STABILIZATION OF COLLOIDAL SYSTEMS BY THE FORMATION OF IONIC LIPID-POLYSACCHARIDE COMPLEXES

Abstract: Stabilization of colloidals systems through the formation of ionic lipid-polisaccharide complexes. There is disclosed a process for the preparation of colloidal systems which includes the incorporation of a water soluble and positively charged amino polysaccharide and a negatively charged phospholipid. The colloidal systems (which comprise polymer nanoparticles, nanocapsules and nano-emulsions) are stabilized through the formation of a ionic complex, at the interface, comprised of the aminopolysaccharide and the phospholipid. The colloidal systems are characterized in that they have a particle size lower than 1 µm, a electric positive charge and an exeptional stability during storage. They are lyophilizable so that they can be dry stored and redispersed subsequently while maintaining the original characteristics of the system. They are useful as pharmaceutical forms for the oral, transdermic, topical, ocular, nasal and vaginal administration of medicaments. They are also useful as forms for cosmetic use.

Description: The present invention relates to a procedure for the preparation of colloidal systems involving a combination of two ingredients: a water soluble and positively charged polyssacharide and a negatively charged phospholipid. The procedure can be applied to the stabilization of colloidal systems of pharmaceutical and cosmetic use. These systems include oil-in-water submicron emulsions, nanocapsules consisting of an oily core surrounded by a polymer coating and polymeric solid nanoparticles. The common feature to all these colloidal systems is that they consist of a dispersed phase -oily nanodroplets, nanocapsules or nanoparticles- and a continuous aqueous phase. The originality of the procedure relies on the incorporation of a negatively charged phospholipid, preferably lecithin (anionic ingredient), as a lipophilic surfactant, in the dispersed phase and of a cationic aminopolysaccharide, selected from chitosan and chitin, as a hydrophilic suspending agent, in the continuous aqueous phase. Lecithin is a natural surfactant composed of a mixture of various phospholipids. The major compound is phosphatidylcholine (phospholipid of a neutral character) and the secondary compounds are phosphatidylethanolamine, phosphatidylserine and phosphatidic acid (phospholipids with a negative charge). Presently, there are several types of lecithin available in the market. They differ in their origin and in their phosphatidylcholine content. Chitosan is a natural polymer obtained by a deacetatilation process of the chitin (compound extracted from the crustacean shells). Chitosan is an aminopolysaccharide and has a positive charge. Presently, there are several types of chitosan available in the market. They differ in their molecular weight, deacetilation degree and the type of salt or acid form. The colloidal systems according to the present invention are characterized in that they contain a negatively charged phospholipid, preferably lecithin and a cationic aminopolysaccharide, selected from chitosan and chitin in their composition and they have a positive charge and an improved stability. The other ingredients will be dependent on the type of system i.e. an oil, in the case of the submicron emulsions; an oil and a hydrophobic polymer, in the case of the nanocapsules, and a hydrophobic polymer in the case of the nanoparticles. Drugs, proteins and other bioactive compounds of interest in medicine and cosmetics can be incorporated in these systems. Consequently, their application as new administration form in humans is extended to the fields of medicine and cosmetics. An important draw back associated to the use of the colloidal systems in the fields of medicine and cosmetics is their unstability following in vivo administration and also during storage. Certainly, the majority of the developped colloidal systems have a negative surface charge and, because of this fact, they interact with definite cationic biologic compounds thus leading to the coalesce and destruction of the system. Additionally, the freeze-drying of these systems, specially the nanocapsules and submicron emulsions entail important difficulties, thus they have to be stored in aqueous suspension. This necessity of storing as a suspension liquid form, leads after a certain period of time (mounths) to the destabilization of the systems. The novel systems presented in this patent have a positive charge and an improved stability upon contact with biological cations Consequently, these systems overcome the limitations mentioned above. There are in the literature an important number of publications and patents describing diverse procedures to produce colloidal systems such as nanoparticles, nanocapsules and submicron emulsions. EP0486959 discloses a pharmaceutical composition which comprises a biodegradable polymer and/or a polyssacharide jellifying and/ or bioadhesive polymer, and an amphiphilic polymer, an agent modifying the interface properties of the particles and a pharmacologically active substance. The compositions are prepared by co-solubilisation of the polymeric constituents with the agent modifying the interface properties in the presence of solvents or suspension of the active substance in the mixture of polymeric compounds, that is, all of the various components are solubilized in a common solvent prior to the preparation of particles. The particles are formed by the techniques of emulsion or extrusion or spray drying or spray congealing. The object of the invention is, the incorporation in such colloidal systems of two specific ingredients: a negatively charged phospholipid, preferably lecithin and a cationic aminopolysaccharide, selected from chitosan and chitin. Namely, the present invention refers to a process for the preparation of pharmaceutical compositions in the form of colloidal particles of a size less than 1 µm, suitable for the delivery of active compounds comprising the steps of: providing an organic solution comprising a negatively charged phospholipid and either a hydrophobic polymer or oil or both substances simultaneously, dissolved in an organic solvent, providing an aqueous solution of cationic aminopolysacharide selected from chitin and chitosan, combining said organic and aqueous solutions so as to simultaneously and spontaneously form and coat said colloidal particles with a film which is the ionic reaction product of said phospholipid and aminopolysacharide, and to provide said particles with a positive surface charge, wherein at least one of said solutions contains said active compounds. The preparation of these systems involves the use of two phases: an oily phase that is dispersed in an aqueous phase. Both phases normally contain surfactants. The most common surfactant introduced in the oily phase is a negatively charged phospholipid, preferably licithin coming from different natural sources. Lecithins contain phosphatidylcholine as principal component and other phospholipids of negative charge. Consequently, colloidal systems containing lecithin have a more or less important negative surface charge. This negative charge normally leads to the destruction of the system, mainly upon contact with biological cations. This limitation inherent to most of the colloidal systems has been recently overcome by using lipophilic surfactants with a positive charge. These positive surfactants are introduced in the dispersed oily phase (S. Benita, oil-in water emulsion of positively charged particles WO 93/18852). The present invention describes a new approach to provide the colloidal particles of a positive charge. This approach is based on the use of a cationic polyssacharide, selected from chitosan and chitin and derivatives thereof, that is dissolved in the continuous aqueous phase and a negatively charged phospholipid such as lecithin, that is introduced in the oily dispersed phase. The positively charged chitosan molecules interact with the negatively charged phospholipids, thus forming a film at the interface of the colloidal system. The interaction process of chitosan with phospholipids was previously described as a way to stabilize emulsions (no submicron emulsions) (P. Faldt, B. Bergenstahl, P.M. Claesson, Stabilization by chitosan of soybean oil emulsions coated with phospholipid and glycocholic acid, Colloids Surfaces A: Physicochem. Eng. Aspects 71, 187-195, 1993) and liposomes (I. Henriksen, G. Smistad and J. Karlsen, Interactions between liposomes and chitosan, Int. J. Pharm., 101, 227-236, 1994). Nevertheless, no reference concerning the application of such interaction (chitosan-phospholipid) to the stabilization of submicron emulsions, nanocapsules and nanoparticles has been found. On the other hand, it is important to mention that the approaches described until now for the freeze-drying of colloidal systems, such as nanocapsules and submicron emulsions, are based on the use of enormous amounts of sugars (RJ. Gautier and R.S. Levinson, Lyophilized emulsion compositions and method, South Africa patent No. 864032) whereas the freeze drying of the nanocapsules covered in this invention require the use of relatively low amounts of sugars (less than 10%). The systems covered in this patent, characterized by the formation of a polysaccharide-lipid complex at the interface, have some relevant advantages: (1) The systems can be stored in a suspension liquid form for extended periods of time, (2) the nanocapsules based on this approach can be freeze dried and the resultant dry product reconstituted upon addition of water (3) the nanocapsules coated with a cationic aminopolysaccharide, selected from chitosan and chitin herewith described are more stable in the presence of biological cations than conventional uncoated nanocapsules, (4) the systems have a positive electrical surface charge that enables their interaction with negatively charged biological surfaces. The present invention describes novel systems of interest in therapeutics and cosmetics. These systems can be presented in a liquid form of variable viscosity or in a semi-solid (cream) or solid form (freeze-dried powder). The dispersed phase of the system consists either of a polymer or an oil or both substances simultaneously. The specific ingredient of this dispersed phase is a negatively charged phospholipid. This phase can contain as well a variable amount of an active ingredient. The oils can be chosen among vegetable oils or semisynthetic polyoxyethylenated oils (Migliol®, Labrafil®, Labrafac®...) of various H.L.B. (hydrophilia lipophilia balance) values. The polymer can be any hydrophobic polymer which is adequate for pharmaceutical or cosmetic use. The proportion of the hydrophobic polymer with respect to the oily phase can vary from 0% (submicron emulsions) up to 100% (nanoparticles). Intermediate proportions lead to the formation of nanocapsules in which the oil is in the polymer forming a reservoir system. The specific ingredient of the external aqueous phase is chitosan. For freeze drying purposes some cryoprotective agents such as dextran and glucose need to be added to this external phase. This phase can incorporate as well ingredients to provide a certain density or viscosity to the preparation, bacteriostatic agents for the prevention of contamination and other hydrophilic agents. These systems can be formulated in different ways in order to incorporate in their structure one or more active ingredients of a hydrophilic or lipophilic character. Active ingredient is the ingredient for which the formulation is destined; in other words, the ingredient which will have an effect following its administration to an organism (humans or animals). The corresponding effect can be curing, minimizing or preventing a disease (drugs, vitamins, vaccines...) or improving the physical appearance and aesthetics (e.g... skin hydration...) and other. Cyclosporin A, an immunossupressive peptide, indomethacin (antiinflammatory drug) metipranolol (beta-blocker) and tiopental (hypnotic agent) are examples of drugs which have been successfully associated to the colloidal systems described in this patent. A common feature to the systems described in this patent is the colloidal nature, which means that their size is lower that 1 µ m. Tables 1 and 2 show the mean particle size of the nanocapsules, submicron emulsions and nanoparticles containing the oil Migliol® 840 and various amounts of polyepsiloncaprolactone, soybean lecithin and dextran. As mentioned above, a relevant property of the systems described here is their positive electrical charge. This positive charge promotes the interaction of the systems with the negatively charged mucosa and epithelia and also improves their stability in the presence of biological cations. As shown in table 3 the zeta potential of the systems varies between +30 and +60 mV, being these values dependent on the molecular weight of chitosan. The inner structure of the systems described here is variable and dependent upon the composition of the system. As indicated before, the composition of the systems can vary substantially being the common ingredients a negatively charged phospholipid, preferably lecithin and chitosan or chitin or their derivatives. Two main inner structures can be described: a reservoir system consisting of an oily core surrounded or not by a polymer wall, and a matrice system consisting of solid particles containing none or little amounts of oil entrapped. The redispersability of the colloidal systems upon freeze-drying is a major advantage of the systems covered in this patent. Tables 3 and 4 show the particle size of the nanocapsules before and after freeze-drying. The procedure described in this invention leads to the formation of novel systems for pharmaceutical or cosmetic applications. In addition, these systems could be administered by various routes: topical, oral, nasal, pulmonary, vaginal and subcutaneous. The specific ingredients, chitosan and lecithin, provide to these systems a positive electrical charge and an improved stability not only during storage but also upon freeze-drying and further rehydration. Table 1 : Particle size of the poly(e-caprolactone) (PECL) nanocapsules containing Migliol ® 840 and a fixed concentration of chitosan (Seacure 123, 0.2% ) % Lecithin (w/v) % Dextran (w/v) % PECL (w/v) 0 1 2 0,5 1 340±23 361 ± 22 353 ± 21 0,5 2 278 ± 43 324 ± 28 292 ± 38 1 1 324 ± 23 384 ± 5 313 ± 19 1 2 313 ± 11 303 ± 28 318 ± 24 1.5 1 314 ± 19 341±18 346±20 1,5 2 284±12 321 ± 10 339 ± 13 Table 2: Particle size of the poly(e-caprolactone) (PECL) nanoparticles prepared with a fixed concentration of chitosan (Seacure 223, 0.2% ) % Lecithin (w/v) % Dextran (w/v) % PCL (w/v) 1 2 0,5 1 290 ± 16 308 ±15 0,5 2 286 ± 12 296 ±20 1 1 330 ±15 330 ± 2 1 2 299 ± 16 317 ±10 1,5 1 337 ±10 355 ± 19 1,5 2 326 ±18 332 ±12 Table 3: Zeta potential of the poly(ε-caprolactone) (PECL) nanocapsules and submicron emulsions containing Migliol ® 840 and a fixed concentration of chitosan (Seacure 320, 0.2%). % Lecithin (w/v) Zeta Potential (mV) Submicron emulsions Nanocapsules PECL 1% PECL 2% 0,5 +52 ±2 +60±1 +60±1 1 +60±1 +61±1 + 60 ±0.07 1,5 + 59 ±0.3 +59 ±2 + 61 ±0.4 Table 4: Particle size of the poly(ε-caprolactone) (PECL) nanocapsules containing Migliol ® 840 and a fixed concentration of chitosan (Seacure 223 viscosity 100 cps and Seacure 320 viscosity 680 cps, 0.2% ). Final concentration of PECL and lecithin in the suspension: 1% and 0.5% respectively. Chitosan viscosity % Dextran (p/v) (cps) Particle size (nm) Before freeze-drying After freeze-drying 100 1 459 ±23 487±19 100 2 472 ±8 462 ±19 680 1 443 ±30 475 ±30 680 2 461 ±13 505 ±16 Example 1: -Preparation of a formulation of nanocapsules containing PECL and Migliol ® 840. The nanocapsules were prepared using the following ingredients (%, w/w): Migliol® 840 oil 0.5 Soybean lecithin 1.0 polyepsiloncaprolactone 1.0 Dextran 1.0 Chitosan 0.2 Water up to 100% Chitosan and dextran were dissolved in an acidic aqueous solution (acetic acid 0.05M, pH 5.0). The oil Migliol® 840, the surfactant soybean lecithin and the polymer poly(ε-caprolactone) were dissolved in 25 ml of acetone. The acetonic solution was then added, upon magnetic agitation, to an aqueous solution. Three min later the system was transferred to a rotavapor for the elimination of the acetone. The size and zeta potential of the nanocapsules were: 385 nm and +45mV respectively. Finally, glucose was dissolved in the aqueous suspending medium and the nanocapsules freeze-dried. The particle size and zeta potential of the nanocapsules was determined again upon freeze-drying and resuspension. Results were: 359 nm and +42mV. Example 2: --Preparation of a formulation of nanocapsules containing PECL and Migliol ® 840. The nanocapsules were prepared as described in example 1 but containing different amounts of lecithin and oil:: Migliol® 840 oil 1.5 Soybean lecithin 0.5 polyepsioloncaprolactone 1.0 Dextran 1.0 Chitosan 0.2 Water up to 100% The particle size and zeta potential of these nanocapsules were: 433 nm and +32 mV, respectively, before freeze-drying and 582 and +43 mV after freeze-drying. Example 3: --Preparation of a formulation of a submicron emulsion containing Migliol ® 840. The emulsion was prepared as described in example 1 but without the polymer PECL: Migliol® 840 oil 1.5 Soybean lecithin 0.5 Dextran 1.0 Chitosan 0.2 Water up to 100% The results of particle size and zeta potential were: 463 nm and +42 mV, respectively.

Claims: Process for the preparation of pharmaceutical and cosmetic compositions in the form of colloidal particles of a size less than 1 µm suitable for the delivery of active compounds comprising the steps of: - providing an organic solution comprising a negatively charged phospholipid and either a hydrophobic polymer or oil or both substances simultaneously, dissolved in an organic solvent, - providing an aqueous solution of cationic aminopolysacharide selected from chitin and chitosan, - combining said organic and aqueous solutions so as to form and coat said colloidal particles with a film which is the ionic reaction product of said phospholipid and aminopolysacharide, and to provide said particles with a positive surface charge, wherein at least one of said solutions contains said active compounds. Process according to claim 1, characterised in that said phospholipid is Process according to any of claims 1 and 2, characterised in that the percentage of the aminopolyssacharide in the final aqueous medium can be up to 2% preferably between 0.05 ad 0.5% by weight. Process according to any of claims 1 to 3, characterised in that the percentage of the phospholipid in the final aqueous medium can be up to 5% preferably between 0.2 and 1% by weight. Process according to any of claims 1 to 4, characterised in that the colloidal particles are nanodroplets which are formed upon the incorporation of a vegetable or semisynthetic oil, dissolved in the organic phase, the oil being in an amount up to 1% with respect to the external aqueous medium. Process according to any of claims 1 to 4, characterised in that the colloidal particles are nanocapsules which are formed upon the incorporation of a vegetable or semisynthetic oil and a polyester, dissolved in the organic phase, the oil and the polyester being in variable proportions up to 1% and 4% respectively. Process according to any of claims 1 to 4, characterised in that the colloidal particles are nanoparticles which are formed upon the incorporation of a polyester, dissolved in the organic phase, the polyester being in variable proportions up to 4%. Process according to claim 6, characterised in that the composition includes complementary ingredients, in particular 1 to 2% in weight of dextran and 5% in weight of glucose, to allow the freeze-drying of the nanocapsules and further resuspension in water. Process according to any of claims I to 8, characterised in that said active ingredient is selected from indomethacin, metipranolol, diazepam and cyclosporin A. Process according to claim 1, characterised in that the composition includes ingredients which are non-toxic and compatible with their application by the topical, oral, nasal, vaginal and pulmonary routes of administration, these particles having a positive charge that facilitates their interaction with mucosas and epithelia.

Inventors: ALONSO FERNANDEZ MARIA JOSE, CALVO SALVE PILAR, REMUNAN LOPEZ CARMEN, VILA JATO JOSE LUIS, ALONSO FERNANDEZ, MARIA JOSE, CALVO SALVE, PILAR, REMUNAN LOPEZ, CARMEN, VILA JATO, JOSE LUIS, ALONSO FERNANDEZ, Maria José Uni de Santiago, CALVO SALVE, Pilar Universidade de Santiago de, REMUNAN LOPEZ, Carmen Universidade de Santiago de, VILA JATO, José Luis Universidade de Santiago de
Applicants: ADVANCELL ADVANCED IN VITRO CE, ADVANCELL - ADVANCED IN VITRO CELL TECHNOLOGIES
Application year: 1996
Main classification: A61K47
IPC codes: A61K9