WO2007138135A1

WO2007138135A1 - Bioadhesive nanoparticles for administration of biologically active molecules

Info

Publication number: WO2007138135A1
Application number: PCT/ES2007/000309
Authority: WO
Inventors: Hesham H. A. Salman; Carlos Gamazo De La Rasilla; Maite AGÜEROS BAZO; Juan Manuel Irache Garreta
Original assignee: Instituto Científico Y Tecnológico De Navarra, S.A.
Priority date: 2006-05-26
Filing date: 2007-05-25
Publication date: 2007-12-06
Also published as: ES2286951B1; ES2286951A1

Abstract

The invention relates to nanoparticles for the administration of biologically active molecules comprising a biodegradable polymer, preferably the copolymer of methyl vinyl ether and maleic anhydride (MVE/MA), and thiamine. Such nanoparticles are simply produced having excellent characteristics of bioadhesion, size and zeta potential, rendering them suitable for the administration of active molecules. The procedure of obtainment of such nanoparticles is realised by simple incubation for a short period of time, the nanoparticles being preformed in an aqueous medium. The biologically active molecule may be incorporated during the process of formation of the nanoparticles by desolvation or through modification with thiamine. By oral route said nanoparticles develop bioadhesive interactions with the gastrointestinal mucous membrane, including the lymphoid tissue associated with mucous membranes (Peyer's patches), and are therefore useful for increasing the oral bioavailability of various drugs or inducing immune responses in mucous membranes. Thus when such nanoparticles contain an antigen within the matrix thereof the resultant mixture is capable of inducing strong immune responses to the encapsulated antigen. Such stimulating effect on immune response is useful in vaccination and immunotherapy.

Description

BIO ADHESIVE NANOPARTICLES FOR THE ADMINISTRATION OF BIOLOGICALLY ACTIVE MOLECULES

FIELD OF THE INVENTION

The invention relates to bioadhesive nanoparticles, based on a biodegradable polymer and thiamine, manufacturing processes, formulations containing them and their applications.

BACKGROUND OF THE INVENTION

In recent years biodegradable polymeric nanoparticles have been proposed as new transport systems for drugs or biologically active molecules. In the case of the administration of drugs through mucous membranes, the most important characteristics that they offer to the biologically active molecule that they incorporate are: (i) protection against possible physical-chemical and / or enzymatic degradation (increase in their half-life in the organism); (ii) controlled release of the incorporated drug (sustained effects that reduce the number of doses); (iii) interaction / adhesion with the surface of the mucosa where absorption or drug action takes place (increased bioavailability); and (iv) in the case of vaccination, they facilitate the presentation of the incorporated antigen to the antigen presenting cells.

The oral route is the most convenient and popular route for drug administration. However, the bioavailability of a certain active molecule depends (i) on the characteristics of the drug molecule and its pharmaceutical form of administration, and (ii) on the physiological conditions present in the gastrointestinal tract, such as the presence of enzymes. proteolytic, peristaltic movements and presystemic metabolism. The use of polymeric nanoparticles can be a good strategy to overcome some of these obstacles. In principle, these transporters have a large specific surface so that their interaction with the biological support (the gastrointestinal mucosa) is facilitated. Likewise, the control of drug release allows the effect of molecules with low biological half-lives to be prolonged over time. On the other hand, nanoparticles can be captured by Peyer's plate cells and lymphoid tissue follicles. This phenomenon allows the drug to be directed towards the lymphatic route and, in the case of vaccines, to facilitate its antigen presentation. However, conventional nanodeparticles based on biodegradable polymers have some important disadvantages with respect to their oral use: (i) some instability in gastrointestinal fluids, (ii) a low degree of intestinal absorption and (iii) a tropism or non-specific adhesion in the gastrointestinal mucosa.

A possible strategy to minimize these inconveniences associated with the use of conventional nanoparticles is based on the use of nanoparticles coated with certain ligands with specificity for certain mucosal receptors. The main ligands proposed for the preparation of these nanoparticle-ligand conjugates have been, so far: (i) antibodies; (ii) adhesins, invasins or other bacterial proteins; (iii) carbohydrates; (iv) vitamin B ₁₂ ; and lectins. Among all these types of ligands, lectins appear to be the most versatile ligands. These molecules are proteins or glycoproteins, of non-immunogenic origin, capable of specifically recognizing sugars located in the glycoconjugates.

Different studies have demonstrated specific adhesion to certain areas of the gastrointestinal tract by conjugates between particles and lectins. Thus, for example, it has been seen that tomato lectin (Lycopersicon esculentum) has a good affinity for the intestine mucosa. However, conjugates with this lectin have very low affinity for Peyer's plaques. After administration in conjugate rats, a delay in intestinal transit has been observed and, in addition, an increase in their absorption. Likewise, conjugates based on polylactic acid and the lectins of L. esculentum and Lotus tetragonobolus have allowed the increase in the fraction of particles attached to the intestinal tract.

Another possible ligand may be thiamine. Thiamine (or vitamin Bl) is essential for the normal development of cell functions and growth. Mammals cannot synthesize this vitamin and, therefore, must be obtained via intestinal absorption from processed foods or after synthesis by microflora of the large intestine. The thiamine absorption process is quite complicated; although a mechanism based on the existence of a receptor (associated with a proton pump) that is located in the basolateral membranes of the jejuno and the ileum. Recently, it has been described that, in humans, the thiamine transporter is expressed throughout the entire gastrointestinal tract, although mostly in the apical membrane of the brush border ^tc ".

The use of thiamine as a nanoparticle ligand has been investigated in recent years. Thus, it has been described that thiamine may be a good ligand to facilitate the binding or association of nanoparticles to the blood brain barrier and to interact with breast tumor cells. However, to date there is no knowledge of the use of thiamine as a ligand to direct nanoparticles intended for oral administration of biologically active molecules, including their use as adjuvants for vaccination and immunotherapy.

Use of adjuvants in vaccination

The use of particulate adjuvants in the form of emulsions, microparticles, ISCOMS or liposomes has been previously evaluated by various research groups [Singh and OΗagan, Int. J. Parasitology, 33 (2003) 469-478].

The capture of antigens by "antigen presenting cells" is increased when they are associated with or included in polymer particles. Biodegradable and biocompatible polyesters have been used in humans and animals for years as controlled antigen release systems. In contrast to aluminum adjuvants, micro- and nanoparticles are effective in inducing cellular and cytotoxic immune responses in mice. In mice, oral immunization with microparticles induces potent mucosal and systemic immune responses against encapsulated antigens. This ability is a consequence of its internalization by specialized cells of the lymphoid tissue of the mucous membranes. Immunization via mucous membranes with different particulate systems has proven effective against different pathogens, such as Bordetella pertussis, Chlamidia trachomatis, Salmonella typhimurium and Brucella sp.

Use of adjuvants in immunotherapy The treatment of allergic diseases can be approached fundamentally in three different ways: (i) avoiding all contact with the allergen; (ii) using antihistamine drugs, and (iii) by immunotherapy. Given that the first two measures are sometimes not applicable, immunotherapy would be the most appropriate method of control.

Specific allergen immunotherapy has been defined as repeated administration of allergens to patients with IgE-mediated health disorders, with the purpose of providing protection against allergic symptoms and inflammatory reactions associated with natural exposure to these allergens.

This treatment alternative is aimed at enhancing a functional predominance of the ThI response with respect to the Th2 response, which will cause allergic symptoms to be inhibited. This modulation towards ThI is also applicable in other processes, such as control by vaccination against bacterial intracellular parasites (e.g., Brucella or Salmonellá).

Nanoparticles of copolymer of polyvinyl methyl ether and maleic anhydride (PVM / MA) [WO02 / 069938], optionally pegylated [ES2246694], useful as drug carriers have been described. Likewise, the use of said PVM / MA nanoparticles as immune stimulating substances has been described.

However, although the use of various vectors of non-biological origin has been described, for example, nanoparticles, as adjuvants in immunotherapy or in vaccines for the administration of antigens and / or allergens, there is still a need to provide alternative adjuvants to existing ones. in order to increase the arsenal of possibilities for the development of vaccines and compositions for immunotherapy. Advantageously, said adjuvants should be useful for use in oral immunization or immunotherapy without having to use very high doses of allergen or antigen. As is known, despite its potential advantages, oral immunization for therapeutic or prophylactic purposes has to face different obstacles, since the dose of immunogenic or allergenic active ingredient required for a beneficial clinical effect is extremely large due to a loss. of immunogen potency. Thus, due to, in general, poor stability of the allergen or antigen in the gastrointestinal tract (pH conditions and presence of hydrolytic enzymes), the doses should always be much higher (up to 200 times) than those normally used subcutaneously . In addition, the gastrointestinal mucosa acts as a barrier very little permeable to the absorption of these macromolecules. SUMMARY OF THE INVENTION

The object of the present invention is to provide nanoparticles that solve the aforementioned problems, that is, that can be administered orally and are stable and specific, that have good bioadhesive characteristics to interact with the mucous membranes, that are capable of transporting a wide group of active molecules that release the active molecule in a controlled manner. As an additional feature, these nanoparticles are intended to act as adjuvants in vaccines and immunotherapy, enhancing the immune response of antigens or allergens. It has now been found, surprisingly, that such problems can be solved by nanoparticles formed by a biodegradable polymer and thiamine. In particular, it has been found that nanoparticles comprising or consisting of (i) a copolymer of polyvinyl methyl ether (PVM) and maleic anhydride (MA) and (ii) thiamine, in addition to being easy to produce, provide excellent bioadhesion characteristics and size, which makes them suitable for administration through different pathways of biologically active compounds or molecules. Likewise, it has been shown that the nanoparticles provided by this invention that, optionally, contain a biologically active molecule, for example, an allergen or an antigen, have the ability to stimulate or enhance the immune response when administered to a subject, which allows its use in immunotherapy and vaccines.

Therefore, in one aspect, the invention relates to nanoparticles comprising a biodegradable polymer and thiamine or its derivatives. In a particular embodiment, said biodegradable polymer is a copolymer of methyl vinyl ether and maleic anhydride (PVM / MA). In another particular embodiment, the thiamine is completely or partially covering the surface of the nanoparticles comprising said biodegradable polymer. In another particular embodiment, the weight ratio between thiamine and the biodegradable polymer is between 1:10 and 1: 500, preferably between 1:10 and 1: 100, more preferably around 1:40. The nanoparticles provided by this invention can be used for the transport of biologically active molecules. In this sense, said nanoparticles can include one or more biologically active molecules, whose Chemical nature may vary within a wide range of possibilities, including, but not limited to, peptides, proteins, nucleic acids (eg, DNA, RNA, etc.), nucleosides, nucleotides, oligonucleotides, polynucleotides, etc. Said biologically active molecule can be a drug or compound with therapeutic or diagnostic activity, eg, an antitumor agent, a central nervous system protector, a glucocorticoid, etc., an antigen for vaccination or an allergen for immunotherapy, among others. Therefore, the nanoparticles provided by this invention can be used in the preparation of pharmaceutical compositions.

In another aspect, the invention relates to a pharmaceutical composition comprising said previously described nanoparticles and a biologically active molecule, wherein said biologically active molecule is a molecule capable of preventing, alleviating or curing a disease or a molecule with diagnostic application, together with a pharmaceutically acceptable carrier or excipient. In a particular embodiment, said composition is a pharmaceutical composition intended for administration orally or parenterally. If desired, said pharmaceutical composition may be in the form of a lyophilisate.

In another aspect, the invention relates to a vaccine or composition for immunotherapy comprising a therapeutically effective amount of the nanoparticles provided by this invention, previously described, and an antigen or allergen, together with a pharmaceutically acceptable carrier, adjuvant or excipient.

In another aspect, the invention relates to a product comprising, separately, a) an antigen or an allergen; and b) a composition comprising said nanoparticles based on a biodegradable polymer and thiamine, as a composition that enhances the immune response against said antigen or allergen, as a combination for simultaneous administration or sequence! to a subject, in the induction or stimulation of an immune response against said antigen or allergen in said subject.

In another aspect, the invention relates to the use of nanoparticles comprising a biodegradable copolymer and thiamine previously described in the preparation of a pharmaceutical composition for the selective stimulation of the ThI immune response, or in the preparation of a pharmaceutical composition for selective stimulation of the Th2 immune response, or in the development of a Pharmaceutical composition for balanced stimulation of Thl and Th2 immune responses.

In a further aspect, the invention relates to a process for the production of nanoparticles comprising a biodegradable copolymer and thiamine previously described comprising: a) the desolvation of an organic solution comprising a biodegradable polymer, with a hydroalcoholic solution, for form the nanoparticles; b) simultaneous incubation of previously formed biodegradable polymer nanoparticles with thiamine in an aqueous solution; and c) the removal of organic solvents, whereby an aqueous suspension of nanoparticles comprising a biodegradable polymer and thiamine is obtained. If desired, said nanoparticles can be stabilized by the use of crosslinking agents. In a particular embodiment, the concentration of the biodegradable polymer in said nanoparticles is between 0.001 and 10% w / v and that of thiamine between 0.001 and 5% w / v. To obtain biologically active molecules loaded nanoparticles, these can be incorporated either in the organic phase where the biodegradable polymer has previously dissolved, before desolvation, or, subsequently, in the aqueous suspension of the nanoparticles already formed so that it Produce your association.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photograph of the result of a scanning electron microscopy analysis for a lyophilized sample of thiamine coated nanoparticles (T-NP). Figure 2 is a diagram showing the fluorescent marker release

(rhodamine B isothiocyanate, RBITC) from thiamine coated nanoparticles (T-NP) and control nanoparticles (NP) after incubation in simulated gastric (FGS) and simulated intestinal (FIS) media. Data are expressed as mean ± SD (n = 3).

Figure 3 is formed by diagrams showing the distribution of nanoparticles in the gastrointestinal tract of laboratory animals, after oral administration of a 10 mg dose of nanoparticles labeled with RBITC. Figure 3 a is a diagram showing the distribution of thiamine coated nanoparticles (T-NP); Figure 3b is a diagram showing the distribution of control nanoparticles (NP); and Figure 3 c is a diagram showing the distribution of thiamine-coated nanoparticles (T-NP) mixed with 5 mg of free thiamine. The X axis represents the different segments of the gastrointestinal tract [Stomach: Sto; portions of the small intestine: II, 12, 13, 14; blind: Ce]; the Y axis represents the adhered fraction of nanoparticles to the mucosa (mg); and the Z axis represents the time after administration (0.5, 1, 3 and 8 hours). Each value is represented by the mean (n = 3; SD was less than 20% of the average). Figure 4 is a graph showing the bioadhesion curves for thiamine-coated nanoparticles (T-NP) (¡), control nanoparticles (NP) (?), And T-NP co-administered with 5 mg of free thiamine (?) The data represent the mean ± SD (n = 3).

Figure 5 is a set of photographs that allow visualization of nanoparticles in normal ileum tissue (M) and in Peyer's plates (PP) by fluorescence microscopy, specifically, thiamine coated nanoparticles (T-

NP) in the normal mucosa (a); control nanoparticles (NP) in normal mucosa (b); the

T-NP in PP (c); and NP in PP (d).

Figure 6 is composed of a set of graphs showing the serum profile of anti-OVA antibodies of type IgG2a and IgGl in BALB / c mice (n = 10). The antibodies were quantified by ELISA starting with a 1:40 dilution of serum, followed by serial double dilutions. Immunization was performed on day 0 by single administration of either 20 µg of OVA in the different formulations subcutaneously (a, b), or 100 µg of OVA in the different formulations orally (c, d). The formulations were OVA-T-NP (?), OVA-NP (?), And free OVA (?). Antibody titers were determined in the animals' serum on days 0, 7, 14, 28 and 42. The titres were determined as the final dilution of the sample with average absorbance values greater than 0.2, compared with those obtained. in serum of non-immunized animals. Figure 7 is composed of a set of graphs showing the fecal profile of anti-OVA secretory IgA in BALB / c mice (n = 10). Immunization was performed on day 0 by single administration either of 20 μg of OVA in the different formulations subcutaneously (a, c), or 100 μg OVA orally (c, d). The formulations were OVA-T-NP (?), OVA-NP (?), And free OVA (?). Antibody titers were determined in the animals' serum on days 0, 7, 14, 28 and 42. The titres were determined as the final dilution of the sample with average absorbance values greater than 0.2, compared with those obtained. in serum of non-immunized animals.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention relates to nanoparticles, hereinafter nanoparticles of the invention, comprising (i) a biodegradable polymer and (ii) thiamine or a derivative thereof.

The nanoparticles of the invention have adequate physicochemical, bioadhesion and specificity characteristics when administered orally, which makes them into drug transport systems or antigen or allergen presenting systems of great interest. In fact, the nanoparticles of the invention can prolong the residence time in the mucosa after oral administration or through another mucosa of the organism. Said nanoparticles can be used to administer biologically active molecules and improve their bioavailability, including drugs with narrow absorption windows, as well as drugs originating in biotechnology and compounds or molecules used to enhance or induce immune responses in a subject.

As used herein, the term "nanoparticle" refers to a structure formed by a biodegradable polymer modified on its surface by thiamine bonding, or a derivative thereof, which, optionally, can be crosslinked by the addition of a crosslinking agent. The binding of thiamine, or a derivative thereof, to the biodegradable polymer can be a covalent bond. The reaction between the biodegradable polymer and the thiamine, and, optionally, the subsequent crosslinking, generates characteristic, independent and observable physical entities, the average size of which is less than 1 micrometer (μm). By "average size" is meant the average diameter of the nanoparticle population that moves together in an aqueous medium. The average size of These systems can be measured by standard procedures known to those skilled in the art, and which are described, by way of illustration, in the experimental part that accompanies the examples described below.

The nanoparticles of the invention are characterized by having an average particle size of less than 1 μm, preferably having an average size between 1 and 999 nm, more preferably between 10 and 900 nm, even more preferably between 100 and 400 nm. The average particle size is mainly influenced by the amount of thiamine, or its derivative, added (the greater the amount of thiamine or the derivative thereof increases the size of the nanoparticle), by the amount and molecular weight of the biodegradable polymer (the greater the size or molecular weight of the biodegradable polymer, the average size of the nanoparticle is increased), and by some parameters of the production process of said nanoparticles, such as the stirring speed and the temperature in the incubation stage with the aqueous phase It contains thiamine. The nanoparticles of the invention comprise a biodegradable polymer and thiamine or a derivative thereof. The term "biodegradable", as used herein, refers to polymers that dissolve or degrade in a period of time that is acceptable for the desired application, in this case in vivo therapy, once they are exposed to a physiological solution. pH between 4 and 9, at a temperature between 25 ° C and 40 ⁰ C. Virtually any biodegradable polymer known in the state of the art that results in the formation of nanoparticles can be used for the implementation of the present invention Illustrative, non-limiting examples of said biodegradable polymers include polyhydroxy acids, such as polylactic acid, polyglycolic acid, etc., and copolymers thereof, eg, poly (lactic-co-glycolic acid) [PLGA], etc .; polyanhydrides; polyesters; and polysaccharides, eg, chitosan, etc. The molecular weight of said biodegradable polymer can vary within a wide range as long as it satisfies the established conditions of forming nanoparticles and being biodegradable.

In a particular embodiment, the biodegradable polymer used is the copolymer of methyl vinyl ether and maleic anhydride in anhydride form (PVM / MA), commercially referred to as Gantrez® AN. Advantageously, said copolymer of

PVM / MA has a molecular weight between 100 and 2,400 kDa, preferably between 200 and 2,000 kDa, more preferably between 180 and 250 kDa. This biodegradable polymer (PVM / MA) is particularly advantageous since it is widely used in pharmaceutical technology due to its low toxicity (LD ₅₀ = 8-9 g / kg orally) and excellent biocompatibility. In addition, it is easy to obtain, both for quantity and price. This biodegradable polymer (PVM / MA) can react with different hydrophilic substances, due to the presence of its anhydrous groups, without having to resort to the usual organic reagents (glutaraldehyde, carbodiimide derivatives, etc.) that possess an important toxicity. In an aqueous medium, the PVM / MA copolymer is insoluble, but its anhydride groups are hydrolyzed giving rise to carboxylic groups. The dissolution is slow and depends on the conditions in which it occurs. Due to the availability of functional groups in PVM / MA, covalent binding of molecules with nucleophilic groups, such as hydroxide or amino, takes place by simple incubation in an aqueous medium.

The nanoparticles of the invention comprise, in addition to the biodegradable polymer, thiamine or a derivative thereof. Thiamine, also called vitamin Bl, or 3 - [(4-amino-2-methyl-5-pyrimidinyl) methyl] -5- (2-hydroxyethyl) -4-methylthiazolium chloride, is a known product. However, it is also possible to use thiamine derivatives, being understood as such, compounds structurally related to thiamine, such as their salts, among which are their addition salts, for example, their acid addition salts. Illustrative, non-limiting examples of said thiamine addition salts include thiamine hydrochloride, thiamine monophosphate chloride dihydrate; thiamine pyrophosphate; oxythiamine chloride hydrochloride; piritiamine hydrobromide, etc., all of them known compounds. In a particular embodiment, the nanoparticles of the invention comprise thiamine or thiamine hydrochloride.

The thiamine, or the derivative thereof, can be found totally or partially coating the surface of the nanoparticles comprising the biodegradable polymer.

The ratio thiamine (or derivative thereof): biodegradable polymer, by weight, in the nanoparticle of the invention may vary within a wide range; however, in a particular embodiment, said weight ratio is comprised between

1:10 and 1: 500, preferably between 1:10 and 1: 100, more preferably around 1:40. In a specific embodiment, nanoparticles of the invention with a ratio of approximately 0.025 mg thiamine / mg biodegradable polymer provides a very efficient adhesion (bonding) capability.

The nanoparticles of the invention may have a surface charge (measured by the Z potential) that varies depending on the structure of the biodegradable polymer and on the proportion thereof with respect to the thiamine or derivative thereof. The contribution of the positive charge is attributed, among others, to the amino groups present in the thiamine or its derivative. Depending on the parameters mentioned, the magnitude of the surface charge of the nanoparticles of the invention can vary within a wide range. In a particular embodiment, nanoparticles of the invention have been obtained, based on PVM / MA copolymer and thiamine, with a Z potential of about -34.0 + 1.9 mV (Example 1, Table 1), while In another particular embodiment, nanoparticles of the invention (based on PVM / MA copolymer and thiamine) loaded with ovalbumin (OVA) have been obtained, with a Z potential of about -28.6 ± 6.2 mV (Example 3, Table 3).

The nanoparticles of the invention can be obtained by conventional methods known to those skilled in the art. In a particular embodiment, the nanoparticles of the invention can be obtained by incubating the previously formed biodegradable polymer nanoparticles with an aqueous solution of thiamine or a derivative thereof, which makes it possible to obtain mostly biodegradable polymer nanoparticles in which the thiamine, or its derivative, is attached to the surface of the nanoparticles. In a particular embodiment, the biodegradable polymer present in the nanoparticles of the invention is a PVM / MA copolymer, the preparation of which is described, for example, in WO 02/069938. In general, the nanoparticles can be obtained from the biodegradable polymer by a process comprising the dissolution of said biodegradable polymer in an organic solvent (eg, acetone, etc.) and subsequent desolvation after the addition of an appropriate solvent, for example, a solvent or a mixture of solvents miscible with the solution of the biodegradable polymer, such as an ethanol-water mixture, obtaining a suspension of nanoparticles from which the organic solvents are removed by conventional methods, for example, by evaporation under reduced pressure, and, then water is added, so an aqueous suspension of nanoparticles is obtained. The ratio, by volume, organic phase: hydroalcoholic solution (ethanol / water) can vary within a wide range; however, in a particular embodiment, said ratio is between 1: 1 and 1: 10 (v: v). After removal of the organic solvents, the biodegradable polymer nanoparticles are modified on their surface efficiently with thiamine or with a derivative thereof, by incubation, at room temperature, for an appropriate period of time. For this purpose, an aqueous solution of thiamine or a derivative thereof is added to said previously obtained suspension of nanoparticles. In a particular embodiment, pharmaceutical grade water is used. Both the concentration of the biodegradable polymer and that of thiamine, or its derivative, can vary within a wide range; however, in a particular embodiment, the concentration of the biodegradable polymer is between 0.001 and 10% w / v and that of thiamine, or between it, between 0.001 and 5% w / v. In general, the thiamine amino groups react with functional groups eventually present in the biodegradable polymer, which leads to the formation of bonds between the biodegradable polymer and thiamine. The association of thiamine with the nanoparticles of the biodegradable polymer is evident due to the significant decrease in the negative surface charge of the nanoparticles (see, for example, Table 1). In a particular embodiment, the thiamine amino groups react with the anhydride groups of the PVM / MA copolymer, a reaction that can easily occur by simply incubating the thiamine with the aqueous suspension of the nanoparticles, which leads to bond formation. amide between the PVM / MA copolymer and thiamine. The nanoparticles of the invention can be purified by conventional methods, for example, by centrifugation, ultracentrifugation, tangential filtration, or evaporation, including the use of vacuum.

Optionally, a crosslinking agent can be added to improve the stability of the nanoparticles of the invention, as described in WO 02/069938. Illustrative, non-limiting examples of crosslinking agents that may be used include diamine compounds, for example 1.3 diaminopropane, simple polysaccharides or saccharides, proteins and, in general, any molecule having functional groups. capable of reacting with the functional groups present in the biodegradable polymer, for example, anhydride groups present in the PVM / MA copolymer.

Also, if desired, the nanoparticles of the invention can be lyophilized by conventional methods. From a pharmacological point of view, it is important to be able to have nanoparticles in lyophilized form since this improves their stability during long-term storage and conservation, in addition to reducing the volume of the product to be handled. The nanoparticles of the invention can be lyophilized in the presence of a usual cryoprotectant agent such as glucose, sucrose, mannitol, trehalose, glycerol, lactose, sorbitol, polyvinyl pyrridone, etc., preferably, sucrose or mannitol; at a concentration within a wide range, preferably between 0.1% and 10% by weight.

The nanoparticles of the invention have a high ability to associate biologically active molecules, which makes them a drug transport system or presentation of very appropriate antigens and allergens. Therefore, in another aspect, the invention relates to a pharmaceutical composition comprising nanoparticles of the invention and at least one biologically active molecule.

In general, said biologically active molecule will be inside the nanoparticle of the invention; however, it could happen that some biologically active molecule was also attached to the surface of the nanoparticle although most of said molecules will be inside (e.g., encapsulated) of the nanoparticles of the invention.

The term "biologically active molecule", as used herein, refers to any substance used in the treatment, cure, prevention or diagnosis of a disease or that used to improve the physical and mental well-being of humans and animals. In general, said term includes both drugs and antigens and allergens. According to the present invention, the nanoparticles of the invention can incorporate one or more biologically active molecules regardless of the solubility characteristics thereof. The association capacity will therefore depend on the incorporated molecule, but, in general, said capacity is high for both hydrophilic molecules and molecules of marked hydrophobic character. In a particular embodiment, the pharmaceutical composition of the invention comprises nanoparticles of the invention containing one or more different drugs. Illustrative, non-limiting examples of such drugs include analgesic, anti-inflammatory, antitumor, neuroprotective, antiallergic, anti-asthmatic, antibiotic agents (eg, antibacterial, antifungal, antiviral, antiparasitic, etc.), pulmonary surfactants, etc.

In another particular embodiment, the pharmaceutical composition of the invention comprises nanoparticles of the invention containing one or more different antigens for vaccination purposes or one or more different allergens for immunotherapeutic purposes.

As used in this description, the term "antigen" refers to any substance capable of being recognized by the immune system of a subject and / or capable of inducing in a subject a humoral immune response or a cellular immune response that leads to the activation of B and / or T lymphocytes when introduced into a subject; by way of illustration, said term includes any immunogenic, native or recombinant product, obtained from a higher organism or from a microorganism, for example, a bacterium, a virus, a parasite, a protozoan, a fungus, etc., which contains one or more antigenic determinants, for example, structural components of said organisms; toxins, for example, exotoxins, etc. Virtually any antigen can be used in the preparation of nanoparticles of the invention loaded with antigen. By way of illustration, not limitation, the term "antigen" includes: "microbial" antigens, that is, microorganism antigens, including, but not limited to, viruses, bacteria, fungi and infectious parasites; said antigens include the intact microorganism as well as parts, fragments and derivatives thereof, either of natural or artificial origin, as well as synthetic or recombinant products that are identical or similar to the natural antigens of a microorganism and induce a specific immune response for that microorganism; in this sense, a compound is similar to a natural antigen of a microorganism if it induces an immune response (humoral and / or cellular) like that of the natural antigen of that microorganism; said antigens are used routinely by those skilled in the art; Y "tumor" antigens, that is, substances, for example, peptides, associated with a tumor or a cancer ("tumor marker"), which is capable of eliciting an immune response, in particular, when presented in the context of a molecule from CMH, eg, Her2 (breast cancer); GD2 (neuroblastoma); EGF-R (malignant glioblastoma); CEA (medullary thyroid cancer); CD52 (leukemia); human melanoma gplOO protein; melanoma-A / MART-1 human melanoma protein; tyrosinase; NA17-A nt protein; MAGE-3 protein; p53 protein; HPV16E7 protein; antigenic fragments of said antigens; etc. As used in this description, the term "allergen" refers to a substance to which a subject is sensitive and causes an immune reaction, for example, allergenic extracts of pollens, allergenic extracts of insects, allergenic extracts of food or products. food, components present in saliva, tweezers or stingers of insects that induce a sensitivity reaction in a subject, components present in plants that induce a sensitivity reaction in a subject, etc., for example, protein extracts of pollens, such as grass pollen, allergic extracts of perennial Lolium, allergic extracts of olea (olive), etc .; Protein extracts of insects, such as dust mites, etc .; allergenic extracts of food components, etc. Virtually any allergen can be used in the preparation of the nanoparticles loaded with allergen of the composition of the invention; however, in a particular embodiment, said allergen is ovalbumin (OVA), a protein widely used as an experimental allergenic model.

Illustrative, non-limiting examples of said biologically active molecules that may contain the nanoparticles of the invention include bacterial: cytoplasmic, periplasmic, cell envelope antigens (eg, inner membrane proteins, outer membrane proteins, lipopolysaccharides and mixed complexes, proteins associated to the cell wall, etc.), etc .; surface structure antigens (eg, fimbriae, glycocalyx, flagellar, etc.), including those of intracellular pathogens, such as Brucella sp., Salmonella sp., etc .; eukaryotic microorganism antigens, both soluble and superficial; viral antigens, for example, matrix, capsid, envelope, internal (including enzymatic), allergens of animal species (mites, etc.), of plants (grasses, etc.), etc. The chemical nature of the biologically active molecule can be very varied. In a particular embodiment, said biologically active molecule is a polysaccharide, a protein, a peptide or a lipid. In another particular embodiment, said biologically active molecule is a nucleic acid (eg, DNA, RNA, etc.), a nucleoside, a nucleotide, an oligonucleotide, a polynucleotide, etc.

The nanoparticles of the invention are used to modify the distribution of the associated biologically active molecule and / or of the conventional nanoparticles when administered by a route that gives access to some mucosa of the organism (including oral, rectal, nasal, vaginal or ocular). Examples of pharmaceutical compositions include any liquid composition (suspension or dispersion of the nanoparticles) for oral, oral, sublingual, topical, ocular, nasal, vaginal or parenteral administration; any composition in the form of gel, ointment, cream or balm for topical, ocular, nasal or vaginal administration; or any solid composition (tablets, capsules) for oral administration. In a particular embodiment, the pharmaceutical composition is administered orally. In another particular embodiment, said pharmaceutical composition is administered parenterally.

The pharmaceutical compositions described will comprise the excipients suitable for each formulation. For example, in the case of oral formulations in the form of tablets or capsules, binders, disintegrants, lubricants, fillers, enteric coating, etc. will be included if necessary. Oral solid formulations are prepared in conventional manner by mixing, dry or wet granulation and incorporating the nanoparticles of the invention. The pharmaceutical compositions may also be adapted for parenteral administration, in the form of, for example, sterile lyophilized solutions, suspensions or products, in the appropriate dosage form; in this case, said pharmaceutical compositions will include suitable excipients, such as buffers, surfactants, etc. In any case, the excipients will be chosen based on the pharmaceutical form of administration selected. A review of the different pharmaceutical forms of drug administration and their preparation can be found in the book "Galenica Pharmacy Treaty", by C. Faulí i Trillo, 10 Edition, 1993, Luzán 5, SA de Ediciones. The proportion of the biologically active molecule incorporated into the nanoparticle of the invention can vary within a wide range, for example, it can be up to 25% by weight with respect to the total weight of the nanoparticles. However, the appropriate proportion will depend in each case on the biologically active molecules incorporated.

The incorporation of the biologically active molecule into the nanoparticles of the invention can be done as described in WO 02/069938, by incorporation into the solution of the biodegradable polymer before the formation of nanoparticles, or subsequently added to the aqueous suspension of the nanoparticles already formed. For example, and depending on the nature of the biologically active molecule, one can proceed as follows: a) Hydrophobic biologically active molecules: addition in the organic phase (eg, acetone) and joint incubation / solubilization with the biodegradable polymer for a period variable time (up to 1 hour) under agitation (mechanical, magnetic or ultrasonic stirrer); and b) Biologically active bi-hydrophilic molecules: either by addition in the organic phase (eg, acetone) and joint incubation with the biodegradable polymer for a variable period of time (up to 1 hour) under stirring (mechanical, magnetic or ultrasonic agitator) until obtaining a fine suspension in the organic solvent [this procedure has been used successfully to encapsulate a model protein (ovalbumin, about 44 kDa protein); incorporation was efficient allowing high encapsulation of the model protein; or by addition in the aqueous phase to incubate with the preformed nanoparticles (this is the case used to encapsulate the fluorescent marker used in the examples that accompany this description, rhodamine B isothiocyanate).

As previously mentioned, the nanoparticles of the invention produce a stimulatory or potentiating effect of the immune response after administration to a subject and can therefore be used as an adjuvant in vaccines or in immunotherapy In fact, the nanoparticles of the invention have the ability to stimulate the two immune response pathways (ThI or Th2), so it can be used in vaccine or immunotherapeutic formulations. By way of illustration, for a vaccine formulation, it is generally required, depending on the pathogenic mechanisms of the organism from which the antigen (intracellular or extracellular, toxin dependent, scourge dependent, etc.), a stimulation of the ThI response ( intracellular, as in the case of Brucella, Salmonella, etc.) or Th2 response (extracellular, as in the case of Staphylococcus, Escherichia coli, enterotoxigenic bacteria, etc.). Also, by way of illustration, a tolerance induction is required for an immunotherapeutic formulation by the presence of the two types of response, that is, inducing ThI and Th2 responses in a balanced manner. In a particular embodiment of the invention, to stimulate an immune response, the formulations will contain nanoparticles of biodegradable polymer and thiamine, and encapsulated therein or totally or partially coating the surface thereof the antigen or the allergen.

Therefore, in another aspect, the invention relates to a vaccine or composition for immunotherapy comprising a therapeutically effective amount of the nanoparticles of the invention and an antigen or allergen, together with a pharmaceutically acceptable carrier or excipient. The antigen or allergen may be contained within said nanoparticles and / or at least partially covering the surface of said nanoparticles. These terms "antigen" and "allergen" have been previously defined.

The allergen or antigen present in the nanoparticles of the invention may be at least partially covering the surface of said nanoparticles and / or contained within said nanoparticles. In a particular embodiment, said allergen or antigen is coating all or part of the surface of said nanoparticles.

In a particular embodiment, said vaccine or composition for immunotherapy can be found in any pharmaceutical form of administration by any appropriate route, for example, oral, rectal, nasal, sublingual, etc. In a particular embodiment, said vaccine or composition for immunotherapy is in a pharmaceutical form of oral administration, while in another particular embodiment, said vaccine or composition for immunotherapy is in a form Pharmaceutical administration parenterally, for example, intramuscularly (im), subcutaneously (s.α), intravenously (iv), intraperitoneally (ip), intradermally (id), etc. A review of the different pharmaceutical forms of administering drugs, in general, and their methods of preparation can be found in the book "Tratado de Farmacia Galenica", C. Fauli i Trillo of, I Edition, 1993, Luzan 5, SA of Editions.

The dose of nanoparticles loaded with an antigen or with an allergen may vary within a wide range, for example, between about 0.01 and about 10 mg per kg of body weight, preferably, between 0.1 and 2 mg per kg of body weight.

In a particular embodiment, the nanoparticles of the invention do not incorporate the antigen or allergen, but these are empty and are administered in combination with vaccine or immunotherapeutic compositions containing the antigen or allergen, respectively, producing a stimulatory effect of the immune response. after administration of said vaccine or immunotherapeutic composition and empty nanoparticles. Therefore, a further aspect of the invention is a product comprising, separately, a) an antigen or an allergen; and b) a composition comprising said nanoparticles based on a biodegradable polymer and thiamine, as a composition that enhances the immune response against said antigen or allergen, as a combination for simultaneous or sequential administration to a subject, in the induction or stimulation of a response immune against said antigen or allergen in said subject.

The combined administration of said vaccine or immunotherapeutic composition and of the empty nanoparticles can be carried out simultaneously or sequentially, separated in time, in any order, that is, the vaccine or immunotherapeutic composition can be administered first and, subsequently, the empty nanoparticles or vice versa. Alternatively, said vaccine or immunotherapeutic composition and said empty nanoparticles can be administered simultaneously. In this case, the vaccine or immunotherapeutic composition and the empty nanoparticles can be administered in the same composition or in different compositions. The invention is described below by examples that are not limiting of the invention, but illustrative.

EXAMPLES The following examples describe the production of nanoparticles based on

PVM / MA and coated with thiamine that, optionally, contain an antigen and demonstrate the ability of said nanoparticles to specifically adhere to the gastrointestinal mucosa and, if necessary, act as an adjuvant in immunotherapy or vaccination.

General procedure for the production of thiamine coated nanoparticles

The general method of production of thiamine coated PVM / MA nanoparticles is a variant of the general procedure described above [Arbos et al., J. Control. Relay, 83 (2002) 321-330]. This process comprises dissolving said copolymer in acetone followed by the addition of ethanol. A similar volume of water was added to the resulting solution, so that nanoparticles formed instantly in the medium, under the appearance of a milky suspension. Then, the organic solvents (ethanol and acetone) were removed by evaporation under reduced pressure, the particles being in a stable aqueous suspension. The thiamine was incorporated into the aqueous phase that facilitates the desolvation of the polymer, leaving the reaction to act for a certain time. Depending on the time at which the drug, active molecule or label was added, different formulations were obtained with different arrangement thereof (see Examples 1 and 3). By way of illustration: A. To obtain formulations containing the compound (drug, active molecule or label) adhered to the surface of the nanoparticles [eg, a fluorescent label], incubation with the compound was carried out after evaporating the organic solvents (Example 1).

B. To obtain formulations containing the compound (drug, antigen, etc.) encapsulated inside the nanoparticles [eg ovalbumin (OVA)], the compound (drug or antigen) dispersed in acetone was incubated before adding ethanol and water (Example 3).

The next step consisted of the incubation between the formed nanoparticles and the thiamine. Eventually, after this incubation process, the crosslinking agent which, in this case, has been 1,3-diaminopropane (10 μg / mg polymer) can be added.

The purification of the nanoparticles was carried out by ultracentrifugation. Finally, the purified nanoparticles were lyophilized for long-term storage and preservation.

Characterization of the nanoparticles

The size and zeta potential of the nanoparticles was determined in a Zetamaster (Malvern Instruments / Optilas, Spain). The morphology of the nanoparticles was studied by scanning electron microscopy in a Zeiss DSM940 (Oberkochen, Germany). The amount of thiamine associated with the nanoparticles was quantified by high performance liquid chromatography (HPLC), according to the procedure described by [Batifoulier et al., J. Chromatogr. B Analyt. Technol Biomed Life Sci., 816 (2005) 67-72]. The analysis was carried out on a model 1100 series LC chromatograph (Agilent, Waldbornn, Germany) coupled to a diode-array ultraviolet (UV) detection system. The data was analyzed on a Hewlett-Packard computer using the Chem-Station G2171 program. For the separation of thiamine a Zobrax NH ₂ reverse phase column (4.6 mm x 150 mm; 5 μm; Agilent) heated to 40 ⁰ C was used. The mobile phase was formed by a mixture of phosphate regulatory solution (PBS) ) (pH = 6; 50 mM) and methanol (in proportion 80/20 by volume), and was pumped at a flow rate of 0.5 mL / min. Detection was performed at 254 nm.

For the analysis of the samples, 1 mL of the supernatants from the centrifugation of the nanoparticles was dosed in HPLC vials. Next, a 10 μL aliquot was injected into the HPLC column. Finally, the amount of thiamine associated with the nanoparticles was calculated as the difference between the initial amount of thiamine and the amount of thiamine quantified in the supernatants. The amount of rhodamine B isothiocyanate (RBITC) incorporated into the nanoparticles was determined by colorimetry at a wavelength of 540 nm (Labsystems iEMS Reader MF, Finland). This amount was estimated as the difference between the initial amount added and the amount quantified in the supernatants collected during the centrifugal purification step. For the calculations, a calibration curve between 5 and 100 μg / mL (r> 0.999) was used.

The release kinetics of RBITC from the nanoparticles was performed in Vivaspin ^® 100,000 MWCO dialysis tubes (VTVASPIN, Hannover, Germany). For this, 5 mg of nanoparticles were dispersed in 1 mL of simulated gastric medium (FGS) or simulated intestinal medium (FIS) [USP XXIII] at 37 ± 1 ° C. At certain times, the nanoparticle suspensions were centrifuged (5,000 xg, 15 minutes) and the amount of RBITC in the dialyzed liquids was quantified by colorimetry (λ = 540 nm).

The protein content [active ingredient (in this case ovalbumin)] encapsulated in the thiamine-coated nanoparticles was determined by testing with microbicinconinic acid (Micro BCA, Pierce, USA). For this, the nanoparticles were digested, at 37 ° C for 24 h, with a solution of sodium lauryl sulfate (3%) in 0.1 M NaOH. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) was used for corroborate the content in OVA. To do this, 5 mg of nanoparticles (OVA-T-NP) were dissolved in 2 mL of a mixture of dimethylformamide (DMF) and acetone (1: 1 v / v), at -20 ⁰ C for 24 h. The amount of OVA was estimated by calculating the average density of the band in the SDS-PAGE gel using the Micro Lnage ^{® program} (Version 4.0; Olympus Optical Co., USA). The calibration line was performed with OVA (0.25-2.5 μg protein / well).

Bioadhesion study

This study was carried out by applying a protocol described above [Arbós et al., Int. J. Pharm., 242 (2002) 129-136], in accordance with the regulations of the Committee responsible for the University of Navarra in line with European legislation on experimental animals (86/609 / EU). Briefly, an aqueous suspension with 10 mg of RBITC-labeled nanoparticles (approximately 45 mg particles / kg weight) was administered orally to male Wistar rats (average weight 225 g; Harían, Spain). The animals were sacrificed by cervical dislocation at different times (0.5, 1, 3 or 8 hours after administration). In each animal, the abdominal cavity was opened, the gastrointestinal tract removed, washed with saline and cut into six anatomical regions: stomach (Sto), small intestine (II, 12, 13 and 14) and blind (Ce). Each of the regions, after their logitudinal opening and washing with PBS (pH = 7.4; 0.15 M), were incubated in a solution of 3 M NaOH for digestion. Subsequently, 2 mL of methanol was added and, after stirring, centrifuged at 2,000 xg for 10 minutes. 1 mL aliquots of the different supernatants were diluted to 3 mL with purified water and analyzed in a spectrofluorimeter (X _6x 540 ran and λ _em 580 nm; GENIOS, TECAN, Groedig, Austria). Calibration lines were prepared by adding RBITC solutions in 3 N NaOH (0.5-10 μg / mL) to control tissue segments, which were subjected to the same extraction steps (r> 0.996).

On the other hand, a competition study was conducted to investigate the effect of free thiamine on the bioadhesive capacity of nanoparticles. For this, the animals received, simultaneously, a mixture of 10 mg of thiamine coated nanoparticles and 5 mg of free thiamine.

For each nanoparticle formulation (NP and T-NP) its bioadhesion curve was determined, from which the different bioadhesion parameters were estimated [Arbós et al., J. Control. Relay, 89 (2003) 19-30; Salman et al., J. Control. Relay, 106 (2005) 1-13]: Qmax, AUCadh, Tn _13x , MRT _to dh and K _to dh. Qmax, represents the maximum fraction adhered to the mucosa and allows to estimate the affinity of the nanoparticles for the biological substrate. K _adh represents the removal rate of the adhered fraction and was calculated with the help of the WinNonlin version 1.5 program (Scientific Consulting, Inc.). AUC _adh or area under the curve, represents the fraction adhered against time (expressed as a quantity of adherent marker with respect to time) and was evaluated by the trapezoid method up to I ₂ (the last sampling point), and allows quantify the intensity of the bioadhesive phenomenon. T _max represents the time at which maximum adhesion occurs. Finally, MRT _adh is the average residence time of the adhered fraction of nanoparticles and allows the relative duration of adhesive interactions to be evaluated, taking as a limit the last sampling point. The calculations were performed using the WinNonlin 1.5 program (Pharsight Corporation, USA).

Fluorescence microscopy studies The visualization of the distribution of thiamine coated nanoparticles was carried out by fluorescence microscopy. For this, the laboratory animals received an oral dose of 10 mg of nanoparticles labeled with RBITC. Two hours after administration, the animals were sacrificed and different portions of the small intestine were removed and washed with PBS. Said portions of approximately 0.5 cm in length were treated with Sakura Tissue-Tek Oct® Compound (Sakura, Holland) and frozen in liquid nitrogen. Different tissue samples were cut in sections of 5 μm in a cryostat (2800 Frigocut E, Germany), fixed to supports coated with poly-L-lysine (Sigma, Spain) and stored at - 20 ⁰ C before viewing by microscopy of fluorescence.

Immunization and sampling protocols

The protocols with animals were applied in accordance with European legislation (86/609 / EU). BALB / c mice (20 ± 1 g) (Harían, Spain) were divided into 6 different groups of 10 animals each. The administration strategy was based on the single administration of a subcutaneous or oral dose. In the case of oral immunization, each animal received a volume of 200 μL containing 100 μg of ovalbumin, free or encapsulated in thiamine coated nanoparticles (OVA-T-NP) or in conventional nanoparticles (OVA-NP). For groups of animals immunized subcutaneously (s.c), the protein dose was 20 μg OVA in 50 μL PBS.

Blood samples were collected from the retroorbital plexus on days 0, 7, 14, 28 and 42 after immunization. The samples were centrifuged (3,000 xg, 10 minutes) and kept as "pool" of each group (10 mice). Finally, each pool was diluted with PBS (1:10) and stored at -80 ⁰ C until analysis. Stool samples and their analysis were performed following a protocol described previously [Maciel et al., Braz J Med Biol Res 37 (2004) 817-826]. In this case, the feces of each group of animals were collected on days 1, 7, 14, 28 and 42 after immunization.

Determination of anti-OVA antibodies in serum and feces by ELISA

Serum anti-OVA antibodies were analyzed by ELISA with anti-IgG _t and anti-IgG _2a conjugates (Sigma-Aldrich Chemie, Germany). Briefly, 96 well plates (EB, Thermo Labsystems, Vantaa, Finland) were used to perform this test, where 1 μg of ovalbumin dissolved in carbonate-bicarbonate regulatory solution (0.05 M, pH 9.6) was fixed ) at 4 ° C for 24 hours. Subsequently, the plates were blocked by incubation for 1 h at 37 ° C with 1% bovine serum albumin in a solution of 0.05% PBS-Tween 20 (PBS-T). The plates were washed and the sera were added in serial dilutions starting from 1: 40, and incubated at 37 ⁰ C for 4 hours. Subsequently, 5 washes of 1 minute were performed. Then, the peroxidase conjugate was incubated for 2 hours, another 5 washes of 1 minute were performed, the substrate [ABTS (2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) was added) ( Sigma-Aldrich Chemie, Germany)] and the reading was carried out at 405 nm in a plate reader (iEMS Reader MF, Labsystems, Finland) after 30 minutes of incubation at room temperature.The final titers were determined by dilution of the sample until obtaining an average of OD = 0.2 with respect to that obtained with non-immunized mouse serum.

Stool anti-OVA antibodies were analyzed by ELISA with anti-IgA conjugates. In this case, after coating the plates with OVA, they were blocked by adding, and incubating for 1 h at room temperature, 200 μL of a PBS-T solution containing 3% skim milk. The stool extract was added to two plates and serial dilutions were made in PBS-T. Said plates were incubated for 4 h at 37 ° C. Finally, after washing, the wells were incubated with the peroxidase-labeled anti-IgA conjugate (Nordic rmmunological Labs, The Netherlands). Statistic analysis

Bioadhesion data and physicochemical characteristics were compared using the Mann-Whitney U-test non-parametric test and the t-test.

Student, respectively. P values <0.05 were considered significant. All calculations were performed using the SPSS ^® statistics program (SPSS ^®

10, Microsoft, USA).

EXAMPLE 1

Preparation and characterization of nanoparticles based on the copolymer of methyl vinyl ether and maleic anhydride (PVM / MA)

The process described below is valid for the preparation of colloidal pharmaceutical forms of nanoparticle type coated with thiamine that can be used to encapsulate or associate molecules of hydrophilic nature.

1.1. Preparation of control nanoparticles (NP)

100 mg of the copolymer of methyl vinyl ether and maleic anhydride (PVM / MA) [Gantrez® AN 119] were dissolved in 5 mL of acetone. Subsequently, on this phase and under magnetic stirring, 10 mL of ethanol and 10 mL of deionized water were added. The resulting mixture was allowed to homogenize for 5 minutes. Then, the nanoparticle suspension was evaporated under reduced pressure until both organic solvents were removed and the final volume was adjusted with water to 10 mL. To the resulting suspension was added 100 µl of a 1% v / v solution of 1,3-diaminopropane, subjecting the whole to magnetic stirring for 5 minutes, and 1.25 mg of rhodamine B-isothiocyanate (RBITC). The resulting suspension was subjected to purification by ultracentrifugation (20 minutes at 27,000 xg). The supernatants were removed and the residue was resuspended in water or in a 5% aqueous sucrose solution. Finally, the resulting nanoparticle suspension was lyophilized, keeping all its properties intact. 1.2. Preparation of thiamine coated nanoparticles (T-NP)

100 mg of the copolymer of methyl vinyl ether and maleic anhydride (PVM / MA) [Gantrez® AN 119] were dissolved in 5 mL of acetone. Subsequently, on this phase and under magnetic stirring, 10 mL of ethanol and 10 mL of deionized water containing 2.5 mg of thiamine were added. The resulting mixture was allowed to homogenize and the nanoparticle suspension was evaporated under reduced pressure until both organic solvents were removed, and the final volume was adjusted with water to 10 mL. The resulting suspension was subjected to magnetic stirring for 1 hour at room temperature. Subsequently, 100 µl of a 1% v / v solution of 1,3-diaminopropane was added, subjecting the whole to magnetic stirring for 5 minutes, and 1.25 mg of rhodamine B-isothiocyanate (RBITC). The resulting suspension was subjected to purification by ultracentrifugation (20 minutes at 27,000 x g). The supernatants were removed and the residue was resuspended in water or in a 5% aqueous sucrose solution. Finally, the resulting nanoparticle suspension was lyophilized, keeping all its properties intact.

1.3. Physicochemical characteristics of nanoparticles

Table 1 summarizes the main physicochemical characteristics of the nanoparticles obtained. The control nanoparticles (NP) show a size close to 200 nm with a negative surface charge of -51 mV. On the other hand, the thiamine-coated nanoparticles (T-NP) were significantly higher (close to 400 nm) and showed a significantly less negative zeta potential. These nanoparticles (T-NP) showed an associated amount of thiamine of 15 μg / mg nanoparticles (Table 1). Finally, it is noteworthy that the presence of thiamine has no effect on the manufacturing performance of the nanoparticles. Similarly, small differences were observed when comparing the association of RBITC for both formulations. Table 1

Physicochemical characteristics of Gantrez ^® AN nanoparticles

(mean ± SD, n = 6)

^a Performance: Percentage of polymer transformed into nanoparticles. * P <0.05 versus control nanoparticles (t-Student test).

The thiamine-coated nanoparticles showed a very homogeneous population of spherical particles (Figure 1).

EXAMPLE 2 Bioadhesive characteristics of nanoparticles and distribution in the gastrointestinal tract

2.1. In vitro release studies of RBITC from nanoparticles

In vivo studies to determine the bioadhesive capacity of the different formulations were performed using RBITC labeled nanoparticles. First, to ensure the stability of the association of the fluorescent marker to the nanoparticles in the conditions of the gastrointestinal tract, gastric and intestinal (FIS) simulated gastric (FGS) release studies were performed. Figure 2 shows the results obtained.

In all cases, it was found that the percentage of RBITC released after 8 hours of incubation was always less than 5% of the amount associated with the nanoparticles. Therefore, it can be assumed that the fluorescence intensity and the spots obtained during the visualization of the images corresponded to the fluorescent marker associated with the nanoparticles. 2.2. Distribution of the adhered fraction of nanoparticles in the gastrointestinal tract of laboratory animals

Figure 3 shows the distribution in the gastrointestinal tract of the adhered fractions of thiamine-coated nanoparticles (T-NP) (Figure 3 a) or control nanoparticles (NP) (Figure 3b). It can be seen that, within 30 minutes of the administration of the nanoparticle formulations, both types (NP and T-NP) showed the same ability to adhere to the mucosa of the gastrointestinal tract, mainly in the stomach area and the upper regions of the small intestine (duodenum). However, 1 h after administration of the nanoparticles, the thiamine-coated nanoparticles (T-NP) showed significantly greater bioadhesive capacity and a more homogeneous distribution than the control nanoparticles (NP). The amount of thiamine-coated nanoparticles (T-NP) found adhered to the mucosa was approximately 45% of the administered dose (Figure 3). At 3 hours after administration, the thiamine-coated nanoparticles (T-NP) showed maximum adhesion (65% of the administered dose) and an important tropism for the animal's ileum (portions 13 and 14 in Figure 3 to).

On the other hand, when the mixture of thiamine-coated nanoparticles (T-NP) and free vitamin (thiamine) was administered to laboratory animals, the ability of the nanoparticles to develop bioadhesive interactions in the gastrointestinal tract was strongly inhibited. (Figure 3c). However, the distribution in the tract and the mucosa-adhered fraction of these thiamine-coated nanoparticles (T-NP) was similar to the profile observed for control nanoparticles (NP). This result clearly indicates that the adhesion and tropism of thiamine-coated nanoparticles (T-NP) is driven by the presence of thiamine on its surface.

2.3. Bioadhesion parameters and curves

Bioadhesion curves were obtained by representing the total amount of nanoparticles adhered to the gastrointestinal tract as a function of time after administration (Figure 4). The comparison of bioadhesion profiles for the different formulations allows us to observe that the control nanoparticles (NP) show a maximum of bioadhesion 30 minutes after their administration [Figure 4, (?)]. Subsequently, the amount of control nanoparticles (NP) attached is falling over time.

In contrast, for thiamine coated nanoparticles (T-NP), the maximum adhesion is observed 3 hours after administration. In addition, this maximum represents an amount close to 65% of the administered dose [Figure 4, (¡)]. From these curves, the bioadhesion parameters that allow comparing the adhesive potential of the different nanoparticles were calculated. The parameters are listed in Table 2.

First, it is worth noting that thiamine-coated nanoparticles (T-NP) are characterized by an AUC _at dh (parameter that measures the intensity of bioadhesive interactions) 3 times higher than that observed for control nanoparticles (NP) . Likewise, the maximum adhesion (expressed as Q _max ) of the T-NPs was also significantly higher than for the NP (p <0.01). In contrast, the adhered fraction of T-NP showed a significantly higher elimination rate (Kadh) than for NP (p <0.05) and an average residence time

(MRT _adh ) of approximately 3.12 hours. Particularly interesting was the fact that the co-administration of thiamine (T-NP) and vitamin coated nanoparticles

(thiamine) free, removed the bioadhesive properties of these nanoparticles (T-NP)

[Figure 4, (?)]. Thus, under these conditions, the bioadhesion parameters studied of T-NP were similar to those calculated for control nanoparticles (NP) (p <0.05).

All these results are consistent with previous studies that have described the presence of thiamine receptors along the small intestine of rats and humans [Casirola et al., J. Physiol. (Lond), 398 (1988) 329-339; Laforenza et al., J. Membr. Biol. 161 (1998) 151-162]. It has also been described that the absorption of thiamine (in laboratory and human animals) is inhibited by the administration of structural analogs of thiamine, such as amprolium, oxythiamine and piritiamine [Laforenza et al.,. J. Membr. Biol. 161 (1998) 151-162; Dudeja et al., Am. J. Physiol. CeIl. Physiol., 281 (2001) C786-C792]. Table 2 Bioadhesion parameters for the different nanoarticle formulations

The results are expressed as mean ± SD (n = 3)

TNP-COM represents the values obtained when the thiamine coated nanoparticles (T-NP) were co-administered with 5 mg of the free vitamin

Qmax (mg): Maximum amount of nanoparticles attached to the mucosa

AUC _a dh (mg.h): Area under the bioadhesion curve

K _a dh (h ^"1 ): Speed of elimination of the adhered fraction

MRT _a dh (h): Average residence time of the adhered fraction of nanoparticles

** P <0.01 vs control nanoparticles (NP and NPT-COM) (Man-Whitney U-test).

2.4. Visualization of the distribution of the nanoparticles attached to the intestinal mucosa The visualization of the nanoparticles labeled with RBITC in the gastrointestinal mucosa was observed by fluorescence microscopy. Figure 5 shows several photographs that allow us to observe the distribution of nanoparticles in ileum samples, 2 hours after oral administration of 10 mg of nanoparticles in rats. Control nanoparticles (NP) were mainly detected in the outer layer of the ileum (mucus that covers the mucosa), confirming the low affinity of these transporters for the intestinal mucosa (Figure 5b). In contrast, thiamine-coated nanoparticles (T-NP) were widely distributed and showed a very large affinity for the micro villi of the intestine (Figure 5a). On the other hand, said T-NP showed an important tropism to adhere to Peyer's plates and to be internalized and / or captured by that lymphoid tissue (Figure 5c). This capture was not observed for these NPs (Figure 5d). EXAMPLE 3 Preparation of nanoparticles containing a model antigen

The adjuvant ability of thiamine-coated nanoparticles (T-NP) was studied using ovalbumin (OVA) as an antigen model.

3.1. Preparation of control nanoparticles with encapsulated ovalbumin (OVA-NP)

5 mg of OVA was dispersed in 2 mL of water with the help of ultrasound (Microson ™) or in an ultrasonic bath for 1 minute under cooling. This suspension was added to a solution of 100 mg of the copolymer of methyl vinyl ether and maleic anhydride (PVM / MA) [Gantrez® AN 119] in 3 mL of acetone. Subsequently, on this phase and under magnetic stirring, 10 mL of ethanol and 10 mL of deionized water were added. The resulting mixture was allowed to homogenize for 5 minutes. Then, the nanoparticle suspension was evaporated under reduced pressure (Büchi R-144, Switzerland) until both organic solvents were removed and the final volume was adjusted with water to 10 mL. The nanoparticles obtained have encapsulated OVA (OVA-NP). To the resulting suspension 100 µL of a 1% v / v solution of 1,3-diaminopropane was added, subjecting the whole to magnetic stirring for 5 minutes. The resulting suspension was subjected to purification by ultracentrifugation (20 minutes at 27,000 x g). The supernatants were removed and the residue was resuspended in water or in a 5% aqueous sucrose solution. Finally, the resulting nanoparticle suspension was lyophilized, thereby keeping all its properties intact.

3.2. Preparation of thiamine coated nanoparticles with encapsulated ovalbumin (OVA-T-NP)

5 mg of OVA was dispersed in 2 mL of water with the help of ultrasound

(Microson ™) or in an ultrasonic bath for 1 minute under cooling. This suspension was added to a solution of 100 mg of the methyl vinyl ether copolymer and maleic anhydride (PVM / MA) [Gantrez® AN 119] in 3 mL of acetone. Subsequently, on this phase and under magnetic stirring, 10 mL of ethanol and 10 mL of deionized water containing 2.5 mg of thiamine were added. The resulting mixture was allowed to homogenize and the nanoparticle suspension was evaporated under reduced pressure (Büchi R-144, Switzerland) until both organic solvents were removed and the final volume was adjusted with water to 10 mL. The resulting suspension was subjected to magnetic stirring for 1 hour at room temperature and purified by centrifugation at 27,000 xg for 20 minutes, collecting the supernatants to quantify the thiamine bound to the nanoparticles. Subsequently, 100 µL of a 1% v / v solution of 1,3-diaminopropane was added, subjecting the whole to magnetic stirring for 5 minutes. The resulting suspension was subjected to purification by ultracentrifugation (20 minutes at 27,000 xg). The supernatants were removed and the residue was resuspended in water or in a 5% aqueous sucrose solution. Finally, the resulting nanoparticle suspension was lyophilized, thereby keeping all its properties intact.

3.3. Physicochemical characteristics of nanoparticles

Table 3 summarizes the main physicochemical characteristics of the resulting nanoparticles (OVA-NP and OVA-T-NP). The OVA-T-NP formulation showed a significantly larger size (412 nm) than the control nanoparticles (280 nm). On the other hand, the presence of OVA slightly affects the amount of thiamine attached to the surface of the nanoparticles. Thus, the presence of this protein (OVA) decreased by 20% the amount of vitamin (thiamine) bound (12 μg / mg vs 15 μg / mg). The amount of encapsulated OVA was determined, in this case, by densitometry of the bands of this protein in the SDS-PAGE gel, using the Microlmage® Version 4 program. The amount of OVA was, for OVA-NP and OVA-T - NP, of 12.1 ± 1.4 and 11.6 ± 2.3 μg / mg nanoparticles, respectively. Table 3

Physicochemical characteristics of Gantrez ^® AN nanoparticles containing ovalbumin as an antigen model (mean ± SD; n = 6)

EXAMPLE 4

Study of the adjuvant capacity of nanoparticles that encapsulate ovalbumin

Figure 6 shows the titers of IgG2a and IgGl after oral or subcutaneous immunization of Balb / C mice with a single dose of the different treatments (OVA-NP, OVA-T-NP and free protein). Table 4 summarizes the values of the areas under the curves of the immune response.

In subcutaneous immunization (sc), administration of OVA-T-NP induced the production of anti-OVA antibody levels of type Th2 (IgGl) similar to those induced by the OVA-NP formulation. However, the IgG2a titers (corresponding to a ThI response) were at least 2 times higher for OVA-T-NP than for OVA-NP. All this demonstrates that with thiamine-coated nanoparticles (T-NP), more balanced levels of ThI and Th2 profiles can be achieved, since their level-inducing activity of IgG2a (ThI) levels is much higher than that induced by control nanoparticles (NP ) not coated with this vitamin (thiamine). In all cases, the levels of anti-OVA antibodies generated by both formulations (OVA-NP and OVA-T-NP) were significantly higher than when immunized with free protein (OVA). Table 4

Values of areas under curves (AUCs) representing the different immune response profiles expressed in Figure 6.

AUCs are expressed as arbitrary units (title x time)

After oral administration, the OVA-NP nanoparticles also showed a predominantly Th2 type profile (Figure 6). The oral ThI response was very low (AUC of 1.5; AUQ _h2 is 7 times higher than AUCa ₁₁ , see Table 4) and, at the same time, the antibody response induced by oral administration was lower than that described after subcutaneous administration.

On the contrary, oral immunization with OVA-T-NP induced ThI and Th2 responses superior to those obtained with OVA-NP. In addition, both responses were of the same order (Table 4). On the other hand, while for the OVA-NP nanoparticles the immune response via s.c. It was significantly more intense than orally, for the OVA-T-NP nanoparticles the induction of anti-OVA antibodies by the s.c. and oral were quite similar, especially the ThI response. These results clearly show the ability of thiamine-coated nanoparticles (T-NP) to increase and enhance ThI type cellular responses. This observation has great value when obtained in an animal model predisposed to the development of Th2 responses [Gieni et al., Int. Hnmunol., 8 (1996) 1511-1520].

These results are consistent with previous ones where it was shown that the presence of microparticles with OVA in the distal areas of the small intestine promoted the induction of ThI-type responses [Cronkhite and Michael, Vaccine, 22 (2004) 2106-2115]. This potentiation of the ThI response may be related to the high tropism of the T-NP nanoparticles by the regions. distal of the small intestine, as well as its capture by the Peyer plates rich in antigen presenting cells (Figures 3-5). In fact, it has recently been established that it is much easier to induce the production of antibodies of the IgG2a type in the distal portion of the gastrointestinal tract where, it seems, there is a greater number of dendritic cells that promote cellular or ThI-type responses [Peng et al., J. Immunol., 176, (2006) 3330-3341; Hattori et al., Biochem. Biophys Res. Commun., 317 (2004) 992-999; Shi et al., Tumori., 91 (2005) 531-538].

Figure 7 shows the evolution of anti-OVA antibodies of the IgA type in the feces of mice immunized by the s.c. or oral In both cases, immunization with nanoparticles, regardless of the route of administration, induced the production of high levels of IgA. However, thiamine-coated nanoparticles (T-NP) induced the secretion of significantly higher levels of intestinal IgA than conventional nanoparticles. Orally, this difference was 64 times greater for the OVA-T-NP nanoparticles than for the OVA-NP nanoparticles. This phenomenon may be related to the effective capture of the T-NP nanoparticles by the Peyer plates of the gastrointestinal tract and their passage to the lymphocytes responsible for the synthesis and secretion at the level of IgA mucous membranes.

In conclusion, thiamine-coated nanoparticles (T-NP) have shown a particular ability to develop adhesive interactions in the gastro-intestinal mucosa. This ability may be interesting to increase the oral bioavailability of numerous drugs. On the other hand, as vaccination adjuvants, these polymeric transporters are capable of reaching Peyer's plates and potentiating a high induction of antibodies against the transported antigen. In addition, the response that is generated is humoral (Th2) and cellular (ThI), which may be of interest for vaccination against numerous pathogens and for immunotherapy for the treatment of allergies.

Claims

1. Nanoparticles comprising a biodegradable polymer and thiamine or a derivative thereof.

2. Nanoparticles according to claim 1, wherein said biodegradable polymer is a copolymer of methyl vinyl ether and maleic anhydride (PVM / MA).

3. Nanoparticles according to any one of claims 1 or 2, wherein the copolymer has a molecular weight between 100 and 2,400 kDa, preferably between 200 and 2,000 kDa, more preferably between 180 and 250 ka.

4. Nanoparticles according to any one of claims 1 to 3, comprising thiamine or thiamine hydrochloride.

5. Nanoparticles according to any one of claims 1 to 4, wherein the thiamine or its derivative is completely or partially coating the surface of the nanoparticles.

6. Nanoparticles according to any one of claims 1 to 5, wherein the weight ratio between the thiamine, or derivative thereof, and the biodegradable polymer is between 1:10 and 1: 500, preferably between 1:10 and 1: 100, more preferably around 1: 40.

7. Nanoparticles according to any of claims 1 to 6, wherein said nanoparticles are crosslinked.

8. Pharmaceutical composition comprising nanoparticles according to any one of claims 1 to 7 and a biologically active molecule capable of preventing, alleviating or curing a disease.

9. Composition according to claim 8, comprising as a biologically active molecule a protein or a peptide.

10. Composition according to claim 8, which comprises as a biologically active molecule a compound selected from the group consisting of a nucleic acid, a nucleoside, a nucleotide, an oligonucleotide, a polynucleotide and mixtures thereof.

11. Composition according to claim 8, comprising as an biologically active molecule an antitumor agent or a tumor antigen.

12. Composition according to claim 8, comprising as a biologically active molecule a protector of the central nervous system or a glucocorticoid.

13. Composition according to claim 8, comprising as a biologically active molecule an antigen for vaccination or an allergen for immunotherapy.

14. Pharmaceutical composition according to any of claims 8 to 12, for oral administration.

15. Pharmaceutical composition according to any of claims 8 to 12, for parenteral administration.

16. Pharmaceutical composition according to any of claims 8 to 12, for administration by a route that gives access to any mucosa of the organism, preferably the rectal, ophthalmic, vaginal, nasal, pulmonary or sublingual route.

17. Use of the nanoparticles according to any of claims 1 to 7 in the manufacture of a medicament.

18. A lyophilisate comprising the nanoparticles according to any one of claims 1 to 7.

19. A vaccine or composition for immunotherapy comprising a therapeutically effective amount of the nanoparticles according to claims 1 to 7 and an antigen or allergen, together with a pharmaceutically acceptable carrier or excipient.

20. Vaccine according to claim 19, wherein the antigen or allergen is contained within said nanoparticles and / or at least partially covering the surface of said nanoparticles.

21. Vaccine according to any of claims 19 or 20, wherein said antigen comprises an immunogenic extract from an organism.

22. Vaccine according to any of claims 19 or 20, wherein said allergen comprises an allergenic pollen extract, an allergenic insect extract or an extract of an allergenic food product.

23. A product comprising, separately, a) an antigen or an allergen; and b) a composition comprising nanoparticles based on a biodegradable polymer and thiamine according to claims 1 to 7, as a composition enhancer of the immune response against said antigen or allergen, as a combination for simultaneous or sequential administration to a subject, in the induction or stimulation of an immune response against said antigen or allergen in said subject.

24. Use of nanoparticles according to any one of claims 1 to 7 in the preparation of a pharmaceutical composition for the selective stimulation of the ThI immune response, or in the manufacture of a pharmaceutical composition for the selective stimulation of the Th2 immune response, or in the manufacture of a pharmaceutical composition for the balanced stimulation of ThI and Th2 immune responses.

25. Process for the production of nanoparticles according to any one of claims 1 to 7 comprising: a) desolvation of an organic solution comprising a biodegradable polymer, with a hydroalcoholic solution to form the nanoparticles; b) simultaneous incubation of the biodegradable polymer nanoparticles formed in step a) and the thiamine in an aqueous solution; and c) elimination of organic solvents obtaining an aqueous suspension of nanoparticles.

26. The method of claim 25, wherein the concentration of the biodegradable polymer is between 0.001 and 10% w / v and that of thiamine between 0.001 and 5% w / v.

27. A method according to any of claims 25 and 26, comprising additional purification steps.

28. A method according to any of claims 25 to 27, further comprising incorporating a biologically active molecule by adding it in the organic phase where the biodegradable polymer is dissolved, prior to its desolvation.

29. A method according to any of claims 25 to 27, further comprising incorporating a biologically active molecule by adding it to the aqueous suspension of already formed nanoparticles.

30. A method according to any of claims 25 to 29, comprising an additional lyophilization step, optionally in the presence of a cryoprotectant, preferably sucrose or mannitol.