EP1778192A2

EP1778192A2 - Microparticulate systems for the oral administration of biologically active substances

Info

Publication number: EP1778192A2
Application number: EP05760593A
Authority: EP
Inventors: Daniele Vigo; Vincenzo Russo; Massimo Faustini; Mario Francesco Pace; Eleonora Munari; Maria Luisa Torre; Ubaldo Conte
Original assignee: Universita degli Studi di Pavia; Universita degli Studi di Milano
Current assignee: Universita degli Studi di Milano
Priority date: 2004-06-22
Filing date: 2005-06-20
Publication date: 2007-05-02
Also published as: WO2005123034A8; WO2005123034A2; WO2005123034A3; ITMI20041255A1

Abstract

The present invention relates to microparticulate systems consisting of a gastroresistant, biocompatible and biodegradable polymer matrix, comprising a gastroresistant and enterosoluble polymer, a cryoprotector or lyoprotector, a divalent or trivalent metal salt of i biocompatible and biodegradable polymer having acid groups, and biologically active substances. Such gastroresistant microparticulate systems are used for the administration, preferably oral, of biologically active substances to animal species. The special composition of such microparticulate systems allows the protection of said biologically active substances from degradation by proteases and gastric acid, allowing their release into the intestine, where they may perform their activities.

Description

"Microparticulate systems for the oral administration of biologically active substances" DESCRIPTION The present invention relates to the preparation, use and formulation of microparticulate systems for the oral administration of biologically active substances. PRIOR ART The proteins, lipids and carbohydrates contained in food must be broken down into their elementary components . in order to be _.absorbed by the intestine. In monogastric animals, non-ruminant polygastric animals (such as: the young of ruminant species with nonfunctional pre-stomachs, birds and fish) and in the posterior (post-omasal) digestive tract of ruminants (such as: bovids, ovicaprids, camelids and buffalos) , digestion is principally enzymatic, assisted by mechanical and microbial processes. In healthy adult animals, digestive enzymes are secreted into the lumen of the digestive system^' from the glands associated with it (salivary glands, exocririe pancreas and the liver) or contained in the mucosa. In the above mentioned animal species, the mechanical processes associated with digestion and, particularly the movements of peristalsis, ,antiperistalsis and segmentation, ensure an effective action of the digestive enzymes by increasing the available surface of the foodstuffs, thus promoting improved distribution inside the intestine. Such mechanical processes are also responsible for the expulsion of undigested or unabsorbed materials. Protein digestion begins in the stomach of monogastric and non-ruminant polygastric animals and in the abomasum of polygastric ruminants. In this case, the digestive enzymes •are prόteolytic enzymes, such as for example pepsin, which are active at acidic pH. Hydrochloric acid, secreted by the oxyntic or parietal cells of the glands of the stomach base, makes the proteins structurally suited to attack by proteolytic enzymes, activates pepsinogen pro-enzymes, transforming them into pepsin, and creating the optimal conditions for their activity (pH = 1-2.5).^' All^' protein^' molecules in the gastric lumen endure such action and, hence, the enzymes and structural proteins present in cell walls and/or in plasma membranes also endure this process. •In zootechnics. (above all for livestock. such as:porcines, bovines, caprines,^' ovines, equines, canines, felines, camelids, lagomorphs, rodents, other mammals, fowl, etc.) solid foods are introduced with the diet even during lactation, since the first days of the newborns life. Weaning is generally very early, for example, for porci es,- it takes place between the third and ^'.fourth weeks, of life with the passage from milk-based liquid foods to solids, in the form of starter feed. The fundamental constituents of weaning feeds for many animal species, particularly porcines, are complex carbohydrates varying in qualitative-quantitative composition depending on the subject's age. The main complex carbohydrates in the feedstuffs for post-weaning subjects are starches. Complex carbohydrates, such as starch, are mainly digested in the intestine thanks to the action of special enzymes, such as for example α-amylase, which are secreted into the intestinal lumen and display maximal activity at pH values about 7. Under physiological conditions, α-amylase is produced by the exocrine pancreas and secreted in an aqueous solution containing sodium bicarbonate and other electrolytes, having the role of buffering the pH of the intestinal tract in the range of maximal enzyme activity (pH 6.5-7.0) . The development and the structural and functional maintenance of the gastroenteric system, and also the development of the enzymatic system, are induced by cellular growth and differentiation factors present in mothers milk: sudden and premature weaning, for example of suckling piglets, induces a regression in the development of such apparatus, which is expressed as a reduction in the length of the villi and an increase in crypt depth (villar atrophy) . This phenomenon is frequently associated with a reduction in voluntary food consumption and impaired immune defences with increased risk of bacterial (e.g. enterotoxic E. coli) or viral (e.g. rotavirus) infections. Such infections, in turn, induce inflammatory reactions (Kenworthy, 1976/ Hampson and Kidder, 1986) . Also, in pigs, exocrine pancreas functional development is progressive and induced by growth factors_, present in mother's milk: chymotrypsin secretion begins around the first week of life, lipase appears around the second week, and α-amylase only appears^' from the fourth week onwards . However, if weaning takes place prior to the appearance of α-amylase, then starch digestion is reduced or compromised due to the lack of this enzyme. Hence, there is ^'severe slowing down of the animal's weight gain with serious health and economic repercussion^'s for breeding. In order to achieve improved health, wellbeing and growth performance, it might be advantageous to administer to ' the animals, orally, dietary supplements containing biologically active substances, such as, for example: enzymes, peptides, polypeptides, aminoacids, yeasts, microorganisms, milk enzymes etc., in order to improve digestion and, hence, absorption of nutritional substances; drugs, vaccines, hormones, drug and hormone precursors etc., in order to treat any diseases which might possibly arise. Obviously the preferred route of administration is orally, in that it is the simplest (said substances are combined with the solid or liquid diet) , the most economical and may even be used for slightly uncollaborative subjects. However, oral administration has problems due to the fact that the aforementioned biologically active substances may be partially degraded by gastric juices and, hence, either partially or totally, loose their activity. For example, enzymes such as α-amylase undergo extensive modifications to their primary, secondary and supersecondary structures in acid environment and, by the time they reach the intestine, they are completely inactivated. Hence, the technical problem addressed by this invention is that of the successful oral administration of biologically active substances to animals, without incurring in the complete loss of their biological activities . DETAILED DESCRIPTION OF THE INVENTION In a first aspect the present invention relates to microparticulate systems consisting of a gastroresistant, biocompatible and biodegradable polymer matrix, comprising a gastroresistant and- enterosoluble polymer, a cryoprotector or lyoprotector, a divalent or trivalent metal salt of a biocompatible and biodegradable polymer having acidic groups and biologically active substances. An additional biocompatible and biodegradable polymer may also optionally be present. By the term biologically active substances is meant animal, plant, bacterial or recombinant derived proteins and peptides and precursors thereof, classified under the following categories: enzymes, coagulation factors, haemopoietic growth factors, interferons, polypeptide and protein ^' antibiotics, immunoglobulins, insulin, growth hormones, gonadotropins, protein molecules, antigenic glycoproteins and lipoproteins, prokaryotic cells and viruses, vaccines, drugs, hormones, phyto- and organo-therapeutic^' extracts and mixtures thereof. Preferably, the enzymes are digestive enzymes selected from: proteases and peptidases, amylases, cellulases, poly- and oligosaccharidases, upases, nucleosidases, phytases. Preferably, the prokaryotic cells and^' viruses are selected from: yeasts, lactobacilli, probiotic bacteria, antigenic bacteria o viruses, (live, attenuated or killed) . Preferred biologically, active substances include: enzymes, vaccines, drugs, antibiotics, phyto- and organo- therapeutic extracts and prokaryotic cells and viruse-s or mixtures thereof. Even more preferably, they are enzymes. .Said microparticulate systems are used for the administration, preferably orally, of biologically active substances to animals selected from: porcines, bovines, caprines, ovines, equines, canines, felines, camelids, lagomorphs, rodents, primates and other animal species, such as fish and fowl. Preferred animals are the young of such species. The special composition of such microparticulate systems allows the protection of said biologically active substances from degradation by proteases and gastric acid, allowing their release into the intestine, where they may fulfil their activities. ^' Preferably, said gastroresistant and enterosoluble polymer is selected from: phthalic acid cellulose esters, (for example: cellulose acetophthalate, hydroxypropyl- methylcellulose phthalate) , trimellitic acid cellulose esters (for example: cellulose trimellitate, hydroxypropylcellulose trimellitate, hydroxypropyl- methylcellulose trimellitate) ; acrylates and polymethacrylates (for example: synthetic anionic and cationic polymers of dimethylaminoethylmethacrylates, of methacrylic acid and ethylmethacrylate) . The more preferred are: hydroxypropyl-methylcellulose phthalate, synthetic anionic and cationic polymers of methacrylic acid and methylmethacrylate. Said synthetic anionic and cationic polymers of polymethacrylic acid and methylmethacrylate are for example: Eudragit LlOO^® and Eudragit L100-55^® or Eudragit S100^®. The cryoprotector or lyoprotector is preferably selected from: glycerol, propanediols, polyethyleneglycols of various molecular weights, sucrose, lactose, mannitol, glycocoll, pentaerythritol, trehalose, sorbitol, xylitol, dimethylsulphoxide, isopropanols, polyvinylpyrrolidones, polyoxyethylenes, hydroxyethylamides, alpha, beta and gamma cyclodextrin and derivatives thereof. More preferably, the cryoprotector or lyoprotector is selected from: sucrose, lactose and mannitol. Even more preferably, it is lactose. The divalent or trivalent metal salt of a biocompatible and biodegradable polymer having acidic groups is a calcium, barium, strontium, zinc, aluminium, iron, or chromium salt of alginic acid, hyaluronic acid or xanthan gum. The additional biocompatible and biodegradable polymer is selected from the group consisting of: glucans, scleroglucans, mannans, galactomannans, gellans, carrageenans, pectins, polyanhydrides, polyaminoacids, polyamines, xanthans_,_ tragacanth gum, guar gnm, xanthan gum, celluloses and derivatives thereof, carboxymethylcellulose, ethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, polyvinylalcohols, polyoxyethylenes, carboxyvinylpolymers, starches, collagens, chitins and chitosans. Carboxymethylcellulose, ethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose hydroxypropylmethylcellulose, polyvinylalcohols, polyoxyethylenes, carboxyvinylpolymers, starches, collagens, chitins, chitosans are preferred. Hydroxypropylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose are even more preferred. The microparticulate systems, described above and obtained by means of the process described below, have dimensions comprised between 30 μm and 1200 μm, preferably between 50 μm and 500 μm. In a second aspect, the present invention relates to a process for the preparation of these gastroresistant microparticulate systems. Said process comprises the following stages: a) combining an aqueous solution of gastroresistant and enterosoluble polymer with an aqueous or physiological solution of cryo- or lyoprotector and the salt of a monovalent metal, prefexably the potassium or sodium salt of a biocompatible and biodegradable polymer, having acidic groups, preferably alginic acid, so as to obtain a colloidal solution or homogeneous suspension; b) solubilising or dispersing the biologically active substance in the solution or suspension from step a); c) nebulising or extruding the solution or suspension from step b) into an aqueous solution of a soluble, inorganic, salt of divalent or trivalent ions. The presence of divalent or trivalent ions leads to the formation of an insoluble matrix consisting of, for example, the alginate salt of calcium and/or barium and/or some other divalent or trivalent cation. This leads to the formation of insoluble microparticulate systems containing the biologically active substances. Step a) includes a step al) for the preparation of the monovalent metal salt solution of a biocompatible and biodegradable polymer, having acidic groups and the cryo- or lyoprotector; and a step a2) for the preparation of the gastroresistant and enterosoluble polymer solution. In step al) a saline solution (0.5-15% sodium chloride in water, preferably about 0.9% NaCl in water) of the monovalent metal salt of a biocompatible and biodegradable polymer, having acidic groups, in concentration ranging from 0.01% to 10% w/v, preferably from 0.1% to 3% w/v is preferably prepared. This is left stirring using a mechanical s^'tirrer unto the salt is completely dissolved. The monovalent metal salt of a biocompatible ^' and biodegradable polymer, having acidic groups, is preferably a sodium or potassium salt of alginic acid, hyaluronic acid or xanthan gum, more preferably, it is sodium alginate. In the case where sodium alginate is used, an (aqueous or physiological) solution of approx. 2% w/v is prepared, having a viscosity of between 200 cP and 20000 cP at 25°C. The alginate has a mean molecular weight of between 20,000 and 240,000 Daltons . To the solution thus obtained is added, while stirring, the cryo- or lyoprotector in such amounts as to obtain a concentration of- from 1 to 40% w/v, preferably from 2% to 20% w/v. Said cryo- or lyoprotector is . as described above. Optionally, to the solution thus obtained ^' may be added an additional biocompatible and biodegradable polymer in such quantities as to obtain a concentration of from 0.1% to 50% w/v, preferably from 2 to 15% w/v. Said biocompatible and biodegradable polymer is as described above . In step a2) , an aqueous solution of the gastroresistant and enterosoluble polymer is prepared with a concentration ranging from 1% to 50% w/v, preferably from 5% to 15% w/v, with a pH from 6 to 8, even better if from 7 to 7.8. As described in step a) , the solution obtained in al) and the solution obtained in a2) are combined in ratios of from 3:1 to; 1:3, preferably in a ratio of' 1:1. .This operation results in the formation of a colloidal solution or a homogeneous suspension. Said colloidal solution or homogeneous suspension is adjusted to a pH. of between 5.5 and 10, preferably between 6.5 and 8.5. In step b) , the biologically active substance is dispersed in the colloidal solution or homogeneous suspension, thus obtained, at concentrations ranging from 0.01% to 20% w/v, preferably from 0.1% to 5% w/v. In step c) , nebulisation takes place with the aid of orifices, nozzles, or syringes having sizes ranging^' from 10 μm to 5000 μm, preferably from 300 μm to 2000 μm..^• Extrusion takes place with the aid of automatic o ^■ semiautomatic microencapsulators, peristaltic or piston pumps or alternatives, or by means of a syringe, manually and/or automatically driven at such a speed as to produce from 10 to 250 drops/minute, preferably from 20 to 120 drops/minute . Nebulisation or extrusion determines the formation of very small drops which are collected in an aqueous solution of a soluble divalent or trivalent inorganic salt, kept stirring at a speed of between 10 and 200 rpm, preferably between 20 and 100 rpm. The volumetric ^' ratio between the extruded solution and the inorganic salt solution is between 1:1 and 1:6,, preferably, the ratio is 1:4. This divalent or trivalent ion inorganic salt is selected from: calcium, barium, strontium, zinc, aluminium, iron or chromium chloride, preferably calcium chloride, barium chloride or aluminium chloride. Even more preferably, it is calcium chloride. The concentration of said inorganic salt solutions is of between 0.1 M and 2.0 M, preferably between 0.2 M and 0.8 M. The presence of a divalent- or trivalent metal salt leads to the formation of a matrix consisting of insoluble salts of the biodegradable and biocompatible polymer, having acidic groups, with the divalent or trivalent metal used, and thus the attainment of rapidly sedimenting microparticulate systems. These microparticulate systems have a spherical shape and are insoluble. They are separated from the solution by aspiration or filtration. Optionally, ■ they may be washed several times with ^' physiological solution (isotonic saline) . All the aforementioned steps in the process are performed at ^• a temperature of between -5°C and 50°C, preferably between 0°C and 30°C. In one preferred aspect, the microparticulate systems thus obtained may be subjected to outer surface cross- linking, by means of interfacial polymerisation of the biocompatible . and biodegradable polymer divalent . or trivalent^' metal salt, using ^■ polyamine-type cross-linking agents such as, for example: protamine sulphate or phosphate, poly-L-lysine hydrobromide (molecular weight range from 1,000 Da to 80,0000 Da), polyvinylamine, chitosans (molecular weight range^' from 15,000 Da to 1,000,000 Da). Said cross-linking agents are preferably used as aqueous solutions at concentrations between 0.01% and 5% w/v. The cross-linking reaction is carried out ^' at a temperature between 5°C and 40°C, preferably about 25°C for times between 1 minute and 120 minutes, preferably between 3 and 30 minutes. The cross-linking^' reaction leads to the hardening of the membrane of the microparticulate systems making them easier to handle. In one preferred aspect, said microparticulate systems may be subsequently subjected to lyophilisation, using techniques known to those skilled in the_. art,- or dried by means- of any method known in the art which is not prejudicial to the activity of the encapsulated biologically active substance. Said microparticulate systems may be stored at temperatures between -200°C and 40°C, preferably between

4°C and 40°C, possibly in a controlled atmosphere, as known to those skilled in the art. In a third aspect, the present invention relates to a kit for the preparation of capsules, according to the invention, comprising previously prepared, pre-measured and pre-packaged raw materials, as well as any relevant disposable, sterile, non sterile or sterilisable materials . Hence, such kit will comprise said gastroresistant and enterosoluble polymer, said monovalent metal salt of a biocompatible and biodegradable polymer having acidic groups, said divalent or trivalent inorganic salt, said cryo- or lyoprotector and said biologically active substance in separate pre-measured packages. Optionally, it may contain said additional biodegradable and biocompatible polymer and said cross-linking agent in separate pre-measured packages. The kit will additionally comprise extrusion devices such as sterile, non-sterile or sterilisable nozzles, needles or syringes. The kit may require storage at temperatures -ranging from -5°C to 40°C, preferably about 0°C, in order to avoid deterioration of the active substance, ' and the immediate use and/or- cryo-desiccation ^■ of the microparticulate systems immediately upon formation. The microparticulate systems forming the subject of the present invention may be administered- orally, by administration with a liquid diet or as supplements in solid feed. In special cases, administration may take place by means of a gastro-oesophageal, rhino-oesophageal and intragastric probe, using surgical and microsurgical techniques, possibly with the aid of an echographic guide. In a fourth aspect, the present invention relates to a pre-packaged animal feed, supplemented with the microparticulate system of the invention. These gastroresistant and enterosoluble microparticulate systems may also be used for the administration of biologically active substances to humans . By biologically active substances is meant those listed previously. This invention provides microparticulate systems having such dimensions as to allow optimal dispersion in solid and liquid foods without any problems involving the particles aggregating and, hence, ^' separating from the solid or precipitating out of the liquid. This allows easy administration to humans and animals. The gastroresistant microparticulate systems of the invention may be administered orally and afford, - in gastric acid environments, effective protection of the biologically active substances vehicularised, and the rapid release of the aforesaid substances, with high biological activity, in the enteric environment (small or^' large intestine) . Said gastroresistant microparticulate systems have significant application potential in the sector of veterinary gastroenterology and nutrition, especially in monogastric animal species, but also in polygastric non- ruminants and in those ruminants with still non-functional pre-stomachs . ^• Such preparations may be classified among the zootechnical feed additives (as described in the 1^st enclosure to Reg. EC N° 1831/2003) . In the rearing of livestock, the invention resolves the essential problem of the administration of digestive enzymes (e.g. α-amylase) in young, where enzyme expression is still not entirely efficient, and in adults with reduced digestive capacity. This way, it is^' possible to- supplement the maternal milk diet of the young, with feeds promoting faster growth, thus obtaining improved quality of life and health for the— animals and significant economic advantages for the grower. GASTRORESISTANCE AND BIOLOGICAL ACTIVITY TESTING In order ^• to assess whether said microparticulate systems are effectively gastroresistant and whether the enzyme or biologically active substance maintains its activity, a sample of the microparticulate systems has been subjected to the assay provided in the Pharmacopoeia (FUI XI) for gastroresistant pharmaceutical, forms. The sample has firstly been incubated at 37 °C in hydrochloric acid at pH 1 for two hours and subsequently transferred to phosphate buffer at pH .6.8 for one hour. Following incubation in hydrochloric acid, the microparticulate systems showed no morphological changes but, after a few minutes of incubation at pH 6.8, they begun to dissolve rapidly, completely solubilising and hence liberating the- vehicularised active substance. The quantity and activity of the remaining active substance has been determined on such solution. The results of various tests have confirmed that the active substances vehicularised in ^■ the microparticulate systems maintain an activity of no less than 25-30% but even^' until 80-90% and, in the case of microparticulate systems containing enzymes, complete denaturation induced by the acidic environment has not been observed. For comparison, an analogous assay, ^• performed under identical conditions, on the free enzyme, i.e. not _. encapsulated within the microparticulate . systems described, . lead to the total denaturation of the enzyme and complete loss of activity. Such result confirms that the finding of the present patent application provides gastroresistant microparticulate systems, inside which it is possible to vehicularise biologically active substances, in a_. form- of administration guaranteeing their stability and_. release- into the intestine. This result fully meets the objectives of the present patent application, as will be further confirmed by the examples . The following examples are reported by way of non-limiting illustration of the microparticulate system preparation process forming the subject of the present invention. In particular, in the examples α-amylase has been used as an enzyme particularly sensitive to acid denaturation, and lactate dehydrogenase has been used as a ubiquitous enzyme, widely used in the field of biochemistry and enzymology. EXAMPLE 1: PREPARATION OF α-AMYLASE CONTAINING> MICROPARTICULATE^' SYSTEMS BY NEBULISATION. Solution A .To isotonic saline (physiological solution) (NaCl 0.9% w/v) is added low viscosity sodium alginate (250 cps, 2% solution, 25°C) (alginic acid, sodium • salt, low viscosity, SIGMA Chemical CO, St.- Louis, Missouri,- USA) so- as to obtain a concentration of 5% w/v and it is left stirring with the aid of a magnetic stirrer at -100 rpm at room temperature until the polymer is completely, dissolved. To the resulting solution is then added hydroxypropylmethylcellulose (Methocel E3- Dow Chemical) so as to obtain a concentration of 1% w/v. As a cryoprotector, lactose (Carlo Erba, Milan, Italy) is then added to the above solution in such quantities as to give a concentration of 4.5% w/v. Solution B A 10% w/v solution of polymethacrylate (Eudragit S100®, Rohm Pharma, GmbH, Darmstadt, D) in phosphate buffer at pH 7.5 is prepared by stirring at room temperature. Solution B is then added to solution A, whilst kept stirring with the aid of a magnetic stirrer, in a volumetric ratio of 1:1, to give a suspension containing 2.5% sodium alginate, 0.5% hydroxypropylmethylcellulose, 2.25% lactose and 5.0 % polymethacrylate ^'. To .the' suspension is then added α-amylase (from Hog Pancreas; Sigma, Milan, Italy) , in the form of a fine powder, in such quantities as to achieve a concentration of 0.3% w/v. With the aid of a peristaltic pump, the resulting suspension is nebulised, by means of a suitable spray system fitted with a 0.5 mm diameter nozzle, into a 0.3 M solution of calcium chloride in physiological solution at 0.9% w/v NaCl, whilst kept stirring at 100 rpm with the aid of a magnetic stirrer. The nebulisation nozzle is positioned approx. 10 cm from the surface of the solution. The volumetric ratio between the nebulised solution and the calcium chloride solution is 1:4; the pressure applied is 4 atm. Microparticulate systems, which sediment rapidly, are obtained: they are separated by removal of the supernatant by means of aspiration, washed twice with physiological solution, filtered and placed in suitable stainless steel containers in order to be subsequently subjected to lyophilisation. The lyophilisation process is carried out in accordance with techniques known to those skilled in the art . The lyophilised product appears as a fine powder, with good properties of flow and flowability. The composition of the lyophilisate, calculated from the composition of the nebulised solution, is as follows: α-amylase: 2.8% Calcium alginate: 23.7% Lactose: 21.3% Hydroxypropylmethylcellulose : 4.7% Polymethacrylate: 47.5% The lyophilisate consists of microparticulate systems, insoluble in water, with normal granulometric distribution and mean diameter of approx. 150 μm, (Coulter LS230 laser scattering granulometer, Beckman-Coulter Inc., Fullerton, CA, USA) . The powder has good free-flow and wetting properties, and is hence particularly suitable to be added to solid and liquid feeds, in order to obtain homogeneous mixtures or suspensions. Following preparation of the microparticulate systems, determination of the activity of the encapsulated α-amylase has been performed using the method reported in Bergmayer. Following encapsulation in the microparticulate systems, the α-amylase maintains practically unchanged levels of enzymatic activity, being equal to 90%, with respect to the activity of the enzyme prior to encapsulation. The 10% loss of enzymatic activity is due to the process to which the enzyme has been subjected in order to incorporate into the microparticulate systems. The microparticulate systems have subsequently been subjected to the above mentioned assay envisaged in the

Pharmacopoeia (FUI XI) for gastroresistant pharmaceutical forms, so as to assess the enzyme's in vitro stability. Mean α-amylase enzyme activity has been evaluated as being equal to 29% . However, the enzyme carried in the microparticulate systems is only partially prone to the denaturation induced by the acidic environment; while for the free enzyme, such denaturation is total. EXAMPLE 2: PREPARATION OF α-AMYLASE CONTAINING, MICROPARTICULATE SYSTEMS BY EXTRUSION. Solution A To isotonic saline (physiological solution) (NaCl 0.9% w/v) is added low viscosity sodium alginate (250 cps, 2% solution, 25°C) (alginic acid, sodium salt, low viscosity, SIGMA Chemical CO, St. Louis, Missouri, USA) so as to obtain a concentration of 5% w/v and it is left stirring with the aid of a magnetic stirrer at 100 rpm at room temperature until the polymer is completely dissolved. As a cryoprotector, lactose (Carlo Erba, Milan, Italy) is then added to the above solution in such quantities as to give a concentration of 4.5% w/v. Solution B A 10% w/v solution of polymethacrylate (Eudragit

S100®, Rohm . Pharma, GmbH, Darmstadt, D) in phosphate buffer at pH 7.5 is prepared by stirring at room temperature. Solution B is then, added to solution A, whilst kept stirring with the aid of a magnetic stirrer, in a volumetric ratio of 1:1, to give a suspension containing 2.5% sodium alginate, 2.25% lactose and 5.0% Eudragit S100®. To this solution is then added α-amylase, in the form of a fine powder, in such quantities as . to give a concentration of 0.3% w/v. The. resulting suspension is then extruded, in the form of drops, through needles (25Gx5/8") with the aid of a peristaltic pump, into a 0.3 M solution of calcium chloride containing 0.9% w/v sodium chloride, kept stirring with the aid of a magnetic stirrer at 100 rpm. The extrusion, needle is positioned approx. 10 cm from the surface of the solution. The volumetric ratio between the extruded solution and the calcium chloride solution is 1:4; the speed of. the peristaltic pump is such so as to produce 60 drops of extrudate/min. Microparticulate systems, which sediment rapidly, are thus obtained: they are separated by removal of the supernatant by means of aspiration, . washed - twice with physiological solution, filtered and placed in suitable stainless steel containers in order to be subsequently subjected to lyophilisation. The lyophilisation process is carried out in accordance with techniques known to those skilled in the art. The lyophilised product appears as an easy flowing, fine powder. The composition of the lyophilisate, calculated from the composition of the extruded solution, is as follows: α-amylase: 3.0% Calcium alginate: 24.8% Lactose: 22.3% Eudragit S100^®: 49.9% The lyophilisate is constituted by microparticulate systems with diameters of approx. 500 μm (Coulter LS230 laser scattering granulometer, Beckman-Coulter Inc.,

Fullerton, CA, USA) , having good free-flow and wettability properties, and is hence particularly suited to being added to solid and liquid feeds, in order to obtain homogeneous mixtures or suspensions. Following preparation of the microparticulate systems, determination of the activity of the encapsulated α-amylase has been performed using the method reported in

Bergmayer. Following encapsulation in the microparticulate systems, the α-amylase maintains practically unchanged levels of enzymatic activity, being equal to 90%, with respect to the activity of the enzyme prior to encapsulation. The 10% loss of enzymatic activity is due to the process to which the enzyme has been subjected in order to incorporate into the microparticulate systems. The microparticulate systems have subsequently been subjected to the assay provided in the Pharmacopoeia (FUI XI) for the above mentioned gastroresistant pharmaceutical forms, so as to assess the enzyme's in vitro stability. Mean α-amylase enzyme activity has been evaluated as being equal to 29.5%. However, the enzyme carried in the microparticulate systems is partially protected from denaturation induced by the acidic environment; while for the free enzyme, such denaturation is total. EXAMPLE 3: PREPARATION OF LACTATE DEHYDROGENASE (LDH) CONTAINING, MICROPARTICULATE SYSTEMS BY NEBULISATION. Solution A To isotonic saline (physiological solution) (NaCl 0.9% w/v) is added low viscosity sodium alginate (250 cps, 2% solution, 25°C) (alginic acid, sodium salt, low viscosity, SIGMA Chemical CO, St. Louis, Missouri, USA) so as to obtain a concentration of 5% w/v and it is left stirring with the aid of a magnetic stirrer at 100 rpm at room temperature until ^• the polymer is ^' completely dissolved. A cryoprotector, i.e. lactose (Carlo Erba, Milan, Italy) is then added to the above solution in such quantities as to give a concentration of 4.5% w/v. Solution B A 10% w/v solution of polymethacrylate (Eudragit S100®, Rohm Pharma, GmbH, Darmstadt, D) in phosphate buffer at pH 7.5 is prepared by stirring at room temperature . Solution B is added to solution A, kept stirring with the aid of a magnetic stirrer, in a volumetric ratio of 1:1, to • give a suspension containing.2.5% sodium alginate, 2.25% lactose and 5.0% Eudragit S100®. LDH, in the form of a fine, easily soluble powder, ^' is added to this , suspension^' in such quantities as to give a concentration of 0.3% w/v. With the aid of a peristaltic pump, the resulting suspension is nebulised by means of a spray system fitted with a 0.5 mm diameter needle into a 0.3 M solution of calcium chloride in 0.9% w/v sodium chloride, kept stirring with the aid of a magnetic stirrer at 100 rpm. The nebulisation nozzle is positioned approx. 10 cm from^' the surface of the solution. The volumetric ratio between the nebulised solution and the calcium chloride solution is 1:4; the pressure applied is 4 atm. Microparticulate systems, which sediment rapidly, are obtained: they are separated by removal of the supernatant by means of aspiration, washed twice with physiological solution, filtered and placed in suitable stainless steel containers in order to be subsequently subjected to lyophilisation. The lyophilisation process is carried out according to techniques known to those skilled in the art. The lyophilised product has good free-flowing and wettability properties. The composition of the lyophilisate, calculated from the composition of the nebulised solution, is as follows: Lactate dehydrogenase: 3.0% Calcium alginate: 24.8% Lactose: 22.3% Eudragit. S100^®: 49.9% The lyophilisate consists of microparticulate systems with normal granulometric distribution and mean diameter of 130 μm, (Coulter LS230 laser scattering granulometer,

Beckman-Coulter Inc., Fullerton, CA, USA). The powder has good . free-flow and wettability properties, and is hence particularly suited to being mixed with other excipients necessary for the formulation of solid pharmaceutical forms, as well as to being used as the dispersed phase in the formulation of suspensions or to being added to solid or liquid feeds. Following preparation^' of the microparticulate systems, determination of the activity of the encapsulated

LDH has been performed using the method reported in

Bergmayer. Following encapsulation in the microparticulate systems, the LDH maintains practically unchanged levels of enzymatic activity, being equal to 90%, with respect to the activity of the enzyme prior to encapsulation-. The 10% loss of enzymatic activity is due to the process to .which the enzyme has been subjected in order to incorporate into the microparticulate systems. The microparticulate systems have subsequently been subjected to the above mentioned assay envisaged in the Pharmacopoeia (FUI- XI) for the ^' gastroresistant pharmaceutical forms, so as to assess the enzyme's in vitro stability. Mean LDH enzyme activity has been evaluated as being equal to 75%. Thus, the enzyme vehicularised in the microparticulate systems is significantly protected from denaturation induced by the acidic environment; while for the free enzyme, such denaturation is total.

Claims

CLAIMS 1. Microparticulate systems . consisting of a gastroresistant, biocompatible and biodegradable polymer matrix containing biologically active substances characterised in that said matrix comprises: • a gastroresistant and enterosoluble polymer selected from^': phthalic acid cellulose esters, trimellitic acid cellulose esters, acrylates and polymethacrylates; • a cryoprotector or^' lyoprotector; • a divalent or trivalent metal salt -. of a biocompatible and biodegradable polymer having acidic groups, selected from: salts of . alginic . acid, of hyaluronic acid and of xanthan gum.

2. The microparticulate systems according to claim 1 having dimensions ranging from 30 μm to 1200 μm, preferably from 50 μm to 500 μm.

3. The microparticulate systems according to claims. 1 or 2 wherein said biologically active substances are animal, plant, bacterial or recombinant derived proteins and peptides and precursors thereof, classified under the following categories: enzymes, coagulation factors,, haemopoietic growth factors, interferons, polypeptide and protein antibiotics, immunoglobulins, insulin, growth hormones, gonadotropins, protein molecules, antigenic glycoproteins and lipoproteins, prokaryotic cells and viruses, vaccines, drugs, hormones, phyto- and organo- therapeutic extracts and mixtures thereof.

4. The microparticulate systems according to any of the claims 1 to 3 wherein said enzymes are digestive enzymes selected from: proteases and peptidases, amylases, cellulases, poly- and oligosaccharidases, upases, nucleosidases and phytases; said prokaryotic cells and viruses being selected from: yeasts, lactobacilli, probiotic bacteria, antigenic bacteria or viruses (live, attenuated or killed) .

5. The microparticulate systems according to any of the claims 1 to 4 wherein said biologically active substances are: enzymes, vaccines, drugs, antibiotics, phyto- and organo-therapeutic extracts and prokaryotic cells and viruses or mixtures thereof.

6. The microparticulate systems according to any of the claims 1 to 5 wherein said biologically active substances are enzymes.

7. The microparticulate systems according to any of the claims 1 to 6 wherein said gastroresistant and enterosoluble polymer is selected from: cellulose acetophthalate, hydroxypropyl-methylcellulose phthalate, cellulose trimellitate, hydroxypropylcellulose trimellitate, hydroxypropyl-methylcellulose trimellitate, synthetic anionic and cationic polymers of dimethylaminoethylmethacrylates, methacrylic acid and methylmethacrylate .

8. The microparticulate systems according to any of the claims 1 to 7 wherein said gastroresistant and enterosoluble polymer is selected from: hydroxypropylmethylcellulose phthalate and synthetic anionic and cationic polymers of dimethylarαinoethylmethacrylates, methacrylic acid and methylmethacrylate.

9. The microparticulate systems according to any of the claims 1 to 8 wherein said cryoprotector or lyoprotector is selected from: glycerol, propanediols, polyethyleneglycols of various molecular weights, sucrose, lactose, mannitol, glycocoll, pentaerythritol, trehalose, sorbitol, xylitol, dimethylsulphoxide, isopropanols, polyv-inylpyrrolidones, polyoxyethylenes, hydroxyethylamides, alpha, beta and gamma cyclodextrin and derivatives thereof.

10. The microparticulate systems according to any of the claims 1 to 9 wherein said cryoprotector or lyoprotector is selected from: sucrose, lactose and mannitol.

11. The microparticulate systems according to any of the claims 1 to 10 wherein said cryoprotector or lyoprotector is lactose.

12. The microparticulate systems according to any of the claims 1 to 11 wherein said divalent or trivalent metal salt of a biocompatible and biodegradable polymer having acidic groups is a calcium, barium, strontium, zinc, aluminium, iron, or chromium salt of alginic acid, hyaluronic acid or xanthan gum.

13. The microparticulate systems according to any of the claims 1 to 12 wherein said divalent or trivalent metal^' salt of a biocompatible and biodegradable polymer having acidic groups, is sodium alginate with a mean molecular weight of between 20,000 and 240,000 Da,^' in aqueous or physiological solution at approx. 2% w/v, having a viscosity at 25°C of between 200 cP and 20,000 cP.

14. The microparticulate systems according to any of the claims 1 to 13 wherein said polymer matrix comprises an additional biocompatible and biodegradable polymer.

15. The microparticulate systems according to. -claim 14 wherein said additional biocompatible and biodegradable polymer is selected from: glucans, scleroglucans, mannans, galactomannans, gellans, carrageenans, pectins, polyanhydrides, polyaminoacids, polyamines, ^' xanthans, .tragacanth gum, guar gum, xanthan gum, celluloses and derivatives thereof, carboxymethylcellulose, ethylcellulose, methylcellulose, hydroxypropylcellulose,- hydroxyethylcellulose, hydroxypropylmethylcellulose, polyvinylalcohols, polyoxyethylenes, carboxyvinylpolymers, starches, collagens, chitins and chitosans.

16. The microparticulate systems according to claims 14 or 15 wherein said additional biocompatible and biodegradable polymer is selected from: cellulose and • derivatives thereof, carboxymethylcellulose, ethylcellulose, methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose hydroxypropylmethylcellulose, polyvinylalcohols, polyoxyethylenes, carboxyvinylpolymers, starches, collagens, chitins and chitosans.

17. The microparticulate systems according to any of the claims 14 to 16 wherein said additional biocompatible and biodegradable polymer is selected - from: hydroxypropylcellulose, hydroxyethylcellulose hydroxypropylmethylcellulose .

18. Use of the microparticulate systems according to any of the claims 1 to 17 as a gastroresistant pharmaceutical formulation for the administration of biologically active substances to animals and humans.^'

19. The use according to claim 18 wherein said animals include: porcines, bovines, caprines, ovines, equines, canines, felines, camelids, lagomorphs, rodents, other mammals, and fowl, preferably the young of said species. 20. The use according to claims 18 or 19 wherein said administration is orally, by means of gastro- oesophageal, rhino oesophageal and intragastric— probe, using surgical and microsurgical techniques, possibly with the aid. of an echographic guide. 21. The use according to claim 20 wherein, in the 5 case of oral administration, said microparticulate systems are combined with a liquid or solid diet and complimentary feeds and even with solid or liquid dietary supplements . 22. A kit for the preparation of microparticulate systems according to any of the claims 1 to 21 comprising

10 disposable, sterile, non-sterile or sterilisable, preset and pre-packaged instrumentation, for single and or multiple preparations. 23. The kit according to claim 22 comprising a gastroresistant and enterosoluble polymer, a monovalent

15 metal salt of a biocompatible and biodegradable polymer having acidic groups, a divalent or trivalent ion inorganic salt, a cryoprotector or lyoprotector and the biologically active substances, in separate pre-measured packages.

20. . 24. The kit according to claims .22 or 23 further comprising a cross-linking agent and an additional biodegradable and biocompatible polymer in separate pre- measured packages. 25. The kit according to any of the claims 22 to 24,

25 further comprising extrusion devices such as sterile, non- sterile or sterilisable nozzles, needles or syringes. 26. Animal feeds supplemented with the microparticulate systems according to any of the claims 1 to 21. 27. A production process for the microparticulate systems comprising the following steps: a) combining an aqueous solution of gastroresistant and enterosoluble polymer with an aqueous or physiological solution of a monovalent metal salt, preferably a potassium or sodium salt of a biocompatible and biodegradable polymer, having acidic groups, and a cryoprotector or lyoprotector, so as to obtain a colloidal solution or homogeneous suspension; b) ^' solubilising or dispersing the biologically active substance in the solution or suspension' from step a); c) nebulising or extruding the solution or suspension from step b) in the presence of an aqueous solution of a water soluble inorganic divalent or trivalent salt, so as to obtain the formation of insoluble microparticulate systems containing the biologically active substances. 28. The process according to claim 27 wherein said step a) includes a step al) for the preparation of the monovalent metal salt solution of a biocompatible and biodegradable polymer, having acidic .groups and the cryoprotector or lyoprotector; and a step a2) for the preparation of the gastroresi-stant and enterosoluble polymer solution. . 29. The process according to claim 28 wherein, ' in step al) a physiological solution of said monovalent metal salt of a biocompatible and- biodegradable polymer is prepared with a concentration ranging from 0.01% to 10% w/v, preferably from 0.1% to 3% w/v. 30. The process according to claims 28 or 29 wherein, in step al), to said solution is added the cryoprotector or lyoprotector in such quantities as to give a concentration ranging from 1% to 40% w/v, preferably from 2% to 20% w/v. 31. The process according, to any of the claims 28.to

30 wherein, in step al), to said solution is added ah additional biocompatible and biodegradable polymer in such quantities as to obtain a concentration ranging from 0.1% to 50% w/v, preferably from 2% to 15% w/v. 32. The process according to any of the claims 28 to

31 wherein, in step a2) , an aqueous solution of gastroresistant and enterosoluble polymer is prepared at a concentration ranging from 1% to 50% w/v, preferably from 5% to 15% w/v. 33. The^' process according to claim_, 32 wherein said solution has a pH ranging from 6 to -8, preferably from 7^' to 7.8. 34. The process according to any of the claims 27 to 33 wherein, in step a), the solution- obtained in step al) and the solution obtained in step a2) are mixed in a ratio of 1:2, preferably 1:1 so as to obtain a colloidal solution or homogeneous suspension. 35. The process according to claim 34 wherein , said colloidal solution or homogeneous suspension is adjusted to a pH value ranging from 5.5 to 10, preferably from 6.5 to 8.5. 36. The process according to any of the^' claims 27 to

35 wherein, in step b) , said biologically active substance is dispersed in said colloidal solution or homogeneous suspension in such quantities as to give a concentration ranging from 0.01% to 20% w/v, preferably ;from 0.1% to 5% w/v. 37. The process according to any of the claims 27 to

36 wherein, in step c) , nebulisation occurs- through the use of orifices, nozzles or needles. 38. The process according to claim 37 wherein said nebulisation occurs through the use of needles having sizes ranging between 10 μm to 5000 μm, preferably from 300 μm to 2000 μm. 39. The process according to any of the claims 27 to 38 wherein, in step c) , said extrusion takes place with the use of automatic, semi-automatic microencapsulators, peristaltic or piston pumps or alternatives, or with a manually and/or automatically operated syringe. 40. The process according to claim 39 wherein said extrusion occurs through the use of a syringe at such a speed as to produce from 10 to 250 drops/minute, preferably from 20 to 120 drops/minute. 41. The process according to any of the claims 27 to 40 wherein, in step c) , said inorganic divalent or trivalent salt solution is an aqueous solution of calcium, barium, strontium, zinc, aluminium, iron or chromium chlorides. 42. The process according to claim 41 wherein said solution is an aqueous solution of calcium chloride, barium chloride or aluminium chloride. 43. The process according to claims 41 or 42 wherein said solution is an aqueous solution of calcium chloride. 44. The process according to any of the claims 41 to 43 wherein said inorganic salt solution has a concentration ranging from 0.1 to 2.0 M, preferably from 0.2 to 0.8 M. 45. The process according to any of the claims 27 to 44 wherein, in step c) , said extruded or nebulised solution and said inorganic salt solution are combined in a ratio of 1:6, preferably 1:4. 46. The process according to any of the claims 27 to 45 wherein said steps are carried out at a temperature of between -5 and 50°C, preferably between 0 and 30°C. 47. The process according to any of the claims 27 to 46 further comprising the steps of separation of the microparticulate systems from the solution by means of aspiration or filtration and the washing of said capsules with physiological solution (isotonic saline) . 48. The process according to any of the claims 27 to 47 additionally comprising the step of cross-linking the outer surfaces of the microparticulate systems through the use of cross-linking agents such as: protamine sulphate or phosphate, poly-L-lysine hydrobromide, polyvinylamine, chitosans. 49. The process according to claim 48 wherein said cross-linking agents are aqueous solutions at concentrations ranging between 0.01 and 5% w/v. 50. The process according to any of the claims 27 to 49 additionally comprising the step of lyophilisation of said microparticulate systems.