WO2008131506A1

WO2008131506A1 - Mucoadhesive granules containing chitosan nano- and/or microspheres and the process of manufacture

Info

Publication number: WO2008131506A1
Application number: PCT/BR2008/000123
Authority: WO
Inventors: Maria Helena Andrade Santana; Classius Ferreira Da Silva; Fernanda Martins
Original assignee: Universidade Estadual De Campinas - Unicamp
Priority date: 2007-04-27
Filing date: 2008-04-28
Publication date: 2008-11-06
Also published as: BRPI0705072B8; BRPI0705072A2; BRPI0705072B1

Abstract

This invention relates to a product, functional heterogeneous granules containing a granulation excipient, and micro- and/or nanoparticles encapsulating a pH-sensitive active compound, and to the production process thereof. These granules incorporate active compounds in chitosan nano- and/or microparticles, which have mucoadhesion properties and sustained release of the incorporated active compound. These properties provide a higher in vivo action efficiency of vehiculated drugs or active compounds when administered through mucosae, reduction of side effects, conservation of the activity of labile active compounds, and reduction of the concentration and frequency of dosages. The production process can be operated in a semicontinuous or discontinuous regimen, under sterile conditions, and can be scaled for application in the industrial sector.

Description

Specification of Patent of Invention for

Mucoadhesive Granules Containing Chitosan Nano- and/or Microspheres and the Process of Manufacture

The present invention relates to the functional product, as well as to the process for obtaining mucoadhesive granules composed of chitosan nano- and/or microspheres associated with a granulation excipient. These granules represent a functional device which is economically feasible and more effective for the administration of drugs and nutraceuticals.

Description of the prior art

Polymeric nano and microspheres have been employed successfully to the incorporation and/or encapsulation of active compounds of various natures, such as scents, enzymes, cosmetic products, drugs, and the like. Encapsulation protects the active compounds, thus bypassing physicochemical and/or biological stability limitations. Some active compounds, when exposed to the oxygen from the air, light, heat, or the action of biological means, are subjected to oxidation and/or breakdown or denaturation, thereby losing their activity. Therefore, in order to render possible the reliable marketing and utilization of these products, their protection is needed. Additionally, it is also possible to produce the controlled release of the active compound incorporated/encapsulated in various means, including the biological means, in which in many cases also occurs a decrease of toxicity and greater action efficiency as compared to the free forms. Incorporation refers to the scattering of the active compound in the entire polymeric matrix of the microspheres (interior and surface), whereas encapsulation refers to its confinement only in the interior of the structure.

The adhesion of molecules to a biological tissue is a simple definition of bioadhesion. The adhesive binding generally occurs in the epithelial cell layer, in the continuous mucus layer, or both. The term mucoadhesion is used whenever adhesion involves specifically the mucus layer. Bioadhesion is a complex phenomenon, which in molecular level involves several physicochemical properties, such as electrostatic forces, hydrophobic interactions, hydrogen bonds and van der Waals interactions. In hydrogels, there are also interpenetration forces and interlacement of the polymer chains.

Bioadhesive polymers belong to the classes of hydrophilics, hydrogels and thermoplastics, which can be either synthetic or natural. Many biocompatible polymers used in bioadhesive formulations include cellulose derivatives, ethylene glycol and copolymers thereof, such as oxyethylene, polyvinyl alcohol, polyvinyl acetate and hyaluronic acid esters. Among biodegradable polymers are included polylactic, polyglicolic, polylactic-polyglicolic acids, polycaprolactones, polyortoesters, polyphosphoesters, and poly anhydrides. Bioadhesion can be nonspecific or specific. Comprised in the main representatives of non-specific bioadhesion are polycarbophil (polyacrylic acid cross-linked with divinyl glycol), carbopol/carbomers (polyacrylic acid cross-linked with alkyl saccharose or alyl pentaerythritol) and chitosans (copolymers of glucosamine and N- acetylglucosamine). Complex glucosamines, like lectins, make up the main representatives of especifc bioadhesive polymers. Chitosan is a natural polymer having mucoadhesive properties. In this case, the electrostatic attraction between the positively-charged chitosan and the negatively- charged mucin, both in physiological pH, is the main responsible for mucoadhesion. Chitosan is the name used for the deacetylated form of chitin, which is composed primarily of glucosamine and 2-amido-2-deoxy-β-D-glucose. Chitin is a glycan having bonds β (1-4), composed of 2-acetamine-2-deoxy-β-D-glucose. The molar mass of chitin ranges from 1.03 x 10⁶ to 2.5 x 10⁶ Da, however, after deacetylation, chitosan exhibits a mean molar mass of 1.0 x 10⁵to 5.0 x l0⁶Da.

Mucoadhesive polymers have been used individually or in admixture with substances of various natures, such as other polymers, lipides, stabilizers, polysaccharides, etc., in formulations of systems for controlled release of active compounds. In in vivo applications through mucosae, the success of such controlled release systems is restricted to its residence time at the absorption site. Mucoadhesive polymers have been studied as a strategy to extend the residence time of such systems on the surface of mucosae. Thus, controlled release systems containing mucoadhesive polymers have the advantages of increasing the bioavailability of the active compounds and reducing the dosages and/or administration frequency thereof.

The most used controlled release systems are the nano- and/or microspheres, in solid form or in liquid dispersion, and solid granules. In nano/microspheres, the active compound is scattered on the whole polymeric matrix.

Nano/microcapsules represent a particular case of nano/microspheres, in which the active compound is confined in a core which is covered by the polymeric matrix. The granules contain the active compound scattered in its entire polymeric matrix, and differ from nano/microspheres in its dimensions and geometric shapes. Such granules, spherical or cylindrical, have dimensions in the order of millimeters (10^" m), while nano/microspheres have a size in the range of nanometers (10^~9 m) or micrometers (10^" m).

Conventional granules (homogeneous matrix or homogeneous mixture), produced from different polymers and excipients, have been applied in pharmaceutical and food industries since the second half of the 20th century. In the pharmaceutical industry in particular, granules have been very useful for the fractioning of doses administered to children and elderly, and also in the case that the conventional presentation of the drug in the form of tablets is of difficult ingestion. Furthermore, granules are a most attractive form of drug presentation, improving patient compliance with drug therapy.

From 1990s up to now, chitosan has been used as a raw material in the manufacturing of microparticles for the encapsulation of drugs and active compounds for various technical fields, such as pharmaceutics, cosmetics, food-related, veterinary, etc. Among documents which disclose drugs encapsulated in chitosan nano- or microparticles for various therapeutic purposes, one could mention: (MITRA, S., GAUR, U., GHOSH, P. C, MAITRA, A. N., Journal of Controlled Release, v.74, p.317-323, 2001); (CHANDY, T., DAS, G. S., RAO, G. H. R., Journal of Microencapsulation, v.17, n.5, p.625- 638, 2000); and (CUI, Z., MUMPER, R. J., Journal of Controlled Release, v.75, p.409-419, 2001). Chitosan particles may be obtained by various techniques, such as spray-drying, coacervation, ionotropic gelation, etc. Among documents which disclose the obtainment of chitosan particles using some of such techniques, we could mention patents US4285819, US4647536 and US5489401. The production of gastroresistant granules for the protection of active compounds sensitive to stomachic pH has been described in recent studies in the form of patents. Among documents which disclose processes for the production of granules containing chitosan, we could mention patents US6413749, US4533557 and US5474989.

The process for the production of gastroresistant homogeneous granules, containing high drug content, is disclosed in the documents US2001051188 and US2001005716. In these documents, the granules are composed essentially of three substances: pH-sensitive drug (80 to 100% by mass), disintegrating agent (about 0 to 10% by mass) and binding agent (0 to 10% by mass). In order to obtain the granules, these compounds are mixed, extruded and spheronized. The obtained granules may or not be subjected to a coating process with a gastroresistant polymer.

The process for the production of stavudine (an anti-HIV drug) controlled release granules is described by the document US 2002002147. The hydrolysis of stavudine is one of the biggest challenges in obtaining controlled release pharmaceutical forms, since most of the processes of granulation and encapsulation may involve water in one of the steps. In this regard, this process utilizes magnesium stearate to grant stability to stavudine during the granulation process, wherein water is added for obtaining a wet mass. This process is similar to the one of the previously mentioned works. After extrusion and spheronization, the granules can be subjected to a coating with gastroresistant polymers. As seen, the developed works comprise the production of chitosan nano- and/or microparticles or granules composed of a homogeneous mixture of ingredients which vehiculate the active compounds. In these works, homogeneous mixtures containing the active compound are responsible for its protection, as well as its controlled release at the target site. Nevertheless, the described works do not suffice when active principles which are pH-sensitive and/or which require an extended release time at the target site are used. Some of these active compound are high value-added and hence protection techniques for the activity thereof, as well as techniques which allow for higher control during the release become necessary.

Objects of the invention

One object of the present invention is to obtain mucoadhesive granules which provide higher protection to the active compound, as well as a higher control during the extended release thereof as compared to the granules hitherto known and used in the administration of drugs and nutraceuticals. Summary of the Invention:

The present invention discloses a composition of mucoadhesive granules comprising chitosan nano- and/or microspheres associated with a granulation excipient, as well as a process for the production of said granules. The mucoadhesive granule to which this invention relates comprises: (a) at least one active compound incorporated in chitosan micro- and/or nanoparticles and (b) a granulation excipient. The invention also relates to pharmaceutical compositions which comprise, in its formulation, the respective mucoadhesive granules described. The invention still relates to a process for obtaining said mucoadhesive granules, comprising the following steps: a) preparing a solution A containing chitosan; b) preparing a solution B containing at least one active compound; c) obtaining a primary mixture comprising chitosan micro- and/or nanoparticles incorporated with the active compound; d) admixing said chitosan micro- and/or nanoparticles incorporated with the active compound with a granulation excipient forming a secondary mixture; and e) obtaining said mucoadhesive granule from said secondary mixture. The subject invention also relates to processes for obtaining mucoadhesive granules which include the process for obtaining the mucoadhesive granules described herein. The invention still relates to mucoadhesive granules obtained according to the process described herein, as well as pharmaceutical compositions containing mucoadhesive granules obtained according to the process described herein.

Brief Description of the Drawings

In the following, reference is made to the Drawings appended to this specification, for better understanding and illustrating the same, in which:

Figure 1 shows a flowchart of the process of production by extrusion and spheronization, of the heterogeneous granules containing nano- and/or microspheres. Figure 2 shows a histogram of size distribution of microspheres obtained from the optical microscopy image upon encapsulation of didanosine (ddl) drug and cross-linking of the microspheres with tripolyphosphate (TPP). (Experiment conditions: 25.00 mg ddl/mL / 2.00 % Chitosan / 10.00 % TPP). Figure 3 shows a micrography obtained by optical microscopy of the chitosan particles cross-linked with tripolyphosphate (TPP) and containing the didanosine (ddl) drug, (magnification: 100-fold): Experiment conditions: 25.00 mg ddl/mL / 2.00 % Chitosan / 10.00 % TPP. Figure 4 shows the release profiles of the didanosine drug from chitosan microspheres prepared with two initial concentrations of didanosine (25.00 and 39.00 mg ddl/mL).

Figure 5 shows release profiles of didanosine from: (a) cylindrical granules from physical mixture, (b) spherical granules containing microspheres and 4.8% of chitosan as an excipient (c) spherical granules containing microspheres without excipient. This Figure also shows a comparison between experimental and adjusted values by means of a mathematical model of diffusion through porous solids.

Figure 6 shows a micrography, obtained by scanning electron microscopy, of the spherical and heterogeneous granules containing chitosan microspheres encapsulating the didanosine drug. Figure 7 shows digital photographs from (a) spherical granules with 4.8% of chitosan (b) spherical granules without excipients; (c) cylindrical granules of VIDEX^®EC (commercially available granules with homogeneous polymeric matrix and enteric coating which vehiculate the drug didanosine). Figure 8 shows the isotherms of mucin adsorption obtained for the in vitro characterization of the mucoadhesion from: (a) lyophilized microspheres (b) spherical granules containing microspheres and chitosan as an excipient (c) spherical granules containing microspheres prepared without excipients.

Figure 9 shows the inverted gut scheme (1) incubated in oxygenated medium TC- 199 (2), used for the ex vivo characterization of the mucoadhesion of granules containing microspheres and chitosan as an excipient and comparison with other pharmaceutical forms.

Figure 10 shows the permeation of didanosine drug through the duodenal membrane when vehiculated in crushed tablets of didanosine (a) in comparison with other pharmaceutical forms: granules containing microspheres and without excipient (b) and granules containing microspheres and chitosan as an excipient (c). (* → p < 0.05; ** — >• p < 0.10; *** → p < 0.01). Figure 11 shows the permeation of didanosine through the jejunal membrane when vehiculated in crushed tablets of didanosine (a) in comparison with other pharmaceutical forms: granules containing microspheres and without excipient (b) and granules containing microspheres and chitosan as an excipient (c). (*→ p < 0.05).

Figure 12 shows the permeation of didanosine through the ileal membrane when vehiculated in crushed tablets of didanosine (a) in comparison with other pharmaceutical forms: granules containing microspheres and without excipient (b) and granules containing microspheres and chitosan as an excipient (c).

Figure 13 shows the release of didanosine in TC- 199 medium (with glucose) containing the duodenum portion when vehiculated in crushed tablets of didanosine (a) in comparison with other pharmaceutical forms: granules containing microspheres and without excipient (b) and granules containing microspheres and chitosan as an excipient (c). (* — > p < 0.05; ** -> p < 0.01 ; *** -> p < 0.10). Figure 14 shows the release of didanosine in

TC- 199 medium (with glucose) containing the jejunum portion when vehiculated in crushed tablets of didanosine (a) in comparison with other pharmaceutical forms: granules containing microspheres and without excipient (b) and granules containing microspheres and chitosan as an excipient (c). (* — > p < 0.05; ** → p < 0.01).

Figure 15 shows the release of didanosine in TC- 199 medium (with glucose) containing the ileum portion when vehiculated in crushed tablets of didanosine (a) in comparison with other pharmaceutical forms: granules containing microspheres and without excipient (b) and granules containing microspheres and chitosan as an excipient (c). (* — > p < 0.05; ** → p < 0.01). Detailed Description of the Invention

The present invention relates to the functional product, as well as to the process for obtaining mucoadhesive granules composed of chitosan nano- and/or microspheres associated with a granulation excipient. These granules represent a functional device which is economically feasible and more effective for the administration of drugs and nutraceuticals, as compared to formulations containing homogeneous microspheres or granules (with physical mixture of its compounds), both used individually. The mucoadhesive granules described herein can be used for the incorporation/encapsulation of active compounds of various natures, such as hydrophilic, hydrophobic, amphophilic, charged or neutral, toxic or susceptible to degradation through the action of temperature, pH, oxygen and light. Initially, the active compound is incorporated into chitosan nano- and/or microspheres, which can be cross-linked by means of conventional agents and methods. The formulation of these microspheres, which involves the molar mass of chitosan, the ratio between chitosan concentrations and cross-linking agent, as well as the association with other biopolymers, is responsible for the modulation of the incorporation/encapsulation capacity and the release of the active compound, preserving the physicochemical properties thereof, reducing its toxicity and increasing its action efficiency in relation to the free form of the active compounds or homogeneous mixture with excipients. Chitosan grants mucoadhesive properties to the microspheres and granules produced, providing, through interaction with the mucus, greater residence time of the formulation on the surface of mucosae in relation to other biopolymers. Mucoadhesion occurs mainly due to the positive charge of chitosan, which promotes the electrostatic interaction with the mucus which is positively charged. The use of chitosan in the form of nano- and/or microspheres increases mucoadhesion in relation to the free polymer, owing to the larger surface area available for interaction with the mucus. Further, if these nano- and/or microspheres are associated with granules which utilize chitosan itself or a different granulation excipient, its mucoadhesion capacity is increased, because mucoadhesion properties of both the free polymer and the polymer in the form of nano- and/or microspheres are contemplated, thus producing a larger surface area for mucoadhesion. As a consequence of the above described properties, these granules perform jointly the functions of increased mucoadhesion and controlled release, improving and extending the absorption of active compounds. Chitosan is a natural polymer, whose use in food diets is widely known, thus ensuring biocompatibility even in its free form for oral use and through oral cavity. Other mucosal administration routes, such as nasal, vaginal and ocular require control of the dosage and concentration of the polymer, which can be attained by means of the formulations of the nano- and/or microspheres. Such microspheres, along with excipients, are used in the production of granules through extrusion and spheronization. The process for producing these functional granules is simple, sterilizable, and scalable, and does not make use of organic solvents. It can operate in a discontinuous or semicontinuous manner. The produced granules can also be coated with gastroresistant polymers, so as to protect and retain the activity of pH-sensitive active compounds, when used in oral administration. Looking at the problems present in the state of art, the mucoadhesive granules described herein (heterogeneous granules), composed of chitosan nano- and/or microspheres and a granulation excipient, are capable of incorporation and controlled release of active compounds and exhibit several advantages, such as: incorporation/encapsulation of active compounds of various natures; modulation and control of the encapsulation and controlled release of active compounds; mucoadhesion superior to homogeneous granules and nano- or microparticles used individually; improved product biocompatibility; preservation of product contamination with organic solvents, as well as the environment, by utilizing aqueous means during its production; easiness of operation, control and scaling of the production process; allow for covering with other polymers so as to meet the requirements of the administration route and preserving active compound properties.

There is no mention of the development of granules consisting of heterogeneous domains compound of nano and/or microspheres and a granulation excipient. Microspheres or nanospheres provide protection and control of the sustained release of the active compound, which is very desirable and not observed in currently existing granules. Moreover, on account of having chitosan in their formulation, the granules produced in the present invention exhibit mucoadhesive properties, not present in the formulations disclosed in the literature. It is highlighted, for explanation purposes, that the usual literature does not discriminate, in terms of nomenclature, between microparticles and homogeneous granules of polymers, whose dimensions are much superior to micrometer (10^~6 m). Therefore, such granules are called microparticles, although they are constituted by just a physical mixture of the polymer with the drug and other excipients, which mixture is properly moistened, extruded and spheronized. In this context, the present invention discloses functional heterogeneous granules containing chitosan microspheres which, in addition to having stronger mucoadhesion properties than homogeneous granules, also have these properties modulated both by the composition of microspheres and the composition of the free chitosan or another granulation excipient used in the constitution of the granules. Additionally, the presence of microspheres provides granules with sustained release properties of the active compounds with grater flexibility of modulation and control. The process for production of these granules must be sterilizable, scalable, in addition to be able to integrate simple unit operations and make use only of aqueous means (exempt from organic solvents).

The existing granules are monolithic or also called of homogeneous domain. The granules produced in the present invention are heterogeneous as they present chitosan microspheres in their composition, which provides improved properties.

The granules of the present invention have a size in the order of millimeters and the nano- or microspheres constituting them have a size in the range of nanometers (10^"9m) and/or micrometers (10^~6 m). They can be produced through a sterilizable process and are susceptible to scaling and application in the industrial sector. They exhibit different physicochemical properties dependent on the formulation and the operational conditions of the production process. Also have various applications in the pharmaceutical, medical/veterinary and food fields. These granules associate functions of mucoadhesion and controlled release, increasing the absorption efficiency of compound actives through mucosae. The mucoadhesive properties of chitosan, a natural polymer present in nano/microspheres and as an excipient in the constitution of the polymeric matrix of the granules, increase the residence time of the system as a whole on the surface of mucosae. Additionally, those granules containing nano/microspheres are able to encapsulate and release in a more slow and controlled fashion any active compounds as compared to conventional granules. These granules can also be coated with gastroresistant polymers, so as to protect and retain the activity of pH-sensitive active compounds, when used in oral administration.

Therefore, those heterogeneous granules compound of nano/microspheres associated with free chitosan or another granulation excipient represent more effective functional devices for the oral administration of drugs and nutraceuticals, when compared to nano/microspheres or homogeneous matrix granules (or monolithic granules) used individually.

The invention described herein relates to a mucoadhesive granule comprising a granulation excipient and at least one active compound incorporated into chitosan micro- and/or nanoparticles. Many are the active compounds which can be incorporated into chitosan micro and/or nanoparticles of the present invention. As an active compound of the present invention it is used, preferably, at least one antiretroviral compound. Among the existing antiretroviral compounds, it is preferably used, in the present invention, at least one reverse transcriptase inhibitor. Among the existing reverse transcriptase inhibitors, it is preferably used didanosine. The micro- and/or nanoparticles present in the mucoadhesive granule of the present invention have a mean size from 100 nanometers to 100 micrometers. In addition to micro- and/or nanoparticles, the mucoadhesive granule of the present invention also comprises a granulation excipient which can consist of a binding agent and/or a bioadhesion agent. The binding agent used in the present invention is selected from the group consisting of starch, gelled starch, pre-gelled starch, sodium carboxymethylcellulose, alginate, chitosan, or any mixture thereof. Preferably, the binding agent used in the present invention is chitosan. The bioadhesion agent used in the present invention is selected from the group consisting of chitosan, alginate, bioadhesive materials, or any mixture thereof. Preferably, the bioadhesion agent used in the present invention is chitosan. The mucoadhesive of the present invention can also be covered with gastroresistant polymers, increasing the protection of the active compound and assuring its release at the target site. The present invention also relates to pharmaceutical compositions comprising the mucoadhesive granule described herein.

Yet, the invention to which concerns this document relates to a process for obtaining the mucoadhesive granule, said granules previously described, comprising the steps of: a) preparing a solution A containing chitosan; b) preparing a solution B containing at least one active compound; c) obtaining a primary mixture comprising chitosan micro- and/or nanoparticles incorporated with the active compound; d) admixing said chitosan micro- and/or nanoparticles incorporated with the active compound with a granulation excipient forming a secondary mixture; and e) obtaining said mucoadhesive granule from said secondary mixture.

In the step (a) of the process for obtaining mucoadhesive granules, the solution A containing chitosan also comprises water and at least one pH-adjusting agent. The pH- adjusting agent can be composed of acids and/or organic and/or inorganic bases and should provide a good solubilization of chitosan, as well as being compatible with the maintenance pH of the activity from the active principle (compound) to be used. The active compound described in step (b) of the process for obtaining mucoadhesive granules preferably comprises at least one antiretroviral compound. Among the existing antiretroviral compounds, the present invention preferably uses at least one reverse transcriptase inhibitor. Among the existing reverse transcriptase inhibitors, the present invention preferably uses didanosine. In step (b) of the process for obtaining the mucoadhesive granule of the present invention, solution B can also comprises, in addition to the active compound, at least one cross-linking agent and at least one pH-adjusting agent. The cross-linking agent used in solution B is selected from the group consisting of sodium tripolyphosphate, glutaraldehyde, glyceraldehyde, epichloridrine, genipin, alginate, compounds which bind to chemical groups available in the chitosan molecule, or any mixture thereof. The pH-adjusting agent can be composed of acids and/or organic and/or inorganic bases and should be compatible with the maintenance pH of the activity from the active principle (compound) to be used.

In step (c) of the process for obtaining mucoadhesive granules, the obtainment of a primary mixture comprising chitosan micro- and/or nanoparticles incorporated with the active compound, occurs through the use of a technique selected from the techniques consisting of coacervation, ionotropic gelation, single emulsion, multiple emulsion, or any combination thereof. The obtainment of a primary mixture comprising chitosan micro- and/or nanoparticles incorporated with the active compound as described in step (c), can occur through addition of solution B (step (b)) to solution A (step (a)). Preferably, solution B is added slowly to solution A. Also preferably, the addition of solution B to solution A occurs under stirring of the formed mixture (primary mixture). During or after the addition of solution B to solution A, occurs the formation of chitosan micro- and/or nanoparticles incorporated with the active compound. The formed mixture containing these particles is called the primary mixture. After forming the primary mixture containing chitosan micro- and/or nanoparticles incorporated with the active compound, the mixture can be subjected to stirring, centrifugation and drying processes, aiming at separating micro- and/or nanoparticles from the remaining components of the mixture. Drying is performed for the purpose of obtaining chitosan micro- and/or nanoparticles having a moisture content from 0.01% to 80%. Preferably, the moisture content of chitosan granules after drying ranges from 70% to 80%.

In step (d) of the process for obtaining the mucoadhesive granule, a granulation excipient is added to the chitosan micro- and/or nanoparticles separated from the primary mixture through the stirring, centrifugation and drying processes. After adding the granulation excipient to the micro- and/or nanoparticles incorporated with the active compound, a secondary mixture is obtained. The granulation excipient utilized can be composed of a binding agent and/or a bioadhesion agent. The binding agent used as a granulation excipient in the present invention is selected from the group consisting of starch, gelled starch, pre-gelled starch, sodium carboxymethylcellulose, alginate, chitosan, or any mixture thereof. Preferably, the binding agent used in the present invention is chitosan. The bioadhesion agent used as a granulation excipient in the present invention is selected from the group consisting of chitosan, alginate, bioadhesive materials, or any mixture thereof. Preferably, the bioadhesion agent used is chitosan. The obtainment of the mucoadeshive granule aimed by the invention (step (e) of the process for obtaining the mucoadhesive granule), occurs through homogenization, extrusion and drying of the secondary mixture obtained in step (d). The obtainment of the mucoadhesive granule may also occur through homogenization, extrusion, spheronization and drying of the secondary mixture obtained in step (d). The granule obtained in step (e) may also be covered with a gastroresistant polymer, which provides a higher resistance and accordingly a higher protection to the active principle, ensuring its release at the target site.

The present invention is illustrated by means of examples which demonstrate the higher mucoadhesion of the heterogeneous granules compound of chitosan microspheres and chitosan as an excipient, the controlled release and the production process thereof. In this example, the incorporation of didanosine was carried out, an anti-HIV drug belonging to the class of reverse transcriptase inhibitors, which is sensitive to the pH of the medium. The examples described herein should be construed as forms of carrying out the invention and, therefore, are not intended to restrain the scope of protection of the same.

The compounds incorporated into the granules, which are the subject matter of the present patent, have a chemically defined composition, are not toxic if ingested, and are able to keep labile substances encapsulated and/or incorporated. The produced granules, when administered orally, can be covered with gastroresistant polymers for protecting drugs which are labile to the pH or drugs having local action on the bowel.

The semicontinuous production process of the granules containing a granulation excipient of chitosan micro- and/or nanoparticles incorporated with the active compound was carried out according to the steps detailed in the following and shown in Figure 1 : initially, Solution A was prepared through solubilization of chitosan under 70% of the total water volume, followed by the addition of glacial acetic acid. After a 24-hours interval under magnetic stirring, the remainder of water was added with continuous stirring for more 24 hours. The pH of this solution should be around 4.8 in order to assure the solubilization of chitosan and the activity of didanosine drug, and its attainment depends on the used acetic acid amount. Accordingly, Table 1 shows the correlation between the concentration of acetic acid required for solubilization of a given chitosan concentration and the attainment of a final pH equal to 4.80.

TABLE 1

Chitosan Acetic Acid

Concentration (%) (m/v) Concentration (%) (v/v)

1.33 0.46

2.00 0.75

3.00 1.00

4.00 1.25

4.67 1.87

For preparing solution B, the drug was mixed with sodium tripolyphosphate (10.00 % in relation to chitosan mass) and magnesium hydroxide for pH maintenance (30.00 % in relation to chitosan mass) at room temperature and in aqueous solution. This solution was subjected to sonication for 20 minutes.

In a subsequent step, solution B was added dropwise to solution A under mechanical stirring. The formed mixture (primary mixture) remained under stirring and, at 90 minutes of stirring, the particle suspension formed was centrifuged for 20 minutes at 3,000 rpm, in order to separate the microspheres and the non-incorporated drug. The chitosan used was produced by Polymar S. A (Fortaleza-Ce), with a deacetylation degree, determined by potentiometric titration, equal to 81.61 + 1.05 % and a molar mass, determined by viscosimetry, equal to 105 kDa. The sodium tripolyphosphate used was from the trademark Synth, whereas magnesium hydroxide was from Sigma. The active compound tested was didanosine, an anti-HIV drug used in AIDS therapy, supplied by company Labojen S.A. (Indaiatuba-SP).

After separation through centrifugation, the sedimented material, containing the microparticles, was oven- dried at 40⁰C (104⁰F) up to 75 % residual moisture for producing the granules. In this step, the excipient granulation was added and mixed, forming a heterogeneous matrix semisolid mass containing the free excipient and the microspheres called the secondary mixture. Said mass (secondary mixture) was then extruded in a device having holes in the order of 1 mm and subsequently spheronized in conventional devices. The yield and size of the remaining granules depend upon operational conditions used in the operations of granulation and spheronization.

The residual moisture of 75% must be adjusted as a function of the excipients and active compounds used. In processes operating in batch (discontinuous), the drying of microspheres can also be carried out in a lyophilizer or by spray- drying, which ensures their stability during storing. The addition of water in order to obtain a wet mass capable of extrusion and spheronization should be carried out upon utilizing these microspheres for producing the granules.

The physicochemical properties of chitosan microspheres can be modulated through composition of the formulation and the operational conditions of incorporation/encapsulation of the active compound and cross- linking. Formulation variables include chitosan concentrations, cross-linking agent, active compound, deacetylation degree, and molar mass of chitosan, residual moisture, kind and concentration of excipients used in the production of the granules. Operational conditions which have influence upon the product properties are: stirring rate used in the production of microspheres, type of device and processing conditions during the extrusion and spheronization of the granules.

Immediately after incorporation/encapsulation, particles were analyzed by optical microscopy. The obtained images were treated using the software Image Tool 3.0 (The University of Texas Health Science Center in San Antonio). From this image treatment, the mean particle size and size distribution histogram were determined. The mean size was obtained in three different micro grafies, wherein 400 to 500 particles had their size measured. The obtained value was very near to the one obtained by LUBBEN et al (LUBBEN, I. M. V. D., VERHOEF, J. C, AELST, A. C. V., BORCHARD, G., JUNGINGER, H. E.βiomaterials, v.22, p.687-694, 2001) for chitosan microspheres prepared through coacervation with sodium sulphate.

The size distribution histogram of the microspheres obtained with 25 mg didanosine/mL is shown in Figure 2. It is noted that the distribution exhibit a behavior very similar to a regular distribution, with a mean diameter of 11.42 ± 4.35 μm. Figure 3 shows a micrography analyzed by the software Image Tool 3.0 for mean size determination and size distribution of chitosan microspheres prepared through the ionotropic gelation technique using tripolyphosphate as a cross- linking agent.

In addition to the microspheres, it can be seen, in Figure 3, the presence of needle-type crystalline structures.

Such crystalline structures occur due to the non-encapsulated drug itself which incorporates into the surface of the granules and the vicinity of the microspheres.

Table 2 shows the effects of varying TPP concentration on the incorporation efficiency, intumescence degree and didanosine loading. It can be noted in Table 2 that the increase on the didanosine concentration promoted a increase on the incorporation efficiency and didanosine loading, wherein for the drug loading, increase was higher than 100 %. The intumescence degree of microspheres was virtually not affected by the incrbase on didanosine concentration.

TABLE 2 ddl Incorporation Loading* Intumescence

Concentration efficiency (%) degree (%)

(mg/mL)

39.00 36.47 1433 465 ± 15

25.00 25.54 643 460 ± 12

*(mg of ddl/g of chitosan)

The release assays of didanosine from the microspheres were carried out through the suspension of 60 mg of particles in 10 mL of simulated enteric juice (SES) without pancreatin under reciprocal stirring (150 rpm and 37⁰C (98.6 F)). SES was prepared according to USP XXIII (United States Pharmacopea). In predefined time intervals samples were taken from the release medium for the quantification of didanosine through spectrophotometry in a wavelength equal to 248 nm. Experimental data were adjusted by means of the solute diffusion model in the interior of solid spheres, described by Fick's law, with a analytical solution obtained by Taylor's series. In adjusting parameter k were utilized 1,000 terms from the series and maximum likelihood method described by DRAPER and SMITH (DRAPER, N. R., SMITH, H. Applied Regression Analysis, Second Edition, John Wiley & Sons, 1981). This method takes into account the uncertainties from all experimental measures in the adjustment. In the limit when t tends to zero, the series is represented by the equation:

where k is the parameter defined by the ratio between the drug diffusion coefficient in the structure and the square of particle radius (D/R ), M(t) is the drug mass released in a given time t and M_∞ is the total released mass.

Table 3 shows values of k (ratio between the drug diffusion coefficient and the square of particle radius, D/R²) adjusted by the model for chitosan microspheres having different didanosine concentrations and granules containing the microspheres and chitosan as an excipient.

The increase on didanosine concentration promoted a slight reduction on constant k, as observed in Table 3 and confirmed by Figure 4 which shows the model adjusted to the experimental data. It is noticed good concordance between the model and the experimental data.

TABLE 3

Particle k (D/R²) (h^"1)

Microspheres with 10 % TPP - 39 mg ddl/mL 2.72 x 10 -3

Microspheres with 10 % TPP - 25 mg ddl/mL 4.00 x 10 -3

Granules having microspheres with 10 % of TPP - 39 mg ddl/mL 2.76 x 10 -3

Excipient: 4.8% of Chitosan

Granules having microspheres with 10 % of TPP - 39 mg ddl/mL 3.51 x 10 -3

Without excipient

Physical mixture granules - without microspheres 22.35 x 10 -3

Granules produced and analyzed through scanning electron microscopy exhibited spherical structures on their surfaces (Figure 6). It should be appreciated that, after centrifugation of the microspheres in the preparation process thereof, the pellet which contains these microspheres was not washed for removing non-encapsulated didanosine, which, under these conditions tends to crystallize on the surface of the particles in the drying process prior to extrusion and spheronization. This crystallization causes microsphere sizes to become larger when compared to the ones obtained in micrographies of isolated microspheres (Figures 3 and 6).

Table 4 shows geometric parameters of granules containing microspheres and 4.8% of chitosan as an excipient, granules without excipients, and granules of Videx^® EC

(commercially available granules with homogeneous polymeric matrix and enteric coating which vehiculate the drug didanosine).

The values from geometric parameters were computed by the software UTSHCSA Image Tool 3.0 from digital photographs of the granules scattered upon a dark surface.

TABLE 4

Geometric 4.8 % of Without Videx* ⁵ EC parameters Chitosan Excipient

Elongation 1.18 ± 0.1 1 1.36 ± 0.30 1.43 + 0.31

Spherocity 0.86 ± 0.09 0.89 + 0.14 0.86 ± 0.10

Feret's Diameter 1.18 ± 0.11 0.91 + 0.22 1.52 ± 0.28

(mm)

Figure 7 shows some photographs used for this purpose. It is noted that there are no substantial differences between granules obtained with the various excipients used.

Spherocity values were higher than 0.80, what is deemed satisfactory. Feret's diameters were very near from 1 mm which is suitable for filling gelatin capsules. Granules containing 4.8% of chitosan exhibited Feret's diameters higher than 1 mm and granules without excipient exhibited a value lower than 1 mm, suggesting that the increase on the diameter is due to the added excipient. Geometric parameters were also very similar to the results for Videx^® EC granules, which are didanosine gastroresistant granules produced by Bristol-Myers Squibb (Princeton - USA), at least the cylindrical form of the granules used in this example.

As a control, granules containing a homogeneous physical mixture of didanosine, whose total chitosan composition was the same as the one present in the microspheres and granules having 4.8% of chitosan as an excipient were obtained. In a vessel, it was carried out the mixture of the following powdered compounds: chitosan, magnesium hydroxide, sodium tripolyphosphate and didanosine (the amount is based on the incorporation efficiency of the assay in which the microspheres were prepared). Powder homogenization was carried out in a reciprocal stirrer for 30 minutes at 500 rpm. Next, distilled water was added until a wet mass capable of extrusion is obtained. It was not possible to carry out spheronization due to the plasticity of the wet mass, and hence cylindrical granules only extruded were obtained. Figure 5 shows release profiles from the cylindrical granules of the physical mixture (Figure 5a), spherical granules containing chitosan microspheres and 4.8% of chitosan as an excipient (Figure 5b) and spherical granules containing microspheres without any excipient. It is noted that the formulation and earlier formation process of the microspheres is very important both in the obtainment of spherical granules and the release of didanosine. The full release of didanosine from physical mixture granules occurred very rapidly (in nearly 10 minutes all didanosine was released), whereas for granules containing chitosan microspheres, the full release occurred in about 100 minutes. It should be appreciated that the model developed for spheres has also exhibited good concordance with the experimental data when adjusted to the release profile from cylindrical granules (Figure 5a), showing low sensitivity to geometry, at the spherocity level of 0.86-0.89.

Table 3 shows that constant k was almost 10 times higher for the cylindrical granules from the physical mixture having 3 mm in height, as compared with microspheres.

In vitro mucoadhesion from the granules was determined by the method developed by HE et al. [HE, P., DAVIS, S. S., ILLUM, L., International Journal of Pharmaceutics, v.166, p.75-68, 1998], though mucin adsorption isotherms in chitosan microspheres. Mucin concentration was measured by means of colorimetric method PAS (Periodic Acid/Schiff). This method was used for the study of mucoadhesion on account of the following reasons: (i) adsorption mimics the process in vivo; (ii) allows for the obtainment of quantitative data; and (iii) simplicity and reproductivity. According to the method, about 20 mg of particles in contact with 5 mL of mucin solution (Type III from pigs and 1 % of sialic acid, trademark Sigma) in different concentrations. After 3.5 h in contact under reciprocal stirring (150 rpm and 37°C (98.6° F), the mucin amount on the supernatant from this suspension was dosed. Experiments were run in triplicate for each concentration of mucin solution. The amount of adsorbed mucin is given by the difference between the initial amount of mucin in solution and the amount of mucin in solution after contact with the particles.

Mucin was dosed by the spectrophotometric method PAS (Periodic Acid/Schiff) described by MANTLE and

ALLEN [MANTLE, M., ALLEN, A., Biochemical Society

Transactions, vol. 6, pp. 607-609, 1978]. This method is based on the measurement of polysaccharides which are oxidized by periodate. It consists in the oxidation of the groups 1 -2 glycol, producing aldehydes. These aldehydes react with unstained fuchsin, called Schiff s reagent, providing a bright-pink-coloured compound which is measured in the wavelength equal to 555 nm.

From mucin quantification, it was possible to compute the concentration of mucin in equilibrium in liquid phase and the concentration of mucin in equilibrium adsorbed in the particles.

Plotting the mucin concentration in the particles as a function of mucin concentration in liquid phase, both in equilibrium, the mucin adsorption isotherm is obtained.

Experimental data were adjusted to Langmuir's model. Equation

(2) represents Langmuir's model:

q* = ^ A .C

^K" ^{+ C} * Eq.(2)

where g* is the concentration of solute in equilibrium in solid phase (in this case, mg of mucin/mg of particles), q_MAX is the maximal amount of solute adsorbed by the particles (in mg of mucin/mg of particles), C* is the concentration of solute in equilibrium in liquid phase (in mg of mucin/mL of solution), and K_D is the Langmuir's constant (in mg of mucin/mL of solution). All produced granules showed good adjustment to Langmuir's model. Table 5 shows coefficients from adsorption isotherms determined for the studied particles. It is found that granules containing microspheres showed values of q_MAX (maximal adsorption capacity) of the same order of magnitude (from 0.40 to 0.80 mg of mucin/mg of particles), of the same order of magnitude from lyophilized microspheres (0.52 mg of mucin/mg of particles), making evident that adsorption is caused by microspheres, that is, the process of spheronization and the addition of the excipient did not change substantially the maximum adsorbed amount.

The values obtained for regression coefficients (r) were as well as the values obtained by HE et al. (1998). On the other hand, the comparison between the values of the coefficients from the models {q^ux and K_D) becomes troublesome as in this work the unit of measure for the solute concentration in solid phase (q*, in this case, mg of mucin / mg of particles) is different from the unit of measure presented by HE et al. (1998), given in (μg of mucin)/(cm² of area from microspheres).

Coefficient K_D, which express the affinity of the adsorbent for mucin, showed substantial variations resulting from spheronization and addition of excipients. The higher is coefficient K_D, lower is the affinity of mucin for the adsorbent, and vice-versa. Upon spheronizing microspheres without excipients, there was a substantial increase of the coefficient K_D, from 5.18 to 18.42 mg of mucin/niL of solution, which showed a reduction of the affinity of mucin for the adsorbent. This phenomenon can be explained by the geometry of the particles. The individual microspheres exhibit a contact surface area much larger than that of its granules. The mucin diffusion resistance in the interior of the granules until the adsorption sites of microspheres promoted a reduction in its affinity.

On the other hand, it was observed that the addition of 4.8% of chitosan promoted a substantial increase on the affinity, that is, a reduction on the coefficient K_D. Mucin is composed of sialic acid which exhibit negative charge in its structure, and electrostatic interactions thereof with positive charges from additional chitosan chains promote the increase of the affinity of mucin for particles, reducing the value of K_D. Figure 8 shows the adsorption isotherms and its respective adjusted models. Table 5 shows coefficients from Langmuir's model for the adsorption isotherm of mucin in the isolated microspheres, in granules containing microspheres without excipients and in granules containing microspheres and 4.8% of chitosan as an excipient. The regression coefficients from adjusted models (r) showed values ranging from 0.94 to 0.99 (Table 5).

TABLE 5

_, . _Λ Langmuir's Model

Particles

Lyophilized Microspheres 0.52 5.18 0.9499

Granules Without Excipients 0.80 18.42 0.9435 Granules with 4.8 % of Chitosan 0.40 1.24 0.9910 * CI_ML-_LX in mg of mucin/mg of particles; K_D in mg of mucin/mL of solution.

Heterogeneous granules were subjected to the inverted intestine assay for comparison of permeation of ddl in its commercial pharmaceutical form and the heterogeneous granules without excipient and containing 4.8% of chitosan as an excipient

Inverted intestine technique shows some advantages like simplicity, speed, good reproductivity and low cost. As a drawback, it could be cited the in vitro nature of the assay, in which the animal model does not reflect the results of real absorption obtained with humans. Other drawbacks result from the role of physiological parameters, such as transit time or the presence of food, the influence from irrigation and the nervous system. The function of these parameters in the absorption can be studied in situ with in vivo models.

In the inverted gut assays, buffer TC- 199 was used as an incubation medium. For one liter of deionized water, 8.470 g of Sodium chloride, 0.340 g of Potassium chloride, 0.126 g of calcium chloride, 0.595 g of dibasic sodium phosphate and 1.801 g of glucose were added in this sequence. According to BARTHE et al [BARTHE, L., WOODLEY, J., HOUIN, G., Fundamental and Clinical Pharmacology, v. 13, p. 154-168, 1999] this solution is capable of keeping the cells viable for up to 2 hours. Adult male rats of Wistar strain, weighing between 250 and 300 g, were anesthetized with ethyl ether and the small intestine was immediately dissected, washed with TC- 199 solution kept at 1O⁰C (50° F). Small gut was gently inverted with the aid of a flexible stem (- 2.5 mm in diameter) with its extremity protected by a thin silk fabric. Next, small intestine was divided in 3 segments, each having 8 cm. The first segment was measured from the pylorus (proximal duodenum), the second segment was measure from the duodenal-jejunal angle (proximal ejunum) and the third segment was measured above the cecum (distal ileum). Each segment had one of its ends closed with a suture thread and immediately filled with TC- 199 without addition of glucose. The other end of the enteral segment was closed in the same manner, in such a way that the intestinal sac is 6 cm in length. Each gut segment 1 was incubated separately (Figure 9) in a system containing TC- 199 with glucose 2 and the drug (0.0875 mg/mL) in the different presentation forms. The medium was kept at 37⁰C (98.6 F⁰) under oxygenation (O₂ : CO₂ - 95 : 5) 3 and gentle stirring (Figure 9). After the predetermined time intervals, intestinal segments were removed from the incubation medium, carefully washed and the content was filtered. Didanosine concentration permeated through intestinal membrane 4 was determined by UV spectrophotometry (λ = 248 nm). Didanosine content in the external medium (not permeated through the membrane) was also determined by spectrophotometry. Studied time intervals were of 5, 10, 20, 40, 80, and 120 minutes. For each time three animals (n = 3) were utilized. Results represent mean ± standard deviation.

Three pharmaceutical forms were compared, which are described in the following:

• Free-form commercial drug (Trademark Farmanguinhos): buffered tablets containing 100 mg of didanosine. The mass of each tablet is of about 850 mg, that is, approximately 750 mg correspond to excipients of which buffer is the main one. Tablets were crushed generally for use in the test;

• Spherical granules containing chitosan microspheres, without any excipient;

• Spherical granules containing chitosan microspheres and 4.8% of chitosan as an excipient.

In order to ensure that any compound present in the medium interferes with the analysis of didanosine, experiments were also carried out for the time interval of 120 minutes, using three intestinal segments of three animals (n = 3), in the absence of any pharmaceutical form. No interfering compounds in the wavelength used for didanosine reading (248 nm) were observed. The choice of didanosine concentration added to the incubation medium was based on the following information:

• Dosage of the commercial drug, Videx^® EC (enteric-coated) is of 400 mg/day, which, for an individual weighing 75 kg (165.34 pounds), correspond to 5.33 mg/Kg of body mass.

• The package insert of the commercial drug reports that for a 7.00 mg/Kg of body mass dosage, absorption is of 33% in adult humans. This dosage was chosen that, for a rat weighing approximately 250 g, corresponds to 1.75 mg of the drug. In this way, we would also have absorption data for comparison purposes.

Figures 10 to 12 show permeation profiles of the drug through intestinal segments. Despite the small number of animals utilized in the experiments, it was possible to find that the deviations were relatively low in terms of in vivo assays.

It can be observed in Figure 10 that the drug absorptions by the duodenum in 120 minutes were of about 34, 20 and 16 %, respectively, for granules containing 4.8% of chitosan as an excipient, granules without excipient and free-form commercial drug (crushed tablets). These results showed that heterogeneous granules containing the microspheres promoted an increase on ddl absorption, thanks to its mucoadhesion. It is important to mention that after the addition of the granules to TC- 199 with the intestinal sac, it was possible to notice adhesion of the granules to the gut sac wall. The permeation of the free-form commercial drug in the course of time reached a baseline where the absorption percentage became virtually constant. On the other hand, the absorption of the drug contained in granules with 4.8% of chitosan did not reach a baseline, becoming evident a trend towards an increase on absorption even at 120 minutes later. It is likely that the drug absorption had been still greater for a period larger than 120 minutes, but the longest time utilized in the kinetic studies was of 120 minutes in order to assure the viability of cells from the intestinal segment. Granules without excipient have also shown this trend towards an increase of absorption after 120 minutes, but in lesser proportion than granules containing 4.8% of chitosan. It is also observed in Figure 10 that after 20 minutes ddl absorption was slower for the granules. Tukey's test showed that until 40 minutes there are no substantial differences between the curves, but for 80 and 120 minutes, granules containing 4.8% of chitosan as an excipient showed substantial differences in respect to the crushed didanosine tablet and the granules without excipient, evidencing the higher mucoadhesion thereof.

According to GUO et al. (GUO, J., PING, Q., JIANG, G., DONG, J., QI, S., FENG, L., LI, C, International Journal of Pharmaceutics, v.278, p.415-422, 2004), chitosan increases drug absorption since it acts on the opening of narrow junctions between cells.

KOTZE et al [KOTZE, A.F., LUEβEN, H.L., LEEUW, B.J., BOER, A.B.G., VERHOEF, J.C., JUNGINGER,

H.E. , Journal of Controlled Release, v.51, p.35-46, 1998] have compared the effect from different chitosan salts and N-trimethyl- chitosan chloride on the permeability of epithelial cells (Caco-2).

Such as GUO et al. (2004), they have also found that chitosan is able to open the narrow junctions of epithelial cells making easier the permeation of hydrophilic drugs.

Figures 11 and 12 showed that there were no significant differences in didanosine absorption by jejunum and ileum according to Tukey's test. Absorption curves are almost overlaid for each presentation form of didanosine. It was also found that under the same conditions, permeation through duodenal membrane was superior to permeation through jejunum and ileum membranes. This fact may be associated to the higher number of vilosities and microvilosities of duodenal membranes. These data are consistent with the information on the package insert of the commercial drug, which mentions that absorption in humans is low, of only 33 % of the administered amount. The comparison of ddl absorption in the same pharmaceutical form, in the three intestinal segments showed that duodenum exhibited higher absorption than jejunum and ileum. SINKO et al. (SINKO₅ P.J., PATEL, N.R., HU, P.D., International Journal of Pharmaceutics, v.109, n.2, p.125-133, 1994) used the intestinal perfusion technique in rats and have also found that didanosine absorption is reduced on each subsequent portion of the bowel.

SINKO et al (SINKO, P.J., SUTYAK, J.P., LEESMAN, G.D., HU, P.D., MAKHEY, V.D., YU, H.S.,

SMITH, C. L., Biopharmaceutics & Drug Disposition, v.18, n.8, p. 697-710, 1997) also made a research on didanosine absorption in dogs through injectable administration of didanosine directly to the intestinal portions. They have also found that didanosine absorption in dogs reduces on each subsequent portion of the bowel.

Figures 13 to 15 show didanosine release on incubation mediums for each presentation form. The analysis of the data demonstrated that heterogeneous granules containing microspheres promoted the slowest release of ddl. The use of 4.8% of chitosan as an excipient delayed even further its release. Figures show that didanosine percentage was slightly lower for the medium in which duodenum is located, due to the higher absorption on this segment. On the other hand, jejunum and ileum showed lower absorption and hence the percentage of didanosine available in the medium was higher.

In the case of duodenum (Figure 13), Tukey's test showed that only granules containing 4.8% of chitosan exhibited significant differences in the entire studied period, since absorption by duodenum is higher, what causes didanosine concentration in the medium to be lower.

Tukey's test for jejunum and ileum (Figures 14 and 15) showed that there is a substantial difference between the curves in the initial points, until 40 minutes. The geometry of the granules made the difference in respect to microspheres, making the release slower. From this time on, there were no significant differences and the curves overlaid each other, because absorption intensities for these segments were nearly the same as for the pharmaceutical forms.

Therefore, the duodenal segment exhibited higher didanosine absorption in relation to the other segments (jejunum and ileum). The absorption on this segment was higher because of the larger number of microvillosities and allows for increased adhesion and bioadhesion. Granules containing 4.8% of chitosan exhibited higher absorption by the duodenal segment. According to some authors, such as GUO et al. (2004); KOTZE et al (1998) and DODANE et al. [DODANE, V., KHAN, M.A., MERWIN, J.R,. International Journal of Pharmaceutics, v.182, p.21-32, 1999], such facts can be assigned to the presence of chitosan which aids in absorbing drugs by opening the narrow junctions of the intestinal membrane.

Claims

1. Mucoadhesive granule, characterized in that it comprises:

(a) at least one active compound incorporated in chitosan micro- and/or nanoparticles; and

(b) a granulation excipient.

2. Mucoadhesive granule, according to Claim

1, characterized in that the active compound incorporated into chitosan micro- and/or nanoparticles comprises at least one antiretroviral compound.

3. Mucoadhesive granule, according to Claim

2, characterized in that the antiretroviral compound comprises at least one reverse transcriptase inhibitor.

4. Mucoadhesive granule, according to Claim 3, characterized in that the reverse transcriptase inhibitor is didanosine.

5. Mucoadhesive granule, according to any one of Claims 1 to 4, characterized in that the chitosan micro- and/or nanoparticles have a mean diameter of 100 nanometers to 100 micrometers.

6. Mucoadhesive granule, according to any one of Claims 1 to 5, characterized in that the granulation excipient comprises a binding agent and/or a bioadhesion agent.

7. Mucoadhesive granule, according to Claim

6, characterized in that the binding agent is selected from the group consisting of starch, gelled starch, pre-gelled starch, sodium carboxymethylcellulose, alginate, chitosan, or any mixture thereof.

8. Mucoadhesive granule, according to Claim

7, characterized in that the binding agent is chitosan.

9. Mucoadhesive granule, according to any one of Claims 6 to 8, characterized in that the bioadhesion agent is selected from the group consisting of chitosan, alginate, bioadhesive materials, or any mixture thereof.

10. Mucoadhesive granule, according to Claim 9, characterized in that the bioadhesion agent is chitosan.

1 1. Mucoadhesive granule, according to any one of Claims 1 to 10, characterized in that it is covered with gastroresistant polymers.

12. Pharmaceutical composition, characterized in that it comprises a mucoadhesive granule as defined in any one of Claims 1 to 1 1.

13. A process for obtaining a mucoadhesive granule as defined in any one of Claims 1 to 11, characterized in that it comprises the following steps: a) preparing a solution A containing chitosan; b) preparing a solution B containing at least one active compound; c) obtaining a primary mixture comprising chitosan micro- and/or nanoparticles incorporated with the active compound; d) admixing said chitosan micro- and/or nanoparticles incorporated with the active compound with a granulation excipient forming a secondary mixture; and e) obtaining said mucoadhesive granule from said secondary mixture.

14. Process for obtaining a mucoadhesive granule, according to Claim 13, characterized in that in step (a), the chitosan-containing solution further comprises water and at least one pH-adjusting agent.

15. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 or 14, characterized in that, in step (b), the active compound comprises at least one antiretroviral compound.

16. Process for obtaining a mucoadhesive granule, according to Claim 15, characterized in that the antiretroviral compound comprises at least one reverse transcriptase inhibitor.

17. Process for obtaining a mucoadhesive granule, according to Claim 16, characterized in that the reverse transcriptase inhibitor is didanosine.

18. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 to 17, characterized in that, in step (b), the solution containing an active compound further comprises at least one cross-linking agent and at least one pH-adjusting agent.

19. Process for obtaining a mucoadhesive granule, according to Claim 18, characterized in that the cross- linking agent is selected from the group consisting of sodium tripolyphosphate, glutaraldehyde, glyceraldehyde, epichloridrine, genipin, alginate, compounds which bind to chemical groups available in the chitosan molecule, or any mixture thereof.

20. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 to 20, characterized in that in step (c), the primary mixture is prepared through a technique selected from coacervation, ionotropic gelation, single emulsion, multiple emulsion, or any combination thereof.

21. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 to 20, characterized in that, in step (c), the obtainment of the primary mixture occurs through the addition of solution B to solution A.

22. Process for obtaining a mucoadhesive granule, according to Claim 21, characterized in that the addition of solution B to solution A occurs under stirring.

23. Process for obtaining a mucoadhesive granule, according to any one of Claims 21 or 22, characterized in that solution B is added slowly to solution A.

24. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 to 23, characterized in that the primary mixture obtained in step (c) is stirred, centrifuged and dried.

25. Process for obtaining a mucoadhesive granule, according to Claim 24, characterized in that the drying occurs until micro- and/or nanoparticles having a moisture content between 0.01% and 80% are obtained.

26. Process for obtaining a mucoadhesive granule, according to Claim 24, characterized in that the drying occurs until micro- and/or nanoparticles having a moisture content between 70% and 80% are obtained.

27. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 to 26, characterized in that, in step (d), the granulation excipient comprises a binding agent and/or a bioadhesion agent.

28. Process for obtaining a mucoadhesive granule, according to Claim 27, characterized in that the binding agent is selected from the group consisting of starch, gelled starch, pre-gelled starch, sodium carboxymethylcellulose, alginate, chitosan, or any mixture thereof.

29. Process for obtaining a mucoadhesive granule, according to Claim 28, characterized in that the binding agent is chitosan.

30. Process for obtaining a mucoadhesive granule, according to any one of Claims 27 to 29, characterized in that the bioadhesion agent is selected from the group consisting of chitosan, alginate, bioadhesive materials, or any mixture thereof.

31. Process for obtaining a mucoadhesive granule, according to Claim 30, characterized in that the bioadhesion agent is chitosan.

32. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 to 31 , characterized in that, in step (e), the obtainment of the mucoadhesive granule occurs through homogenization, extrusion, and drying of the secondary mixture obtained in step (d).

33. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 to 31 , characterized in that, in step (e), the obtainment of the mucoadhesive granule occurs through homogenization, extrusion, spheronization, and drying of the secondary mixture obtained in step (d).

34. Process for obtaining a mucoadhesive granule, according to any one of Claims 13 to 33, characterized in that the mucoadhesive granule obtained in step (e) is covered with a gastroresistant polymer.

35. Process for obtaining a mucoadhesive granule, characterized in that it comprises the use of the process for obtaining a mucoadhesive granule as defined in any one of Claims 13 to 34.

36. Mucoadhesive granule, characterized in that it is obtained by the process as defined in any one of Claims 13 to

35.

37. Pharmaceutical composition, characterized in that it comprises a mucoadhesive granule obtained by means of a process as defined in any one of Claims 13 to 35.