2

++ 0.6 +++ 0.8 ++++ 1

In the control mice (to which nonspecific Ig were administered), a viral replication localized in the nose is observed from day D1 (Figures IA, IB and IC), which is amplified on day D4 (Figures 2A, 2B and 2C). We also note that, for one of the controls, the infection begins to spread to the lung (Figure 2B, mouse 2B2), which is correlated by the data obtained on the lung lysates by directly adding the bioluminescence substrate. (Figure 3B).

Conversely, the RSV virus is not detected (signal extinct) in mice which received preventively, by the nasal route, the solution enriched with anti-RSV Ig, from day D1 (Figures

IA, IB and IC) and until day D4 (Figures 2A, 2B and 2C), regardless of the dose (lmg / kg or 0, lmg / kg). These results are confirmed by those obtained on the lysates of the nasal cavities where no virus is significantly detected (above 200 radiance units) for the two doses (Figure 3A).

These results demonstrate the effectiveness of a preventive treatment by nasal administration of the anti-RSV Ig-enriched composition. The anti-RSV Ig administered is capable of effectively recognizing and neutralizing the RSV virus, locally at the level of the nose, namely upon entry of the virus into the body.

Claims (10)

1. Composition of immunoglobulins (Ig) hyper-enriched in immunoglobulins anti-respiratory syncytial virus (RSV), for its use in the prophylactic treatment of an infection by the virus RSV in a subject, said composition being in a form suitable for an application to the mucous membranes of the upper respiratory tract, said composition further comprising a muco-adhesive. 2. Composition of immunoglobulins (Ig) hyper-enriched in immunoglobulins anti-respiratory syncytial virus (RSV) for its use according to claim 1, characterized in that the mucous membranes of the upper respiratory tract consist of the mucous membranes of the nasal cavities and / or nasopharynx. 3. Ig composition hyper-enriched in anti-RSV immunoglobulins for its use according to claim 1 or 2, said muco-adhesive being selected from the group consisting of: polyacrylic acid or its derivatives, carbomer, polycarbophyl, polymethyl methacrylate or its derivatives, cellulose or its derivatives, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, chitosan or its derivatives and its pharmaceutically acceptable salts, chitosan glutamate, alginates, alginate microparticles conjugated to lectins, pregelatinized starches, pectins, hyaluronic acid, hyaluronic acid chondroitin, polyvinylpyrrolidone, gelatin, dextrans and its derivatives such as amino dextrans or DEAE-dextran, poly hydrogel nanoparticles (methacrylic acid grafted with poly (ethylene glycol)), thiol polymers, natural oligosaccharide gums, xanthan gums, carbopol® 974 , scleroglucans. 4. An Ig composition hyper-enriched in anti-RSV immunoglobulins for its use according to any one of the preceding claims, said composition further comprising a bacteriostatic compound. 5. Composition of Ig hyper-enriched in anti-RSV immunoglobulins for its use according to claim 4, said bacteriostatic compound being selected from the group consisting of: benzalkonium chloride, benzyl alcohol, chlorobutanol, parabens, phenylethyl alcohol, benzoic acid, EDTA, 2-pyrrolidone, and their derivatives. 6. Ig composition hyper-enriched in anti-RSV immunoglobulins for its use according to any one of the preceding claims, comprising a concentration of anti-RSV immunoglobulins of between 10 and 250 g / L, preferably between 10 and 100 g / L. 7. Ig composition hyper-enriched in anti-RSV immunoglobulins for its use according to any one of the preceding claims, characterized in that it is intended to be administered in the form of particles or droplets having a size between 2 and 100 pm, more preferably between 5 and 50 pm. 8. Ig composition hyper-enriched in anti-RSV immunoglobulins for its use according to any one of the preceding claims, characterized in that it is intended to be administered in the form of nasal drops, suspension, powder, d emulsion, gel, ointment, cream, aerosol. 9. Ig composition hyper-enriched in anti-RSV immunoglobulins for its use according to any one of the preceding claims, characterized in that it is intended to be administered using a device, said device being selected in the group consisting of: a powder dispersion system, a drip, an atomization or spraying system, a nasal spray, a nebulizer. 10. Ig composition hyper-enriched in anti-RSV immunoglobulins for its use according to any one of the preceding claims, characterized in that it is intended to be administered in the nostril of the subject, preferably with a volume of administration between 50 and 250pL per nostril. 1/5 AT. Non-specific Ig (10 mg / kg) Non-specific Ig (10 mg / kg) Anti-RSV Ig (lmg / kg) [Mouse 1A3] Anti-RSV Ig (lmg / kg) [Mouse 1A4] Luminescenc Γ 0. Anti-RSV Ig (0.1 mg / kg) θ [Mouse 1A5] ’xlO 0. Anti-RSV Ig (0.1 mg / kg) [Mouse 1A6] -0. Color scale: Min = 6.00e3 Max Radiance = 1.00e5 (p / sec / cm / sr) B. Non-specific Ig (10 mg / kg) [Mouse 1B11 Non-specific Ig (10 mg / kg) [Mouse 1B2] Anti-RSV Ig (lmg / kg) [Mouse 1B3] Anti-RSV Ig (lmg / kg) [Mouse 1B4]