ES2286951B1 - BIOADHESIVE NANOPARTICLES FOR THE ADMINISTRATION OF BIOLOGICALLY ACTIVE MOLECULES. - Google Patents

Abstract

The invention relates to nanoparticles for the administration of biologically active molecules, which comprise a biodegradable polymer, preferably the copolymer of methyl vinyl ether and maleic anhydride (PVM / MA), and thiamine. These nanoparticles are easy to produce, and give excellent characteristics of bioadhesion, size and zeta potential that makes them suitable for the administration of active molecules. The procedure for obtaining the nanoparticles is performed by simple incubation for a short period of time with the preformed nanoparticles in an aqueous medium. The biologically active molecule can be incorporated during the nanoparticle formation process by desolvation or after thiamine modification. Orally, these nanoparticles develop bioadhesive interactions with the gastrointestinal mucosa, including lymphoid tissue associated with mucous membranes (Peyer's plaques), which is useful for increasing the oral bioavailability of various drugs or for inducing immune responses at the mucosal level. Thus, when the nanoparticles contain an antigen, inside the matrix of the nanoparticles, the resulting mixture is capable of inducing strong immune responses against the encapsulated antigen. This stimulating effect of the immune response is useful for vaccination and immunotherapy.

Description

Bioadhesive nanoparticles for administration of biologically active molecules.

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Field of the Invention

The invention relates to nanoparticles bioadhesive, based on a biodegradable polymer and thiamine, manufacturing procedures, formulations containing them and its applications

Background of the invention

In recent years nanoparticles biodegradable polymers have been proposed as new systems transporters of biologically active drugs or molecules. In In the case of the administration of drugs through mucous membranes, most important features offered to the molecule Biologically active they incorporate are: (i) protection against a possible physical-chemical degradation and / or enzymatic (increased half-life in the body); (ii) controlled release of the incorporated drug (sustained effects which allow reducing the number of shots); (iii) interaction / adhesion with the surface of the mucosa where it has place the absorption or the action of the drug (increase of its bioavailability); and (iv) in the case of vaccination, they facilitate presentation of the incorporated antigen to the presenting cells of antigen.

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 form pharmaceutical administration, and (ii) of the conditions physiological present in the tract gastro-intestinal, such as the presence of proteolytic enzymes, peristaltic movements and the Presystemic metabolism The use of polymeric nanoparticles can  Be a good strategy to overcome some of these obstacles. In principle, these conveyors have a large area specific with what your interaction with the biological support (the gastrointestinal mucosa) is facilitated. Similarly, the control of the release of the drug allows the effect to be prolonged over time of molecules with low biological half-lives. By On the other hand, nanoparticles can be captured by cells Peyer's plaques and lymphoid tissue follicles. This phenomenon allows to direct the drug towards the lymphatic route and, in the case of vaccines, facilitate its antigen presentation. Without However, conventional polymer-based nanoparticles biodegradable present some important disadvantages with regarding its use orally: (i) some instability in gastrointestinal fluids, (ii) a low degree of absorption intestinal and (iii) a non-specific tropism or adhesion in the mucosa  gastrointestinal.

A possible strategy to minimize these drawbacks associated with the use of conventional nanoparticles, It is based on the use of nanoparticles coated with certain ligands with specificity for certain receptors of the mucosa. The main ligands proposed for preparation of these conjugates Nanoparticle-ligand have been, so far: (i) antibodies; (ii) adhesins, invasins or other proteins bacterial; (iii) carbohydrates; (iv) vitamin B12; Y lectins Among all these types of ligands, lectins They seem to be the most versatile ligands. These molecules are proteins or glycoproteins, of non-immunogenic origin, capable of specifically recognize 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. The Thiamine (or vitamin B1) is essential for the normal development of Cellular functions and growth. Mammals can't synthesize this vitamin and, therefore, must be obtained via intestinal absorption from processed foods or after synthesis by microflora of the large intestine. The process of Thiamine absorption is quite complicated; although it has proposed a mechanism based on the existence of a receiver (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 of majority form in the apical membrane of the "edge in brush".

The use of thiamine as a nanoparticle ligand It has been investigated in recent years. Thus, it has been described that the Thiamine can be a good ligand to facilitate binding or association of nanoparticles to the blood brain barrier and to interact with breast tumor cells. However, until the date is not known of the use of thiamine as ligand to direct nanoparticles intended for administration oral biologically active molecules, including their use as adjuvants for vaccination and immunotherapy.

Use of adjuvants in vaccination

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The use of particulate adjuvants in the form of Emulsions, microparticles, ISCOMS or liposomes have been evaluated previously by various research groups [Singh and O'Hagan, 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 It can be approached fundamentally in three different ways: (i) avoiding all contact with the allergen; (ii) using drugs antihistamines, and (iii) by immunotherapy. Taking into account that the first two measures are sometimes not applicable, the Immunotherapy would be the most appropriate method of control.

Allergen-specific immunotherapy has been defined as the 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 Th1 response over the Th2 response, which will cause allergic symptoms to be inhibited. This modulation towards Th1 is also applicable in other processes, such as control by vaccination against bacterial intracellular parasites (eg, Brucella or Salmonella ).

Polyvinyl methyl ether and maleic anhydride (PVM / MA) copolymer nanoparticles have been described [WO02 /
069938], optionally pegylated [ES2246694], useful as drug vehicles. 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, for example, nanoparticles, as adjuvants in immunotherapy or vaccines for the administration of antigens and / or allergens, the 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 their use.  in immunization or oral immunotherapy without having to use very high doses of allergen or antigen. How is it known, despite its potential advantages, oral immunization for therapeutic or prophylactic purposes it has to deal with different obstacles, since the dose of active substance immunogenic or allergenic required for a clinical effect beneficial is extremely large due to a loss of immunogen potency. So, due to the, in general, little stability of the allergen or antigen in the tract gastrointestinal (pH conditions and presence of enzymes hydrolytic), the doses should always be much higher (up to 200 times) to those normally used subcutaneously. Further, the gastrointestinal mucosa acts as a very little barrier permeable to the absorption of these macromolecules.

Compendium of the invention

The object of the present invention is provide nanoparticles that solve problems mentioned above, that is, that can be administered orally and be stable and specific, have good bioadhesive characteristics to interact with the mucous membranes, that are capable of transporting a large group of molecules active and release the active molecule in a controlled manner. How additional feature is intended that said nanoparticles act as adjuvants in vaccines and immunotherapy enhancing the immune response of antigens or allergens.

Now it has been found, surprisingly, that these problems can be solved by nanoparticles formed by a biodegradable polymer and thiamine. In particular, it has found that nanoparticles that comprise or consist 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 and size characteristics, which makes them suitable for administration through different routes of biologically active compounds or molecules. It also has could show that the nanoparticles provided by this invention that optionally contain a biologically molecule active, 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 is relates to nanoparticles that comprise a polymer biodegradable and thiamine or its derivatives. In one embodiment in particular, said biodegradable polymer is a methyl copolymer vinyl ether and maleic anhydride (PVM / MA). In another embodiment In particular, thiamine is completely or partially the surface of the nanoparticles that comprise said biodegradable polymer. In another particular embodiment, the weight ratio between thiamine and 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 molecules biologically active In this sense, said nanoparticles they 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 (e.g., DNA, RNA, etc.), nucleosides, nucleotides, oligonucleotides, polynucleotides, etc. That molecule biologically active can be a drug or compound with activity therapeutic or diagnostic, e.g., an antitumor agent, a protector of the central nervous system, a glucocorticoid, etc., an antigen for vaccination or an allergen for immunotherapy, between others. Therefore, the nanoparticles provided by this invention can be used in the preparation of compositions Pharmaceuticals

In another aspect, the invention relates to a pharmaceutical composition comprising said nanoparticles previously described and a biologically active molecule, in where said biologically active molecule is a molecule capable of prevent, relieve or cure a disease or a molecule with diagnostic application, together with a vehicle or excipient pharmaceutically acceptable. In a particular embodiment, said composition is a pharmaceutical composition intended for your administration orally or parenterally. If desired, said pharmaceutical composition may be in the form of a lyophilized

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

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

In another aspect, the invention relates to the use of nanoparticles comprising a copolymer biodegradable and thiamine previously described in the preparation of a pharmaceutical composition for the selective stimulation of the Th1 immune response, or in making a composition Pharmaceutical for selective stimulation of the immune response of Th2, or in the preparation of a pharmaceutical composition for balanced stimulation of Th1 immune responses and Th2.

In a further aspect, the invention is relates to a procedure for the production of nanoparticles comprising a biodegradable copolymer and thiamine previously described comprising: a) the desolvation of a organic solution comprising a biodegradable polymer, with a hydroalcoholic solution, to form the nanoparticles; b) the simultaneous incubation of polymer nanoparticles biodegradable previously formed with thiamine in a solution watery; and c) the elimination of organic solvents, with that an aqueous suspension of nanoparticles is obtained which They comprise a biodegradable polymer and thiamine. If desired, said nanoparticles can be stabilized by using agents crosslinkers In a particular embodiment, the concentration of biodegradable polymer in said nanoparticles is comprised between 0.001 and 10% w / v and that of thiamine between 0.001 and 5% w / v.

To obtain charged nanoparticles biologically active molecules, these can be incorporated well in the organic phase where the polymer has previously dissolved biodegradable, before its desolvation, or, later, 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 sample lyophilized 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 media (FGS) and simulated intestinal (FIS). Data are expressed as mean ± SD (n = 3).

Figure 3 is formed by diagrams that show the distribution of nanoparticles in the tract gastrointestinal laboratory animals, after administration orally a 10 mg dose of nanoparticles labeled with RBITC. Figure 3a is a diagram showing the distribution of thiamine coated nanoparticles (T-NP); the Figure 3b is a diagram showing the distribution of control nanoparticles (NP); and Figure 3c is a diagram that shows the distribution of thiamine coated nanoparticles (T-NP) mixed with 5 mg of free thiamine. The axis X represents the different segments of the gastrointestinal tract [Stomach: Sto; portions of the small intestine: I1, I2, I3, 14; blind: Ce]; the Y axis represents the adhered fraction of mucosal nanoparticles (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 half).

Figure 4 is a graph showing the curves bioadhesion for thiamine coated nanoparticles (T-NP) (\ sqbullet), the control nanoparticles (NP) (?), And T-NP co-administered with 5 mg of free thiamine (\ blacktriangle). 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 on Peyer plates (PP) by microscopy of fluorescence, specifically, the nanoparticles coated with thiamine (T-NP) in the normal mucosa (a); the 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 IgG1 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 (iangle), OVA-NP (,), and free OVA (Δ). 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 faecal profile of secretory IgA anti-OVA in BALB / c mice (n = 10). Immunization it was performed on day 0 by single administration well of 20 \ mug of OVA in the different formulations subcutaneously (a, c), or either 100 µg OVA orally (c, d). Formulations they were OVA-T-NP (\ blacktriangle), OVA-NP (?), And free OVA (?). The antibody titers were determined in the serum of the animals on days 0, 7, 14, 28 and 42. Titles were determined as the final dilution of the sample with absorbance values media 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 derived from it.

The nanoparticles of the invention possess some adequate physical-chemical characteristics of bioadhesion and specificity when administered orally, which turns 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 at through another mucosa of the organism. These nanoparticles can be used to administer biologically active molecules and improve your bioavailability, including drugs with windows narrow absorption, as well as drugs originating in the 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 bonding  of thiamine, 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 polymer Biodegradable can be a covalent bond. The reaction between biodegradable polymer and thiamine, and, optionally, the subsequent crosslinking, generates physical entities characteristics, independent and observable, whose average size is less than 1 micrometer (\ mum).

"Medium size" means the diameter average population of nanoparticles that moves together in an aqueous medium. The average size of these systems can be measured by standard procedures known to experts in the field, and which are described, by way of illustration, in the experimental part that accompanies the examples described more down.

The nanoparticles of the invention are characterized by having an average particle size less than 1 µm, preferably have an average size comprised between 1 and 999 nm, more preferably between 10 and 900 nm, even more preferably between 100 and 400 nm. The average size of the particles is mainly influenced by the amount of thiamine, or its derivative, added (to a greater amount of thiamine or derived from it increases the size of the nanoparticle), by the amount and molecular weight of the biodegradable polymer (the higher  amount or molecular weight of the biodegradable polymer the size nanoparticle average is increased), and by some parameters of the production process of said nanoparticles, such such as stirring speed and temperature at the stage of incubation with the aqueous phase containing 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 25ºC and 40ºC. Virtually any biodegradable polymer known in the state of the art that results in the formation of nanoparticles can be used for the practice 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 polymer Biodegradable used is the methyl vinyl ether copolymer and maleic anhydride in anhydride form (PVM / MA), called commercially 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) results particularly advantageous since it is widely used in pharmaceutical technology due to its low toxicity (DL_ {50} = 8-9 g / kg orally) and excellent biocompatibility In addition, it is easy to obtain, both by Quantity as per its 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 usual organic reagents (glutaraldehyde, derivatives of carbodiimide, etc.) that have an important toxicity. In a medium aqueous, the PVM / MA copolymer is insoluble, but its groups anhydride 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, the union  Covalent molecules with nucleophilic groups, such as hydroxide or amino, takes place by simple incubation in a medium aqueous.

The nanoparticles of the invention comprise, in addition to the biodegradable polymer, thiamine or a derivative of the same. Thiamine, also called vitamin B1, or 3 - [(4-amino-2-methyl-5-pyrimidinyl) methyl] -5- (2-hydroxyethyl) -4-methyl-thiazolium Chloride is a known product. However, it is also possible employ thiamine derivatives, understood as such, compounds structurally related to thiamine, such as its salts, among which are its addition salts, for example, its 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 known compounds. In a particular embodiment, The nanoparticles of the invention comprise thiamine or thiamine hydrochloride

Thiamine, or the derivative thereof, can be completely or partially covering the surface of the nanoparticles comprising the biodegradable polymer.

The thiamine ratio (or derivative of same): 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 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 It provides a very efficient adhesion (bonding) ability.

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

The nanoparticles of the invention can Obtained by conventional methods known to technicians in The matter. In a particular embodiment, the nanoparticles of the invention can be obtained by incubating the nanoparticles of biodegradable polymer previously formed with an aqueous solution of thiamine or a derivative thereof, which allows to obtain mostly biodegradable polymer nanoparticles in the that thiamine, or its derivative, is attached to the surface of the nanoparticles. In a particular embodiment, the polymer Biodegradable 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, nanoparticles can be obtained from the biodegradable polymer by a process that comprises dissolving said biodegradable polymer in a organic solvent (e.g., acetone, etc.) and subsequent desolvation after the addition of an appropriate solvent, for example, a solvent or solvent mixture miscible with the solution of the biodegradable polymer, such as a mixture ethanol-water, obtaining a suspension of nanoparticles from which organic solvents are removed by conventional methods, for example, by evaporation under pressure reduced, and then water is added, which is obtained an aqueous suspension of nanoparticles. The relationship, in volume, organic phase: hydroalcoholic solution (ethanol / water) may vary within a wide range; however, in one embodiment In particular, said ratio is between 1: 1 and 1:10 (v: v).

After solvent removal Organic, biodegradable polymer nanoparticles are modify on its surface efficiently with thiamine or with a derivative thereof, by incubation, at room temperature, for an appropriate period of time. To do this, about said previously obtained nanoparticle suspension is added a aqueous solution of thiamine or a derivative thereof. In a particular embodiment, pharmaceutical grade water is used. Both the concentration of the biodegradable polymer and that of the thiamine, or its derivative, may vary within a wide range; however, in a particular embodiment, the concentration of biodegradable polymer is between 0.001 and 10% w / v and the of thiamine, or derivative thereof, between 0.001 and 5% w / v.

In general, thiamine amino groups react with functional groups eventually present in the biodegradable polymer, which leads to bond formation between the biodegradable polymer and thiamine. The association of the thiamine to biodegradable polymer nanoparticles is made evident due to the significant decrease in the negative charge surface 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, reaction which can be easily given by the simple incubation of thiamine with the aqueous suspension of the nanoparticles, which leads to the formation of amide bonds between the PVM / MA copolymer and the thiamine

The nanoparticles of the invention can purified by conventional methods, for example, by centrifugation, ultracentrifugation, tangential filtration, or evaporation, including the use of vacuum.

Optionally, an agent can be added crosslinker to improve the stability of the nanoparticles of the invention, as described in WO 02/069938. Examples illustrative, not limiting, of crosslinking agents that can be use include diamine compounds, for example 1.3 diaminopropane, polysaccharides or simple saccharides, proteins and, in In general, any molecule that has capable functional groups to react with the functional groups present in the polymer biodegradable, for example, anhydride groups present in the PVM / MA copolymer.

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

The nanoparticles of the invention have high ability of association of molecules biologically active, which makes them transport systems for drugs or presentation of antigens and allergens very appropriate.

Therefore, in another aspect, the invention is relates to a pharmaceutical composition that comprises nanoparticles of the invention and at least one molecule biologically active

In general, said biologically active molecule it will be inside the nanoparticle of the invention; no However, it could happen that some biologically active molecule was also attached to the surface of the nanoparticle although most of these molecules will be inside (e.g., encapsulated) of the nanoparticles of the invention.

The term "biologically molecule active ", as used herein, refers to any substance used in the treatment, cure, prevention or diagnosis of a disease or that used to improve well-being Physical and mental human and animal. In general, said term It includes both drugs and antigens and allergens. According to In the present invention, the nanoparticles of the invention can incorporate one or more biologically active molecules regardless of the solubility characteristics of the same. The association capacity will therefore depend on the incorporated molecule, but in general, said capacity is high both for hydrophilic molecules and for labeling molecules hydrophobic character

In a particular embodiment, the composition pharmaceutical of the invention comprises nanoparticles of the invention containing one or more different drugs. Examples illustrative, not limiting, of said drugs include agents analgesics, anti-inflammatory, anti-tumor, neuroprotective, antiallergics, anti-asthmatics, antibiotics (e.g., antibacterials, antifungals, antivirals, antiparasitic agents, etc.), surfactants lungs, etc.

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

As used in this description, the term "antigen" refers to any substance capable of be recognized by the immune system of a subject and / or capable of induce a humoral immune response or response in a subject immune system 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 a microorganism, by 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 it can be used in the elaboration of nanoparticles of the invention loaded with antigen. By way of illustration, not limitation, The term "antigen" includes:

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"microbial" antigens, that is, microorganism antigens, including, but not limited to, viruses, bacteria, fungi and infectious parasites; said antigens they 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; sayings antigens are routinely used by experts in the matter; Y

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"tumor" antigens, that is, substances, for example, peptides, associated with a tumor or a cancer ("tumor marker"), which is capable of causing immune response, in particular, when presented in the context of a CMH molecule, e.g., Her2 (breast cancer); GD2 (neuroblastoma); EGF-R (malignant glioblastoma); CEA (medullary thyroid cancer); CD52 (leukemia); gp100 protein from human melanoma; protein melan-A / MART-1 of human melanoma; tyrosinase; NA17-A nt protein; protein MAGE-3; p53 protein; HPV16E7 protein; fragments antigens 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 molecule Biologically active can be very varied. In one embodiment in particular, said biologically active molecule is a polysaccharide, a protein, a peptide or a lipid. In other particular embodiment, said biologically active molecule is a nucleic acid (e.g., 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 biologically active molecule associated and / or conventional nanoparticles to be 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 nanoparticles) for oral, oral, sublingual, administration topical, ocular, nasal, vaginal or parenteral; any composition in the form of gel, ointment, cream or balm for administration topical, ocular, nasal or vaginal; or any solid composition (tablets, capsules) for oral administration. In a particular embodiment, the pharmaceutical composition is administered orally. In another particular embodiment, said composition Pharmaceutical is administered parenterally.

The pharmaceutical compositions described They will comprise suitable excipients for each formulation. By example, in the case of oral formulations in the form of tablets or capsules will be included if necessary binder agents, disintegrants, lubricants, fillers, coating enteric, etc. Solid oral formulations are prepared from conventional way by mixing, dry or wet granulation and incorporating the nanoparticles of the invention. The compositions Pharmaceuticals can also be adapted for administration parenteral, in the form of, for example, solutions, suspensions or lyophilized, sterile products, in the dosage form appropriate; in this case, said pharmaceutical compositions will include suitable excipients, such as buffers, surfactants, etc. In any case, the excipients will be chosen depending on the pharmaceutical form of administration selected. A review of the different pharmaceutical forms of drug administration and its preparation can be found in the book "Treaty of Galenic Pharmacy", by C. Faulí i Trillo, 1st Edition, 1993, Luzán 5, S.A. of Editions.

The proportion of the molecule biologically active incorporated into the nanoparticle of the invention may vary within a wide range, for example, it can be up to a 25% by weight with respect to the total weight of the nanoparticles. Do not However, the appropriate proportion will depend in each case on the biologically active molecules incorporated.

The incorporation of the molecule biologically active to the nanoparticles of the invention can be done as is described in WO 02/069938, by incorporation into the solution of biodegradable polymer before nanoparticle formation, or either later add it to the aqueous suspension of the nanoparticles already formed. For example, and depending on the nature of the biologically active molecule, you can proceed as follows:

to)

Biologically active molecules hydrophobic: addition in the organic phase (e.g., acetone) and joint incubation / solubilization with the polymer biodegradable for a variable period of time (up to 1 hour) under agitation (mechanical, magnetic or ultrasonic stirrer); Y

b)

Biologically active molecules hydrophilic: well by

addition in the organic phase (e.g., acetone) and joint incubation with the polymer biodegradable for a variable period of time (up to 1 hour) under stirring (mechanical, magnetic or ultrasonic stirrer) until obtain a fine suspension in the organic solvent [this procedure has been used successfully to encapsulate a model protein (ovalbumin, about 44 kDa protein); the incorporation was efficient allowing high encapsulation of the model protein; or by

addition in the aqueous phase to incubate with the preformed nanoparticles (it 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 immunotherapy. In fact, the nanoparticles of the invention have the ability to stimulate the two immune response pathways (Th1 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, dependent toxin, dependent scourge, etc.), stimulates the Th1 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 Th1 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 is relates to a vaccine or composition for immunotherapy that it comprises a therapeutically effective amount of nanoparticles of the invention and an antigen or allergen, together with a pharmaceutically acceptable carrier or excipient. The antigen or allergen may be contained inside said nanoparticles and / or at least partially coating 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 coating at least partially the surface of said nanoparticles and / or content inside said nanoparticles. In one embodiment in particular, said allergen or antigen is covering the entire or part of the surface of said nanoparticles.

In a particular embodiment, said vaccine or Immunotherapy composition can be found in any form Pharmaceutical administration by any appropriate route, by example, oral, rectal, nasal, sublingual, etc. In one embodiment in particular, said vaccine or composition for immunotherapy is found in a pharmaceutical form of oral administration, while in another particular embodiment, said vaccine or composition for immunotherapy is in a form parenteral administration pharmaceutical, for example, by intramuscular (i.m.), subcutaneous (s.c.), intravenous (i.v.) route, intraperitoneal (i.p.), intradermal (i.d.), etc. A review of the different pharmaceutical forms of drug administration, in  general, and its preparation procedures can be found in the book "Treaty of Galenic Pharmacy", by C. Faulí i Trillo, 1st Edition, 1993, Luzán 5, S.A. of Editions.

The dose of nanoparticles loaded with a antigen or with an allergen can vary within a wide range, for example, between about 0.01 and approximately 10 mg per kg 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 administered in combination with vaccine or immunotherapeutic compositions that contain 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, an additional aspect of the invention constitutes a product that comprises, in a way separated, a) an antigen or an allergen; and b) a composition that comprises said nanoparticles based on a polymer biodegradable and thiamine, as an enhancer composition of the immune response against said antigen or allergen, such as 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.

The combined administration of said composition vaccine or immunotherapeutic and empty nanoparticles can be carried out simultaneously or sequentially, separated in time, in any order, that is, the vaccine or immunotherapeutic composition and, subsequently, the empty nanoparticles or vice versa. Alternatively said vaccine or immunotherapeutic composition and said nanoparticles empty can be administered simultaneously. In this case, the vaccine or immunotherapeutic composition and empty nanoparticles they 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 which, optionally, they contain an antigen and reveal the ability of said nanoparticles to adhere so specific to the gastrointestinal mucosa and, if necessary, act as an adjuvant in immunotherapy or vaccination.

General procedure of nanoparticle production thiamine coated

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. Release, 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:

TO.

For obtain formulations containing the compound (drug, molecule active or marker) adhered to the surface of the nanoparticles [for example, a fluorescent marker], incubation with the compound was carried out after evaporating the solvents organic (Example 1).

B.

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

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

The purification of the nanoparticles led to out through ultracentrifugation. Finally, the nanoparticles purified were lyophilized for storage and preservation at long term.

Characterization of the nanoparticles

The size and potential zeta 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 device (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 NH2 reverse phase column (4.6 mm x 150 mm; 5 µm; Agilent) heated to 40 ° 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 centrifugation of nanoparticles were dosed in HPLC vials. Then 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 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 quantity quantified in the supernatants collected during the purification stage by centrifugation For the calculations, a curve of calibrated between 5 and 100 µg / mL (r> 0.999).

The release kinetics of RBITC from the nanoparticles were performed in Vivaspin® 100,000 dialysis tubes MWCO (VIVASPIN, Hannover, Germany). To do 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. TO certain times, nanoparticle suspensions are centrifuged (5,000 x g, 15 minutes) and the amount of RBITC in the dialyzed liquids were quantified by colorimetry (λ = 540 nm).

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

Bioadhesion study

This study was performed applying a protocol described previously [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; Harlan, 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 (I1, I2, I3 and I4) 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 (λ ex 540 nm 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 study of competition to investigate the effect of free thiamine on the bioadhesive capacity of nanoparticles. To do 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. Release, 89 (2003) 19-30; Salman et al. , J. Control. Release, 106 (2005) 1-13]: Q_ {max}, AUC_ {adh}, T_ {max}, MRT_ {adh} and K_ {adh}. Q_max represents the maximum fraction adhered to the mucosa and allows estimating 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 the amount of marker attached with respect to time) and was evaluated by the trapezoid method up to t_ {z} (the last sampling point ), and allows quantifying 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 performed by fluorescence microscopy To do this, 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 intestine were removed thin and washed with PBS. Such 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 into 5 µm sections in a cryostat (2800 Frigocut E, Germany), fixed to supports coated with poli-L-lysine (Sigma, Spain) and stored at - 20ºC before viewing by microscopy of fluorescence.

Immunization and sampling protocols

The protocols with animals were applied in according to European legislation (86/609 / EU). BALB / c mice (20 ± 1 g) (Harlan, Spain) were divided into 6 different groups of 10 animals each. The management strategy was based on the single administration of a subcutaneous or oral dose. In 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 nanoparticles conventional (OVA-NP). For groups of animals immunized subcutaneously (s.c.), the dose of Protein was 20 µg OVA in 50 µL of PBS.

Blood samples were collected from the plexus retroorbital on days 0, 7, 14, 28 and 42 after immunization. The samples were centrifuged (3,000 x g, 10 minutes) and preserved as "pool" of each group (10 mice). Finally, Each pool was diluted with PBS (1:10) and stored at -80 ° C until subsequent 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 stool by ELISA

Anti-ova antibodies in serum were analyzed by ELISA with conjugates anti-IgG1 and anti-IgG2a (Sigma-Aldrich Chemie, Germany). Briefly, for performing this test 96 well plates were used (EB, Thermo Labsystems, Vantaa, Finland), where it was set 1 ovalbumin dissolved in regulatory solution carbonate bicarbonate (0.05 M, pH 9.6) 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 PBS-Tween 20 0.05% (PBS-T). The plates were washed and added sera in serial dilutions starting at 1:40, and incubated at 37 ° C for 4 hours. Subsequently, 5 washes of 1 were performed  minute. Then, the conjugate was incubated for 2 hours peroxidase, another 5 washes of 1 minute were performed, the substrate [ABTS (acid 2,2'-azino-bis (3-ethylbenzo-thiazolin-6-sulfonic acid) (Sigma-Aldrich Chemie, Germany)] and was carried out reading at 405 nm on a plate reader (iEMS Reader MF, Labsystems, Finland) after 30 minutes of temperature incubation ambient. Final titers were determined by dilution of the sample until obtaining an average of OD ≥ 0.2 with respect to the obtained with non-immunized mouse serum.

Anti-ova antibodies in feces were analyzed by ELISA with conjugates anti-IgA. In this case, after the coating of the OVA plates, these were blocked by adding, and incubation for 1 h at room temperature of 200 µL of one PBS-T solution containing 3% milk skim The stool extract was added to two plates and was they were making serial dilutions in PBS-T. Said plates were incubated for 4 h at 37 ° C. Finally, after the washed, the wells were incubated with the conjugate peroxidase-labeled anti-IgA (Nordic Immunological Labs, The Netherlands).

Statistic analysis

Bioadhesion data and physicochemical characteristics were compared using the Mann-Whitney U-test non-parametric test and the t-Student test, respectively. P values <0.05 were considered significant. All calculations were performed using the SPSS® statistics program (SPSS® 10, Microsoft, USA).

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Example 1 Preparation and characterization of nanoparticles based on copolymer of methyl vinyl ether and maleic anhydride (PVM / MA)

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

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 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.

1.2. Preparation of nanoparticles coated with thiamine (T-NP)

100 mg of the methyl vinyl ether and maleic anhydride (PVM / MA) [Gantrez® AN 119] copolymer 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 the nanoparticles

Table 1 summarizes the characteristics. main physicochemical of nanoparticles obtained. Control nanoparticles (NP) show a size close to 200 nm with a negative surface charge of -51 mV. On the other hand, thiamine coated nanoparticles (T-NP) were significantly older (close to 400 nm) and showed significantly less zeta potential negative. These nanoparticles (T-NP) showed a associated thiamine amount of 15 µg / mg nanoparticles (Table one). Finally, it is noteworthy that the presence of thiamine does not exerts no effect on the manufacturing performance of the nanoparticles. Similarly, small differences were observed at compare the association of RBITC for both formulations.

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TABLE 1 Physicochemical characteristics of Gantrez® AN nanoparticles (mean ± SD, n = 6)

100

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

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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 nanoparticles.  Therefore, it can be assumed that the fluorescence intensity and spots obtained while viewing images corresponded to the fluorescent marker associated with the nanoparticles.

2.2. Distribution of the adhered fraction of nanoparticles in the gastrointestinal tract of animals of laboratory

Figure 3 shows the distribution in the tract Gastrointestinal adhesion of nanoparticles coated with thiamine (T-NP) (Figure 3a) or control nanoparticles (NP) (Figure 3b). It can be seen that, at 30 minutes after administration of the formulations of nanoparticles, both types (NP and T-NP) showed the same ability to adhere to the mucosa of the tract gastrointestinal, mainly in the stomach area and upper regions of the small intestine (duodenum). However, 1 h after administration of the nanoparticles, the thiamine coated nanoparticles (T-NP) showed a significantly higher bioadhesive capacity and a distribution more homogeneous than the control nanoparticles (NP). The amount of thiamine coated nanoparticles (T-NP) found attached to the mucosa was approximately 45% of the administered dose (Figure 3). At 3 hours after administration, the nanoparticles coated with thiamine (T-NP) showed the maximum adhesion (65% of the administered dose) and an important tropism by the animal's ileum (portions I3 and I4 in the Figure 3a).

On the other hand, when the animals of laboratory were administered the nanoparticle mixture coated with thiamine (T-NP) and vitamin (thiamine) free, the ability of nanoparticles to develop bioadhesive interactions in the tract Gastrointestinal was strongly inhibited (Figure 3c). Do not However, the distribution in the tract and the fraction adhered to the mucosa of those thiamine coated nanoparticles (T-NP) was similar to the profile observed for control nanoparticles (NP). This result clearly indicates that adhesion and tropism of nanoparticles coated with Thiamine (T-NP) is directed by the presence of Thiamine on its surface.

2.3. Bioadhesion parameters and curves

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

On the contrary, for nanoparticles Thiamine coated (T-NP), the maximum of Adhesion is observed 3 hours after administration. Further, this maximum represents an amount close to 65% of the dose managed [Figure 4, (\ sqbullet)]. From these curves you they calculated the bioadhesion parameters that allow comparing the adhesive potential of the different nanoparticles. Parameters They are listed in Table 2.

First, it is worth noting that thiamine-coated nanoparticles (T-NP) are characterized by an AUC_ {adh} (a 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 removal rate (K_ {adh}) 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 nanoparticles coated with thiamine (T-NP) and free vitamin (thiamine), eliminated the bioadhesive properties of these nanoparticles (T-NP) [Figure 4, (black triangle)]. Thus, under these conditions, the bioadhesion parameters studied in
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 thiamine structural analogues, such as amprolium, oxythiamine and pyrithiamine [Laforenza et al. . J. Membr. Biol. 161 (1998) 151-162; Dudeja et al. , Am. J. Physiol. Cell Physiol., 281 (2001) C786-C792].

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TABLE 2 Bioadhesion parameters for the different nanoparticle formulations

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2.4. Display of the distribution of nanoparticles attached to the intestinal mucosa

The visualization of the labeled nanoparticles with RBITC in the gastrointestinal mucosa was observed by microscopy of fluorescence. Figure 5 shows several photographs that allow us to observe the distribution of nanoparticles in samples of ileum, 2 hours after oral administration of 10 mg of nanoparticles in rats.

The control nanoparticles (NP) were mainly detected in the outer layer of the ileum (mucus that upholster the mucosa), confirming the low affinity of these transporters through the intestinal mucosa (Figure 5b). For him on the contrary, thiamine coated nanoparticles (T-NP) were widely distributed and showed a very large affinity for gut microvilli (Figure 5a). On the other hand, said T-NPs showed a 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).

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Example 3 Preparation of nanoparticles containing an antigen model

The adjuvant capacity of nanoparticles Thiamine coated (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 was added 100 µL of a 1% v / v solution of 1,3-diaminopropane, subjecting the assembly 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 nanoparticles coated with thiamine with encapsulated ovalbumin (OVA-T-NP)

5 mg of OVA was dispersed in 2 mL of water with ultrasonic support (Microson ™) or in a bath of ultrasound for 1 minute under cooling. This suspension is 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, added 10 mL of ethanol and 10 mL of deionized water containing 2.5 mg thiamine The resulting mixture was allowed to homogenize and the nanoparticle suspension was evaporated under reduced pressure (Büchi R-144, Switzerland) until both are eliminated organic solvents 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 x g 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, subjecting the assembly to magnetic stirring for 5 minutes. The resulting suspension was subjected to purification by ultracentrifugation (20 minutes at 27,000 x g). The supernatants are removed and the residue was resuspended in water or in a solution 5% aqueous sucrose. Finally, the suspension of The resulting nanoparticles were lyophilized, thereby allowing keep all its properties intact.

3.3. Physicochemical characteristics of the nanoparticles

Table 3 summarizes the characteristics. main physicochemical of nanoparticles resulting (OVA-NP and OVA-T-NP). Formulation OVA-T-NP showed a size significantly higher (412 nm) than control nanoparticles (280 nm). On the other hand, the presence of OVA slightly affects the amount of thiamine bound to the surface of the nanoparticles. Thus, the presence of this protein (OVA) decreased by 20%. amount of vitamin (thiamine) bound (12 µg / mg vs 15 \ mug / 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 Microimage® program Version 4. The amount of OVA was, for OVA-NP and OVA-T-NP, 12.1 ± 1.4 and 11.6 ± 2.3 / mug / mg nanoparticles, respectively.

TABLE 3 Physicochemical characteristics of Gantrez® AN nanoparticles containing ovalbumin as antigen model (mean ± SD: n = 6)

102

Example 4 Study of the adjuvant capacity of nanoparticles that encapsulate ovalbumin

Figure 6 shows the titles of IgG2a and lgG1 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). The board 4 summarizes the values of the areas under the response curves immune.

In subcutaneous immunization (s.c.), the administration of OVA-T-NP induced the production of anti-OVA antibody levels of type Th2 (IgG1) similar to those induced by the formulation OVA-NP. However, IgG2a titles (corresponding to a Th1 type response) were at least 2 times upper for OVA-T-NP than for OVA-NP. All this shows that with nanoparticles Thiamine coated (T-NP) can be achieved more balanced levels of Th1 and Th2 profiles, since their activity inducer of IgG2a (Th1) levels is well above the induced by control nanoparticles (NP) not coated with this vitamin (thiamine)

In all cases, antibody levels anti-OVA generated by both formulations (OVA-NP and OVA-T-NP) were significantly older than when immunized with the free protein (OVA).

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TABLE 4 Values of areas under curves (AUCs) that represent the different immune response profiles expressed in Figure 6

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After oral administration, the nanoparticles OVA-NP also showed a profile predominantly of type Th2 (Figure 6). The oral type response Th1 was very low (AUC of 1.5; AUC_2 is 7 times higher than AUC_ {th1}, see Table 4) and, at the same time, the response of Antibodies induced by oral administration was lower than the described after subcutaneous administration.

On the contrary, oral immunization with OVA-T-NP induced Th1 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 sc was significantly more intense than orally, for the OVA-T-NP nanoparticles the induction of anti-OVA antibodies by the sc and oral routes were quite similar. , especially the answer Th1. These results clearly show the ability of thiamine-coated nanoparticles (T-NP) to increase and enhance Th1 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. Immunol., 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 Th1 type responses [Cronkhite and Michael, Vaccine, 22 (2004) 2106-2115]. This potentiation of the Th1 response may be related to the high tropism of the T-NP nanoparticles by the distal regions of the small intestine, as well as their capture by 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 Th1 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 antibodies IgA anti-OVA in mouse feces immunized by the s.c. or oral In both cases, the immunization with nanoparticles, regardless of the route of administration, induced the production of high levels of IgA. However, thiamine coated nanoparticles (T-NP) induced level secretion significantly higher intestinal IgA than nanoparticles conventional. Orally, that difference was 64 times superior for nanoparticles OVA-T-NP that stops OVA-NP nanoparticles. This phenomenon may be related to the effective capture of nanoparticles T-NP by the Peyer plates of the tract gastrointestinal and its passage to the lymphocytes responsible for the synthesis and secretion at the level of IgA mucous membranes.

In conclusion, the nanoparticles coated with Thiamine (T-NP) have shown a capacity particular to develop adhesive interactions in the mucosa gastro-intestinal This ability can be interesting for Increase the oral bioavailability of numerous drugs. For other part, as vaccination adjuvants, these transporters Polymers are able to reach Peyer's plates and enhance high induction of antibodies against the antigen transported In addition, the response that is generated is humoral (Th2) and cell (Th1), which may be of interest for vaccination against to numerous pathogens and for immunotherapy for the treatment of allergies

Claims (30)

1. Nanoparticles comprising a polymer biodegradable and thiamine or a derivative thereof. 2. Nanoparticles according to claim 1, in which said biodegradable polymer is a methyl copolymer vinyl ether and maleic anhydride (PVM / MA). 3. Nanoparticles according to any of the claims 1 or 2, wherein the copolymer has a weight molecular 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 of the claims 1 to 3, comprising thiamine or thiamine hydrochloride 5. Nanoparticles according to any of the claims 1 to 4, wherein the thiamine or its derivative is find covering all or part of the surface of the nanoparticles. 6. Nanoparticles according to any of the 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 the claims 1 to 6, wherein said nanoparticles are They find crosslinked. 8. Pharmaceutical composition comprising nanoparticles according to any one of claims 1 to 7 and a biologically active molecule capable of preventing, relieving or curing an illness. 9. Composition according to claim 8, which it comprises 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 their mixtures 11. Composition according to claim 8, which comprises as a biologically active molecule an antitumor agent or an antigen for tumors. 12. Composition according to claim 8, which comprises as a biologically active molecule a protector of central nervous system or a glucocorticoid. 13. Composition according to claim 8, which it comprises as a biologically active molecule an antigen to 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 which gives access to some mucosa of the organism, preferably the pathway rectal, ophthalmic, vaginal, nasal, pulmonary or sublingual. 17. Use of nanoparticles according to any of claims 1 to 7 in the preparation of a medicine. 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 vehicle or pharmaceutically excipient acceptable. 20. Vaccine according to claim 19, wherein the antigen or allergen is contained within said nanoparticles and / or at least partially coating the surface of said nanoparticles. 21. Vaccine according to any of the claims 19 or 20, wherein said antigen comprises a immunogenic extract from an organism. 22. Vaccine according to any of the claims 19 or 20, wherein said allergen comprises a allergenic pollen extract, an allergen insect extract or a  extract of an allergenic food product.

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23. A product that includes, in a way separated, a) an antigen or an allergen; and b) a composition that it comprises nanoparticles based on a biodegradable polymer and thiamine according to claims 1 to 7, as an enhancer composition of the immune response against said antigen or allergen, such as 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. 24. Use of nanoparticles according to any of claims 1 to 7 in the preparation of a composition Pharmaceutical for selective stimulation of the immune response of Th1, or in the manufacture of a pharmaceutical composition for selective stimulation of the Th2 immune response, or in the manufacture of a pharmaceutical composition for stimulation balanced of the immune responses of Th1 and Th2. 25. Procedure for the production of nanoparticles according to any one of claims 1 to 7 which understands:

to)

desolvation of an organic solution comprising a biodegradable polymer, with a solution hydroalcoholic to form the nanoparticles;

b)

simultaneous incubation of biodegradable polymer nanoparticles formed in step a) and thiamine in an aqueous solution; Y

C)

solvent removal organic obtaining an aqueous suspension of nanoparticles.

26. Method according to claim 25, in which the concentration of the biodegradable polymer is comprised between 0.001 and 10% w / v and that of thiamine between 0.001 and 5% w / v. 27. Procedure according to any of the claims 25 and 26, comprising additional steps of purification. 28. Procedure according to any of the claims 25 to 27, further comprising incorporating a biologically active molecule by adding it in the phase organic where the dissolved biodegradable polymer is, prior to its desolvation 29. Procedure according to any of the claims 25 to 27, further comprising incorporating a biologically active molecule by adding it to the aqueous suspension of already formed nanoparticles. 30. Procedure according to any of the claims 25 to 29, comprising an additional step of lyophilization, optionally in the presence of an agent cryoprotectant, preferably sucrose or mannitol.