COMPOSITIONS OF DIETARY METALS FOR SUPPORT OF THE PHYSIOLOGICAL INTESTINAL DEVELOPMENT AND PREVENTION OF DIARRHOEA FIELD OF THE INVENTION The present invention relates to a novel concept of supporting the normal physiological development of the intestinal mucosa and/or preventing diarrhoea in animals, in particularly in pigs, chickens and calves, by increasing the local intraluminal bioavailability of dietary metal ions, such as zinc ions and copper ions, in the intestinal. tract of an animal. The present invention further relates to pharmaceutical compositions formulated for controlled delivery of metal ions such as to prevent such dietary metal ions from being absorbed systemically from the stomach and duodenum, but to increase intraluminal concentrations of zinc ions and/or copper ions throughout the small intestinal tract of an animal.

BACKGROUND OF THE INVENTION A number of dietary metals, such as zinc and copper, are reported as being of great importance for the proper growth and development of the digestive tract and for support of development of intestinal immune system of an animal. In particularly, supplemental administration of dietary metals is of importance to young animals where the small intestine is far from fully developed at the time of birth. After birth the small intestine still need to be developed such that proper conditions for effective uptake of nutrients is present. For example, the surface area of the small intestinal mucosa as determined by the number and length of intestinal villi should be as large as possible.

Normally, the growth and development of the intestinal tract of young animals is supported by nutrients in the milk during the suckling period. However, the intensive farm animal production of today requires the early separation of the suckling animal from its mother at a very young age. Therefore, weaned animals may suffer from improper development of the small intestine resulting in risk of low growth, poor feed uptake, diarrhoea and improved susceptibility to infections. For many years it has been common to treat weaned

pigs or other animals affected by poor development of the intestinal tract with antibiotic growth promoters. However, risk of public health problems because of resistant bacteria has been considered and antibiotic growth promoters have been forbidden in many countries.

Today, it has become routine in the swine industry to use sparingly soluble zinc salts, such as zinc oxide added to feed in high dosages (2000-3000 ppm of element zinc in feed) for supplement to pigs at weaning so as to promote growth, prevent weaning diarrhoea and decreasing the susceptibility to infectious bacteria. Further alternatives to antibiotic growth promoters have been reported, such as feeding pigs in the suckling period with fermentable dietary fibres and elemental iron (WO 99/62355). WO 98/16218 relates to the use of dietary metals, such as zinc or copper, complexed to a suitable ligand for treating gastro- intestinal infections.

As a sparingly soluble zinc salt, the absolute bioavailability of zinc oxide is estimated to be 10-20%. Thus, the majority of the dietary intake of zinc oxide is not absorbed systemically, but recovered in the manure. Unfortunately, this induces a risk to the environment in that zinc salts may accumulate in cultivated soil via the use of manure as fertiliser.

The majority of zinc ions is believed to be readily absorbed in the proximal part of small small intestine, either the duodenum or the proximal jejunum. For example, it is reported that soluble inorganic zinc salts, such as zinc sulfate, as well as organic zinc salts, such as zinc methione, has a higher systemic bioavailability than zinc oxide (llczlzsz, Joseph D et al.

- Growth and Plasina zinc Responses of Young Pigs Fed Pharmacologic Levls of Zinc, J. anion. Sci., 1993, vol 71, pp. 3020-3024).

However, high dosages of easily soluble zinc salts have proven to be toxic to the animal.

Therefore, according to EU legislation it is only allowable to supplement animal feed with less than 250 ppm of elemental zinc using zinc sources such as zinc oxide, zinc carbonate, zinc lactate, zinc acetate, zinc sulfate or zinc chloride.

Thus, there is a need in the art to provide zinc in lower dosages, although still in sufficient doses to effectively support the normal physiological development of the intestinal tract of young animals, and to prevent any adverse events caused by poor development of the small intestine.

Recently, newer approaches of supplementing zinc to animals have been reported. It has been speculated that organic sources of zinc may be more effective than inorganic sources due to higher bioavailability of zinc associated with an organic molecule, such as an amino acid. Accordingly, lower doses of zinc can be applied to feed. For example, by providing the dietary metal in complexed form with amino acid hydrolysates a higher systemic bioavailability of dietary metals in live stock animals was observed in comparison to zinc sulfate (US 5, 698,724).

Upon feeding post-weaned pigs with feed supplemented with 500 ppm of elemental zinc in the form of a polysaccharide complex of zinc, the growth performance during the first 4 weeks after weaning was improved (Case C. L. et al.-Effect of feeding organic and inorganic sources of additional zinc on growth performance and zinc balance in nursery pigs, J. Ani71z, Sic., 2002, vol. 80, pp. 1917-1924).

In chickens fed with corn-soybean meal diets, the relative bioavailability of zinc in the form of zinc methionine was 206 % and in the form of zinc oxide it was 61% in relation to zinc sulfate (100%) (Wedekind, K. J. et al. - Methodology for Assessing zinc Bioavailability : Eicacy Estimates for Zinc-Met71ionine, Zi71C Sulfate, and Zinc Oxide, J.

Aniiii. Sci, 1992, vol. 70, pp. 178-187).

However, organic sources of zinc may not always result in higher systemic bioavailability of zinc. Cheng J. et al. shows that zinc sulfate 100 ppm and zinc lysine 100 ppm did not differ in respect of bioavailability in pigs (Cheng J. et al.-Influece of Dietary Lysine ofz the Utilization of Zinc from Zinc Sulfate and a Zinc-Lysine Complex by Young Pigs, J. anim. Sci., 1998, ovl. 76, pp. 1064-1074).

In a recent study, the incidence of diarrhoea in weaned pigs in the first 4 weeks after weaning was studied upon feeding the weaned pigs with either organic zinc in a concentration equivalent to elemental zinc of 250 ppm in feed or zinc oxide in a concentration equivalent to elemental zinc of 2500 ppm in feed. (Conradsen, Pia, et al.

Danish article entitled"250 ppm organic zinc hits 2500 ppm of zinc oxide. Hyologisk tidsslcrift, 11,2002, p. 11-12.

An alternative approach of supporting small intestinal development and immune function was suggested by Caine and co-workers. They demonstrated a positive effect on small intestinal development and immune function of newly weaned pigs, when the pigs before weaning were fed by milk from sows that from day 80 of gestation until farrowing had been supplemented with zinc amino complex oxide in a concentration equivalent to elemental zinc of 2500 mg/kg in feed. (Cai7le W. et al-Intestinal Development of Pigs from Sows fed a Zinc Amino Acid Complex, Advances in Pork Production, 2001, vol. 12, Abstract No. 159.

The present inventor has provided a novel concept of supplementing dietary metals to animals. The concept aims at maximising intraluminal bioavailability of dietary metals in the small intestine of an animal, which may improve the likelihood of increasing the pool of dietary metals in intestinal mucosa cells. Advantageous, the novel concept implies that lower doses of zinc may be supplemented to feed, such as doses equivalent to elemental zinc of at most 250 ppm in feed, while still being able of supporting proper intestinal development.

SUMMARY OF THE INVENTION The present invention relates, at least in part, to novel compositions of dietary metals, such as zinc and copper, for minimised release of dietary metal ions in the gastric fluid, but for maximised release of dietary metals along the small intestine of an animal. Thus, compositions with controlled release of dietary metals are contemplated. Such compositions may, even in low doses, be effective in supporting the normal physiological

development of the intestinal tract in an animal in need thereof and preventing the adverse effects of poor intestinal development such as diarrhoea in an animal.

Advantageous, compositions with controlled release of a dietary metal, such as easily soluble zinc salts, may upon per-orally administration to an animal result in high intraluminal concentrations of zinc throughout the small intestine. Thus, inducing high concentration of intracellular metallothionein throughout the small intestine, improving zinc absorption into small intestinal mucosa cells so as to provide high intracellular zinc concentration, 'supporting normal cellular proliferation and differentiation of mucosa cells lining the villus and the crypts throughout the small intestinal canal, and preventing weaning diarrhoea and improving growth performance.

Accordingly, a first aspect of the invention relates to a composition comprising dietary metals suitable formulated for per-oral administration and suitable formulated for limited release of said dietary metals in the stomach, said composition comprises; i) a source of dietary metal, wherein the metal is selected from the group consisting of zinc and copper; and ii) one or more acceptable excipient and/or carrier, said composition releases less than 50 % of its content of said dietary metal when subjected to Dissolution Testing Method I for 0.5 hour, said Method I applies to a Basket Apparatus according to the European Pharmacopoeia Supplement 4.4 04/2003 as the dissolution apparatus, 0.001 N HC1 as the dissolution medium, 39°C as the temperature and 100 rpm as the rotation speed. Preferably, the release of said dietary metals from the composition is even more limited, such as less than 40%, more preferably less than 30%, such as less than 25%, 20%, 15%, 10% or 5% % of its content of said dietary metal is released when being subjected to said Dissolution Testing Method I.

The composition may further be formulated so as to ensure the controlled release of dietary metals during the transit of the small intestinal tract. Thus, a composition according to the

invention may be formulated such that more than 30%, preferably more than 40%, such as more than 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of its content of said metal selected from the group consisting of zinc and copper is released when subjecting the composition to Dissolution Testing Method II for 1.0 hour, said Method II applies to a Basket Apparatus according to the European Pharmacopoeia Supplement 4.4 04/2003 as the dissolution apparatus, 0.1 M phosphate buffer pH 7 as the dissolution medium, 39°C as the temperature and 100 rpm as the rotation speed.

A second aspect of the invention relates to the use of the composition as mentioned herein as a feed/food additive or as a dietary supplement to animals.

In a third aspect, the invention relates to a method for supporting the physiological gastro- intestinal development in an animal comprising administering to said animal an effective dose of a source of dietary metal, wherein the metal is selected from the group consisting of zinc and copper, said method further comprises that upon administering said effective dose of said source of dietary metal per-orally to said animal, less than 50% of the dose of said metal is recovered in the gastric juice during gastric residence time, and more than 50% of the dose of said metal is released into the small intestinal fluid during small intestinal transit time.

In still further aspects, the invention relates to a method for preventing and treating diarrhoea in an animal comprising administering to said animal an effective dose of a source of zinc, such as ionised zinc, said method further comprises that upon administering said effective dose of said source of zinc to said animal, less than 50% of the dose of said metal is recovered in the gastric juice during gastric residence time, and more than 50% of the dose of said metal is released into the small intestinal fluid during small intestinal transit time.

Another aspect of the invention relates to the use of a source of zinc for the preparation of a medicament for preventing and/or treating diarrhoea in an animal, said medicament releases less than 50 % of its content of said zinc when the medicament is subjected to Dissolution Testing Method I for 0.5 hour, said Method I applies to a

Basket Apparatus according to the European Pharmacopoeia Supplement 4.4 04/2003 as the dissolution apparatus, 0.001 N HCl as the dissolution medium, 39°C as the temperature and 100 rpm as the rotation speed.

DETAILED DESCRIPTION OF THE INVENTION Contrary to conventional methods of supplementing zinc and other dietary metals to animals, the present inventor has recognised the importance of providing sufficient local intraluminal available amounts of dietary metals, such as copper and zinc, throughout the small intestine of an animal. Furthermore, the present inventor has recognised that high systemic bioavailability of dietary metals may not support normal physiological development of the small intestine of animal.

Therefore, the present invention relates, at least in part, to limiting the systemic bioavailability of dietary metals and to provide sufficient concentrations of dietary metals in the intestinal fluid throughout the small intestine of an animal so as to increase the likelihood of providing high intracellular concentrations of zinc in small intestinal mucosa cells.

Without being bound to a particular theory, the present inventor put forth that upon targeting the release of dietary metals, such as zinc, in the small intestine of the gastro- intestinal tract, the intracellular concentration of the metal binding protein, metallothionein, increases. This may in turn result in higher uptake of zinc into mucosa cells since metallothionein, an intracellular metal binding, maintains zinc homeostasis by controlling cellular zinc uptake, distribution and excretion. Thus, the pool of zinc in mucosa cells may increase, which is of great importance for cell proliferation of intestinal cells.

Accordingly, a first aspect of the invention relates to a composition for controlled release of dietary metals. Such compositions may be suitable formulated so as to limit high systemic bioavailability of zinc, for example by limiting the release of dietary metals in the gastric juice of the stomach. Thus, compositions of the invention are formulated in a

manner such that upon oral administration to an animal less than 50%, such as less than 40%, 30%, 25%, 20%, 15%, 10% or 5% of its content of dietary metal is reco-vered in the gastric juice during its gastric residence time. In interesting embodiments thereof, less than 30%, preferably less than 25%, more preferably less than 20%, such as less t : han 15%, 10 or 5% of its content of dietary metal is recovered in the gastric juice during gastric residence time. Furthermore, the controlled release of dietary metals include the suitable formulation of the compositions of the inventions, such that upon oral administration to an animal more than 50%, such as more than 60%, 70%, 75%, 80%, 85%, 90% or 95% of its content of dietary metal is released in the small intestinal fluid during its small. 1 intestinal transit time. Preferably, more than 75%, such as more than 80%, 85%, 90% or 95% of its content of dietary metal is released in the small intestinal fluid during its small intestinal transit time.

In specific terms, a first aspect of the invention relates to a composition intended for per- oral administration comprising ; i) a source of dietary metal, wherein the metal is selected from the group consisting of zinc and copper; and ii) one or more acceptable excipient and/or carrier, said composition is further characterised by releasing less than 50 %, preferably less than 40%, more preferably less than 30%, such as less than 25%, even more preferably less than 20%, such as 15%, 10% or 5% of its content of said dietary metal when being subjected to Dissolution Testing Method I for 0.5 hour, said Method I applies to a Basket Apparatus according to the European Pharmacopoeia Supplement 4.4 04/2003 as the dissolution apparatus, 0.001 N HCl as the dissolution medium, 39°C as the temperature and 100 rpm as the rotation speed.

In further embodiments of the invention less than 50%, preferably less than 40%, such as less than 30%, 25%, 20%, 15%, 10% or 5% of its content of metal selected fnom the group consisting of zinc and copper is released when subjecting the composition to said Dissolution Testing Method I for 1.0 hour.

As stated above, the present invention relates to controlled release of dietary metals for improving the local bioavailability of dietary metals in the small intestine of the gastro- intestinal tract. That is to say that in further embodiments of the invention, the compositions releases more than 20%, preferably more than 30%, such as more than 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of its content of said dietary metal when being subjected to Dissolution Testing Method II for 1.0 hour, said Method II applies to a Basket Apparatus according to the European Pharmacopoeia Supplement 4.4 04/2003 as the dissolution apparatus, 0.1 M phosphate buffer pH 7 as the dissolution medium, 39°C as the temperature and 100 rpm as the rotation speed.

In still further embodiments thereof, more than 20%, preferably more than 30%, such as more than 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of its content of said dietary metal is released when subjecting the composition to said Dissolution Testing Method II for 0,5 hour.

As used herein, the term"dietary metal"encompasses metals that form part of normal dietary requirements for mammals, such as animals and humans including zinc, copper, cobalt, manganese and iron. In the presently preferred context of the invention, the dietary metal is copper and/or zinc. Furthermore, in presently interesting embodiments of the invention, the dietary metal is zinc, which has a beneficial effect in preventing diarrhoea in animals, such as in animals with poor developed small intestine.

The term"ionised form"encompasses various oxidation states of a dietary metal, such as a monovalent, a divalent or a trivalent kation of a dietary metal, e. g, a kation selected from the group of elemental dietary metals consisting of Zn+, Zn2+, Cu+, Cu2+, Mn+, Mn2+, Fe2+, Fe3+. In a presently preferred context of the invention, the term"ionised form"is denoted to include kations of dietary metals in oxidation states that are normally present in the gastro-intestinal tract, such as Zn2+, Cu2+ and Fe3+.

The term"a dose equivalent to elemental zinc"is denoted to mean the dose of elemental zinc in a source of zinc.

The term"acceptable carriers and excipients"is intended to mean substances, which are substantially harmless to the individual to which the composition will be administered.

Such excipients normally fulfil the requirements given by national drug agencies and/or feed stuff legislation, Official pharmacopeias such as the United States of America Pharmacopeia and the European Pharmacopeia set standards for well-known pharmaceutically acceptable excipients.

The term"peroral administration"is intended to mean administration to an individual of a composition trough the mouth, preferably where the release and absorption of the therapeutically active principle is not intended to occur in the oral cavity, but rather after passing the oral cavity, such as in the gastro-intestinal tract.

As used herein the term"an animal"characterises an animal, including a human, in need of supplementary doses of dietary metals, such as zinc and copper. The animal may further be characterised by having or being prone to poor development of the small intestine. Signs on poor development of the small intestine include presence of adverse changes in intestinal morphology, including reduced villus height (villus atrophy), increased villus width with oedema, increased crypt dept and reduced absorptive capacity and brush border enzyme activity. The animal may also be characterised by being in growth phase, such as a young animal before being fully matured as an adult animal. Presently interesting examples of animals include pigs, in particularly weaned pigs, pigs in growth phase; chickens such as chickens in growth phase; and calves such as calves in growth phase.

In the context of the present invention, the term"controlled releases denoted to mean any release of dietary metals from a composition, including the metal on ionised form, in complexed form or any other suitable form, wherein the release rate is modified in a controlled, retarded, sustained, delayed and/or in any other manner in comparison to the release of dietary metals from a composition consisting of animal feed products.

Furthermore, the term"controlled release"may in its broadest sense relate to the release of dietary metals from a composition such that a) when subjecting the composition to Dissolution Testing Method I for 0.5 hour, less than 50 % of its content of said dietary metal is released,

said Method I applies to a Basket Apparatus according to the European Pharmacopoeia Supplement 4.4 04/2003 as the dissolution apparatus, 0.001 N fIC1 as the dissolution medium, 39°C as the temperature and 100 rpm as the rotation speed, and/or b) when subjecting the composition to Dissolution Testing Method II for 1.0 hour more than 20% of its content of said dietary metal is released, said Method II applies to a Basket Apparatus according to the European Pharmacopoeia Supplement 4.4 04/2003 as the dissolution apparatus, 0.1 M phosphate buffer pH 7 as the dissolution medium, 39°C as the temperature and 100 rpm as the rotation speed.

As used herein the term"small intestine"is denoted to include duodenum, proximale jejunum, middle jejunum, distale jejunum and ileum. However, in some embodiments of the invention, where it is desirable to limit the absorption of dietary metals from duodenum, the term"small intestine"or the phrase"intestinal part of the gastro-intestinal tract"is denoted to include proximale jejunum, middle jejunum, distale jejunum and ileum, preferably to include middle jejunum, distale jejunum and ileum.

The term"systemic bioavailability"is in generally terms denoted to mean the percentage of a dose of dietary metals absorbed into the circulating blood upon administering a single dose or after administering several doses such as at steady state conditions. The systemic bioavailability may be determined on the basis of plasma AUC. However, dietary metals may only shortly be present in the circulating blood after absorption due to high distribution into other tissues. Therefore, systemic bioavailability may be determined by measuring the recovered amount of dietary metals in storage tissues such as liver, kidney, bones and/or excretion in urine or combinations thereof.

The term"intraluminal bioavailability"or"local bioavailability"is intended to denote the percentage of a dose of dietary metals released into the small intestine of an animal. The uptake of dietary metals in small intestinal mucosa cells may be a measure of"intraluminal bioavailability"or"local bioavailability".

As mentioned, compositions of the invention comprise a source of a dietary metal. In the context of the invention, the term"source of a dietary metal"is denoted to mean

substances with content of dietary metal, preferably zinc and/or copper, wherein the dietary metal may be in various oxidation stages in the form of a salt or a chelate.

Furthermore, it is anticipated that interesting sources of dietary metal relates to those easily soluble in water. Thus, the source of dietary metal according to the invention may be characterised by having a solubility of at least 0.02 mg/ml, preferably at least 0.05 mg/ml, more preferably at least 0.1 mg/ml, still more preferably at least 0,5 mg/ml, most preferably at least 0,7 mg/ml, such as at least 1 mg/ml, 2 mg/ml, 3 mg/ml and 4 mg/ml. when subjected to water at 25 °C.

In suitable embodiments of the invention, the source of dietary metal is a metal salt, such as a zinc salt and/or a copper salt, of an inorganic acid, wherein the inorganic acid is selected from the group consisting of hydrobromic acid, hydrochloric acid, hydroiodic acid, nitric acid and sulfuric acid. Thus, in interesting embodiments of the invention, the salt is a zinc salt and/or copper salt of an inorganic acid, such as zinc bromide, zinc chloride, zinc iodide, zinc nitrate, zinc sulfate, copper bromide, copper chloride, copper iodide, copper nitrate or copper sulfate.

Furthermore, salts of organic acids may be suitable for use in compositions of the invention. Therefore, in still interesting embodiments, the source of a dietary metal is a dietary metal salt, such as a zinc salt and/or a copper salt, of an organic acid, wherein the organic acid is selected from the group consisting of acetic acid, citric acid, formic acid, lactic acid, glucoronic acid, oxalic acid and propionic acid. Thus, interesting zinc salts of organic acids relate to zinc acetate, zinc citrate, zinc lactate, zinc gluconate, zinc oxalate, zinc propionate, copper acetate, copper citrate, copper lactate, copper gluconate, copper oxalate or copper propionate. Preferably, the zinc salt is zinc sulfate, zinc chloride, zinc acetate or hydrates thereof and the copper sulfate, copper chloride, copper acetate or hydrates thereof.

Complexes of dietary metals, such as chelates of dietary metals with amino acids and polysaccharides may be of great importance for achieving high systemic bioavailability.

However, in the context of the present invention, the use of such chelates may not be

anticipated as long as such chelates are prevented from being absorbed in the proximal parts of the gastro-intestinal tract, such as the stomach and duodenum.

The composition according to any one of claims 1 to 8, wherein the Thus, in further embodiments of the invention, the dietary metals is complexed to a ligand, wherein said ligand is selected from the group consisting of saccharides, amino acids, di- carboxylic acids, and (3-hydroxy acids and/or EDTA. Interesting complexes of dietary metals include complexes with ascorbate, aspartate, citrate, histidine, malate, maltol, gluconate, glutamate, succinate, tartrate, lysine or methionine.

It is further contemplated that compositions of the invention may include mixtures of one or more dietary supplements, such as for example sources of zinc in combination with sources of copper or other dietary metals.

In embodiments, wherein the source of dietary metal is a source of zinc, such as ionised zinc, the source of zinc is in an amount equivalent to elemental zinc in the range of 2% to 50% w/w, such as 2-30%, such as 25-25% w/w.

In further embodiments of the invention, the composition is added to feed. Such embodiments comprises said source of zinc, such as a source of ionised zinc, in an amount equivalent to elemental zinc in the range from about 25 to 1000 ppm in feed, such as from about 50 to 500 ppm, 50 to 400 ppm, 50 to 300 ppm, preferably from about 50 to 250 ppm, more preferably from about 100 to 250 ppm in feed.

As stated the compositions may be suitable formulated so as to limit the absorption of dietary metals from the upper part of the gastro-intestinal tract, such as the stomach and/or the duodenum or a part of duodenum, and to release the dietary metals throughout, or at least to the main part of, the small intestine of an animal. It is however not anticipated that the release of dietary metals may continue during the transit of large bowel.

Thus, in preferred embodiments of the invention, the composition further comprises a release-rate controlling agent. Such an agent is described in details infra. In generally

terms, the release-rate controlling agent may be selected from the group consisting of waxes, fats, proteins, polymeric organic compounds, such as celluloses and polysaccharides, and combinations thereof.

In presently interesting embodiments of the invention, the release-rate controlling agent include ethyl cellulose, which may be provided as an outer coating or in an core or combinations thereof.

Compositions of the invention may be formulated in a number of manners as long as the per-oral administration of the composition results in minimised release of dietary metal ions in the gastric fluid, as indicated by a limited systemic bioavailability, and maximised release of dietary metal ions in the small intestinal fluid, preferably such that dietary metal ions is continuously released throughout the intestinal tract, preferable the small intestinal tract.

In presently interesting embodiments of the invention, the compositions are formulated as solids. Thus, such embodiments have a moisture content of less than 50%, such as less than 40%, 30%, 25%, 20%, 15%, 10% and 5%. However, other forms of the composition, such as semi-solids or liquids are not anticipated.

Furthermore, compositions of the invention may be provided as crystals, granules, pellets, spheres, beads, matrixes or other types of agglomerates that allow for oral administration to an animal. In some interesting embodiments of the invention, the compositions is pre- mixed with feeds and/or nutrients or incorporated into feed substances or nutrients, for example animal feed or feed substances such as fibers approved according to Feeding Stuffs Regulations. Alternatively, the composition of the invention may be added to feed, such as provided as top dressing. In suitable embodiments of the invention, said granules, pellets, spheres, beads, matrixes or other types of agglomerates are essentially spherical.

The compositions of the invention may further be characterised by including one or more core (s), wherein the source of dietary metal may be incorporated or not. Such cores as well as compositions of the invention in general may comprise one or more of acceptable

carriers and excipients such as diluents, binders, disintegrants, wetting agents, lubricants, glidants, flow aids and/or flavours. In presenting interesting embodiments of the invention, the core may comprise a polysaccharide such as chitosan.

In the present context, the term"core"is denoted to mean a matrix formed by dry, melt or wet granulation or by blending of acceptable excipients.

Typical examples of diluents include lactoses, starches, mannitol, sorbitol, xylitol, dextroses, phosphates, sucroses, calcium sulfates, calcium lactates, dextrates, inositol, celluloses, calcium carbonates, glycine, bentonite, polyethylenglycol, polyvinylpyrrolidone or mixtures thereof.

Typical examples of binders include acacia, tragacanth, sucrose, gelatin, glucose, starches, pregelatinized starches, celluloses including methylcellulose and sodium carboxymethylcellulose, alginic acid and salts of alginic acid, magnesium aluminum silicate, polyethylenglycol, guar gum, polysaccharides, bentonites, polyvinylpyrrolidone, polymethacrylates, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose or mixtures thereof.

Examples of disintegrants include starches, clays, celluloses, croscalnellose sodium, alginates, crospovidone, gums or mixtures thereof.

Typical examples of wetting agents include quaternary ammonium compounds, phenyl ethers, polyoxyethylene fatty acid/sterates/glycerides and oils, sorbitan esters, propylene glycol fatty acid esters, glyceryl fatty acid esters.

Typical examples of lubricants, glidants or flow aids, include glyceryl behapate, stearic acid and salts thereof, including magnesium, calcium and sodium stearates, hydrogenated vegetable oils, colloidal silica, talc, waxes, boric acid, sodium benzoate, sodium acetate, sodium fumarate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, sodium lauryl sulfate, and magnesium lauryl sulfate, and stabilisers such as desiccating amorphous silica or mixtures thereof.

Suitable flavours are those typically used by a person skilled in the art.

In one embodiment of the invention the core essentially consists of a diluent selected from the group consisting of lactose, starches, sugars, polysaccharides metal salts, celluloses, minerals and polymers. In an interesting embodiment of the invention, the core is in the form of a sugar sphere. In another embodiment, the core is made of excipients that may not substantially affect the release rate of the dietary metal ions such as for example easily soluble excipients such as sucrose. In yet another embodiment, the core comprises one or more agents that may control the release rate of dietary metal ions, such as chitosan. In further embodiments thereof, the core may be coated with a release-rate controlling agent.

According to the invention, the minimised release of dietary metal ions in gastric fluid and the maximised release of dietary metal ions in the intestinal tract of an animal is provided, at least in part, by incorporating one or more release-rate controlling agent (s) in the composition.

Typical principles of incorporating a release-rate controlling agent in a composition of the invention include: 1. providing the source of a dietary metal in an core, wherein the core is coated with an outer coating comprising one or more release-rate controlling agent (s).

Thus, in one typical embodiment of the invention, the source of a dietary metal is mixed with one or more suitable excipients so as to incorporate the source of a dietary metal in one or more cores, and coating the resulting core (s) with an outer coating comprising a release-rate controlling agent. In still further embodiments thereof, the release of metal ions from the composition may in addition be controlled by incorporating one or more release-rate controlling agent (s) in the core.

2. providing cores or matrixes made of one or more excipients, wherein the source of a dietary metal is applied onto the surface of said cores or matrices in mono or multiple

layers and coating the resulting cores with an outer coating comprising a release-rate controlling agent. Optionally, said core may comprise a release-rate controlling agent.

3. providing the source of a dietary metal in a mixture of one or more release-rate controlling agent (s), optionally coated with a film with substantially no effect on the release of metal ions from the composition In the present context a"release-rate controlling agent"is denoted to characterise an agent, which controls the release of a source of dietary metal by means of acting as a pH-dependent barrier. affects the diffusion rate of dietary metal through a coating or out of an core; acts as a erodable and/or degradable agent so that the release of a source of dietary metal will be controlled by the rate of removing, dissolving or eroding of said agent when the source of dietary metal is enclosed in an core together with said erodable and/or degradable agent or is coated with said erodable and/or degradable agent. controls the release of a source of dietary metal by means of acting as a temperature dependent barrier.

Typical pH-dependent rate limiting barriers relates to agents, which is substantially insoluble in gastric juices, e. g. pH below 4, thus preventing release of dietary metal ions in gastric fluid. However, in order to release dietary metal ions in the intestinal tract, such agents should dissolve or otherwise be removed upon being contacted with the intestinal fluids. Such agents are generally termed enteric coatings, which is denoted to mean any pharmaceutically acceptable agent that has a low solubility or is practically insoluble at low pH, such as below pH 4, but may dissolve at higher pH values, such as from pH 4 to 7.5, more preferably from pH 6 to 7.5, or may be destroyed by enzymatically processes.

Various in vitro tests are cited in various Pharmacopoeias for the determination of whether or not an agent may be classified as an, enteric coating. As used herein, an enteric coating is provided by agents that remains intact for at least 60 minutes, preferably at least 80 minutes, more preferably at least 100 minutes when being contacted with artificial gastric

juices such as HC1 of pH 3 at 37°C or 39°C. Furthermore, suitable agents should dissolve or disintegrates upon being contacted with artificial intestinal juices such as a KH2PO4 buffered solution of pH 6.8 or higher such as 7 or 7.5 within the first 30 minutes of contact.

The most effective enteric polymers are polyacids having a pKa of 3 to 5.

Typically examples of enteric coatings include pH-sensitive enteric polymers, preferably those that dissolves at pH levels above pH levels of the gastric fluid such as pH above 4, 4.5, 5,5. 5,6, 6.5, 7.0 or pH 7.5 and include Cellulose acetate phthalate (CAP).

Typical examples of cellulose acetate phthalates have an acetyl content of 17-26% and phthalate content of from 30-40%. An example of an appropriate cellulose acetate phthalate is the marketed product CAP@.

Cellulose acetate trimellitate (CAT).

Typical examples of cellulose acetate trimellitates have an acetyl content of 17-26%, a trimellityl content from 25-35%. An example of an appropriate cellulose acetate trimellitate is the marketed product CAT@.

Hydroxypropyl methylcellulose phthalate (HPMCP).

Hydroxypropyl methylcellulose phthalates typically have a molecular weight of from 20,000 to 100,000 daltons, e. g. a hydroxypropyl content of from 5 to 10%, a methoxy content of from 18 to 24% and a phthalyl content from 21 to 35%.

Methacrylic/acrylic acid copolymers and esters thereof.

Methylacrylates include those of molecular weight above 100,000 daltons based on, e. g. methylacrylate and methyl or ethyl methylacrylate in a ratio of about 1: 1. Typical products include Endragit L, e. g.

Eudragit@ L 100-55 spray dried Eudragit (E) L 30 D-55 which can be reconstituted for aqueous formulations for targeted drug delivery in the duodenum;

Eudragit L 30 D-55 pH dependent anionic polymer solubilizing above pH 5.5 ; Eudragit O L 100 pH dependent anionic polymer solubilizing above pH 6.0 and Eudragit (D S 100 pH dependent anionic polymer solubilizing above pH 7.0.

Hydroxypropyl methylcellulose acetate succinate (HPMCAS).

Hydroxypropyl methylcellulose acetate succinate include polymers in aqueous preparations such as Aqoat@.

Polyvinyl acetate phthalate (PVAP).

Polyvinyl acetate phthalate include, aqueous coating formulation of the polymers such as Opadry-entric (D, SureleaseX and Sureteric@.

Carboxy methyl ethyl cellulose (CMEC).

# Styrol maleic acid copolymers.

In some interesting embodiments, the enteric coating are made from cellulose acetate phthalate and trimellitate, methacrylic acid copolymers e. g. copolymers derived from methylacrylic acid and esters thereof, hydroxypropyl methylcellulose phthalate, and polyvinyl acetate phthalate.

The enteric coating may be applied using conventional techniques implying that the cores or compositions are sprayed with an aqueous or non-aqueous coating suspension or solution of the enteric-coating to form a film coating. In general, satisfactory results are obtained with a coating of 5-100 um, preferably 20-80 Am thickness. Suitable solvents for dissolving or suspending the enteric-coating include organic solvents, e. g. alcohols such as methanol, ethanol and isopropanol, a ketone such as acetone, halogenated hydrocarbons such as methylenchloride or mixtures of such solvents. Other suitable solvents are mixtures of the above mentioned solvents and water.

The enteric coating may also be used together with a plasticizer, such as esters of citric acid, tartaric acid and succinic acid; glycerol and glycerol esters; phthalic acid esters;

glycols such as ethylene glycols, propylene glycols, polyethylene glycols and esters thereof; adipate ; benzophenone; polysorbates; or sorbitan monooleate.

The enteric coating may also include an anti-tack agent such as talc, silica or glyceryl monostearate. The quantity of plasticizer and anti-tack agent may be generally conventional to the art. Typically the coating may include about 10-25 wt % of plasticizer and up to about 50 wt % of anti tack agent, e. g. 5-20 wt % of anti-tack agent.

As mentioned, interesting release-rate controlling agents of the invention relates to those agents that controls the release of dietary metal through simple diffusion, independent of the pH in the surrounding environment, in that the fluid may be able to slowly diffuse into coatings or cores comprising such agents, which in turn may dissolve the dietary metals upon where the metals escapes from the composition by diffusion through the coating membrane or out of the core. Thus, in the present context, an agent that controls the release rate by diffusion is denoted to mean agents that may form coatings that are fully permeable to fluids such as water and any substance dissolved herein. Typical interesting agents relates to film-forming polymeric substances characterised by being substantially water- insoluble, but water-diffusible and substantially pH-independent, or the agent may be a hydrophobic agent: Typical examples of film-forming polymeric substances contemplated for the purpose of the present invention are cellulose derivatives, for instance ethylcellulose, acrylic polymers, vinyl polymers, and other high molecular synthetic polymers such as ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, cellulose valerate, cellulose acetate propionate, polyvinyl acetate, polyvinyl formal, polyvinyl butyral, polymethyl methacrylate, polycarbonate, polystyrene, polyester, coumaroneindene polymer, polybutadiene, vinyl chloride-vinyl acetate copolymer, ethylene-vinyl acetate copolymer and vinyl chloride-propylene-vinyl acetate copolymer.

In one preferred embodiment of the invention, the film-forming polymer is ethylcellulose.

Typical examples of hydrophobic substances are waxes or wax-like substances. Examples of wax-like substances are carnauba wax, beeswax, paraffin wax, ceresine, shellac wax, castor wax, and higher fatty acids such as myristic, palmitic, stearic and behenic acids and esters thereof.

In suitable embodiments, the above-mentioned film-forming and/or hydrophobic substance is mixed with an acceptable water-soluble substance so as to modify the desired diffusion properties of the film, e. g. so as to achieve continuous release of dietary metal ions from the composition.

Typical examples of such acceptable water-soluble substance are selected from polyethylenglycol, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, methylcellulose and polyvinylpyrrolidone and other water-soluble polymers known by a person skilled in the art.

As mentioned, suitable embodiments of the invention relate to those wherein the source of a dietary metal is incorporated into a core.

It may be desirable, to first apply one or more sub-coats to the core, before application of the coating of a release-rate controlling agent. Thus, a sub-coat consequently lies beneath the coat of a release-rate controlling agent. Suitable sub-coat materials include hydroxypropylmethyl cellulose. It may also be desirable to apply one or more over-coats after application of the coat of a release-rate controlling agent, the over-coat consequently lies over the coat of a release-rate controlling agent. The over-coat may be of the same material as the sub-coat. Typically such coatings may be applied by known techniques of aqueous film coating.

In embodiments wherein the dietary metal ions is enclosed in an core together with release-rate controlling agents, the release-rate controlling agent is preferably controlling the release rate by diffusion of erosion or combinations thereof. Such release-rate controlling agent is meant to include without restriction polymers, such as ethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, hydroxypropyl methylcellulose,

polyethylenglycol, methacrylates, polymethylmethacrylate, carbomer, alginates and xanthan gums.

Furthermore, the release-rate controlling agent is meant to include hydrophilic or hydrophobic polymers, such as gums, cellulose ethers, protein derived materials, nylon, acrylic resins, polylactic acid, polysaccharides such as starches, cellulose acetate phthalate.

Of these polymers, cellulose ethers especially substituted cellulose ethers such as alkylcelluloses, in particularly ethylcellulose, Ci-Ce hydroxyalkylcelluloses (such as hydroxypropylcellulose and especially hydroxyethyl cellulose) and acrylic resins (for example methacrylates such as methacrylic acid copolymers) and polysaccharides (such as Chitosan) are preferred.

Additionally, a number of non-polymeric agents may act as release-rate controlling agents Thus, in still interesting embodiments, the release-rate controlling agent is meant to include digestible, long chain (C8-C50, especially C8-C4o), substituted or unsubstituted hydrocarbons, such as fatty acids, hydrogenated vegetable oils, hardened rapeseed oil, castor oil, coconut oil, beef tallow, palm oil, palm kernel oil and soya bean oil, fatty alcohols (such as lauryl, myristyl, stearyl, cetyl or preferably cetostearyl alcohol), glyceryl esters of fatty acids for example glyceryl esters of fatty acids for example glyceryl monostearate, monoacyl and diacylglycerols esterified with acetic-, lactic-, citric-, tartaric-, monoacetyltartaric-and/or diacetyltartaricacid, mineral oils and waxes, e. g. , carnauba wax, castor wax, beeswax, paraffin wax, ceresin, shellac wax. Hydrocarbons having a melting point of between 25°C and 90°C are preferred. Of these long chain hydrocarbon materials, fatty (aliphatic) alcohols are preferred.

In preferred embodiments of the invention, the core comprises a polysaccharide such as chitosan. The term"chitosan"encompasses a series of chitosan polymers with different molecular weights and degree of acetylation. The release rate of dietary metal ions from the chitosan core is determined, at least in part, by the degree of swelling of the chitosan in that the release rate may become constantly and continuos when the degree of swelling has reached its equilibrium state.

The core or matrix may conveniently comprise between 1 wt % and 99 wt % of said hydrophilic or hydrophobic polymer.

In another presently interesting embodiment of the invention, the composition is formulated a semisolid preparation, such as a paste, gel, cream or ointment, wherein the release of the dietary metal ions is targeted to the intestinal tract. The semisolid preparations can be prepared by techniques known by the person skilled in the art.

In other interesting embodiments of the invention, the composition is formulated as liquid preparations. Preferred liquid preparations include suspension and emulsion, wherein the release of dietary metal ions is targeted to the intestinal tract. The suspensions and emulsions can be prepared by techniques known by the person skilled in the art.

As stated, the compositions may comprise a release-rate controlling agent for controlled release. In suitable embodiments thereof, the in vitro release of dietary metals from the composition when being subjected to said Dissolution Testing Method II as described herein is as follows: more than 40%, preferably more than 50%, such as more than 60%, 70%, 75%, 80%, 85%, 90% or 95% of the content of said dietary metal is released following in vitro testing for 2. 0 hours.

# more than 50%, preferably more than 60%, such as more than 70%, 75%, 80%, 85%, 90% or 95% of the content of said dietary metal is released following in vitro testing for 4. 0 hours.

20-40%, preferably 20-50% such as 20-60%, 20-70% or 20-80% of the content of said dietary metal is released following in vitro testing for 1.0 hour.

25-40%, preferably 25-50% such as 25-60%, 25-70% or 25-80% of the content of said dietary metal is released following in vitro testing for 1.0 hours.

30-50%, preferably 30-60% such as 30-70% or 30-80% of the content of said dietary metal is released following in vitro testing for 1.0 hours.

40-60%, preferably 40-70% such as 40-80% or 40-90% of the content of said dietary metal is released following in vitro testing for 1.0 hours.

30-50%, preferably 30-60% such as 30-70%, 30-80%, 30-90% or 30-100% of the content of said dietary metal is released following in vitro testing for 2.0 hours.

40-60%, preferably 40-70% such as 40-80%, 40-90%, 40-100% of the content of said dietary metal is released following in vitro testing for 2.0 hours. wherein 50-70%, preferably 50-75% such as 50-80%, 50-90% or 50-100% of the content of said dietary metal is released following in vitro testing for 2.0 hours.

60-80%, preferably 60-80% such as 60-90% or 60-100% of the content of said dietary metal is released following in vitro testing for 2.0 hours.

# 70-90%, preferably 75-90% such as 75-100% of the content of said dietary metal is released following in vitro testing for 2.0 hours.

60-80%, preferably 60-90% such as 60-100% of of the content of said dietary metal is released following in vitro testing for 4.0 hours.

# 70-90%, preferably 75-90%, such as 75-95 or 75-100% of the content of said dietary metal is released following in vitro testing for 4.0 hours.

It is further contemplated that the in vitro release of dietary metals from a composition may be investigated at pH 6.5 and 7.0, in particularly when aiming at simulating the pH values in the middle and distale part of the small intestine of an animal. Thus, said Dissolution Testing Method II may be applied with alternative pH values such as pH 6.5 and pH 7. 0.

As stated above, the intended use of compositions of the invention mainly relates to supplementing live stock animals, in particularly animals in growth phase, with dietary metals, in particularly copper and zinc, for supporting and improving the normal proliferation of intestinal cells, thus enhancing the absorptive area of the small intestine. In absence of the normal development of the small intestine in an animal in growth phase, the animal may suffer from diarrhoea, anorexia, villus atrophy, poor growth, improved susceptibility to infectious bacteria and poor recovery from virus diarrhoea.

Accordingly, a second aspect of the invention relates to the use of compositions of the invention as a feed/food additive or as a dietary supplement to animals, e. g for supporting the physiological gastro-intestinal development and in the event where the composition

comprises zinc, the feed/food additive or dietary supplement may be used in the prevention of diarrhoea in an animal. Also the use of the feed/food additive or dietary supplement for supporting the recovery from virus diarrhoea is anticipated.

Furthermore, a further aspect of the invention relates to a method of supporting the physiological gastro-intestinal development in an animal, comprising administering to said animal an effective dose of a source of a dietary metal, preferably zinc and/or copper, wherein said method further comprises that upon administering said effective dose of said source of dietary metal per-orally to said animal, less than 50% of the dose of said metal is recovered in the gastric juice during gastric residence time, and more than 50% of the dose of said metal is released into the intestinal fluid during small intestinal transit time.

As reported herein, it has been common use to supplement zinc oxide in admixture with feed in high dosages, such as doses equivalent to 1500-3000 ppm of elemental zinc in feed, in order to prevent diarrhoea in animals, such as growing animals, including weaned pigs.

Furthermore, the utilisation of easily soluble zinc salts and amino acid chelates have been reported. However, it is the current expectations of the present inventor that the administration of zinc oxide, easily soluble zinc salts or amino acid chelates may not result in effective intraluminal concentrations of zinc in the small intestine of an animal. Mainly, because zinc oxide is practical insoluble at neutral pH. Another reason is that easily soluble zinc salts or amino acid chelates are pre-dominantly absorbed in the duodenum and proximal part of the jejunum. However, higher dosages of zinc oxide, such as doses equivalent to elemental zinc that are higher than 2000 ppm in feed may result in sufficient levels of available zinc in the small intestine.

Thus, a still further aspect of the invention relates treating and/or preventing of diarrhoea in an animal, wherein a source of zinc is provided in a form that limits the absorption from the duodenum and proximal part of the jejunum and maximises the local bioavailability of zinc throughout the small intestinal tract. Accordingly, in a still further aspect, the invention relates to a method of preventing and treating diarrhoea in an animal comprising administering to said animal an effective dose of a source of zinc,

said method further comprises that upon administering said effective dose of said source of zinc per-orally to said animal, less than 50% of the dose of said metal is recovered in the gastric juice during gastric residence time, and more than 50% of the dose of said metal is released into the small intestinal fluid during small intestinal transit time.

In terms of maximising the local bioavailability of dietary metals in the small intestine of an animal, it is desirable to effectively limit the release of dietary metal ions in the stomach. Thus, in further embodiments of the invention, the methods imply that less than 40%, preferably less than 30%, more preferably less than 25%, such as less than 20%, 15%, less than 10% or less than 5% of the dose of said metal is recovered in the gastric juice during gastric residence time. Moreover, more than 60%, preferably more than 70%, more preferably more than 75%, such as more than 80%, more than 85%, more than 90% or more than 95% of the dose of said metal is released into the small intestinal fluid during small intestinal transit time.

In further embodiments thereof, the method of preventing and treating diarrhoea in an animal comprises administering said source of zinc for the first 10 weeks after weaning, preferably for the first 9 weeks, such as 8 weeks, 7,6, 5,4, 3 weeks or 2 weeks after weaning.

Moreover, in still another aspect the invention relates to the use of a source of zinc for the preparation of a medicament for preventing and/or treating diarrhoea in an animal, said medicament releases less than 50 % of its content of zinc when the medicament is subjected to Dissolution Testing Method I for 0.5 hour, said Method I applies to a Basket Apparatus according to the European Pharmacopoeia Supplement 4.4 04/2003 as the dissolution apparatus, 0.001 N HCl as the dissolution medium, 39°C as the temperature and 100 rpm as the rotation speed.

The diarrhoea as referred to herein include diarrhoea related to animals in growth phase; weaning diarrhoea; neonatal coli diarrhoea (for example caused by E. coli); proliferative enteropathy (for example caused by Lawsonia intracellularis). Furthermore, generally speaken, the diarrhoea relates to diarrhoea of animals with poor development of the

intestinal tract as defined by presence of villus atrophy. In further embodiments, wherein the animal is a pig, the diarrhoea is Swine dysentery that may be caused by Serpulina hyodysenteriae.

Thus, animals infected with bacteria capable of provoking diarrhoea, such as E. coli, Lawsonia intracellularis, Serpulina hyodysenteriae, may be subject to the uses and methods as described herein.

According to the invention, said methods and uses as described herein, further comprises that the source of a dietary metal or a source of zinc may be provided in the form of a composition of the present invention, as described supra.

As stated the methods and uses described herein include the administration to an animal. In presently interesting embodiments, the uses and methods include an animal selected from the group consisting of pigs, calves and chickens. Moreover, uses and methods as described herein relates to weaned pigs or pigs in the period of weaning.

Furthermore, in the uses and methods as described herein, the source of zinc may be supplemented to the animal in admixture with feed such that the dose equivalent to elemental zinc in feed is from about 25 to 1000 ppm, such as from about 50 to 500 ppm, 50 to 400 ppm, 50 to 300 ppm, preferably from about 50 to 250 ppm, more preferably from about 100 to 250 ppm.

EXAMPLES Example 1 Compositions of the invention comprises the following ingredients: 10-50% Zinc (zinc sulphate mono hydrate; zinc chloride, zinc acetate, zinc sulphate hepta hydrate, zinc lactate dihydrate or zinc lactate trihydrate); and 25-80% Ethyl cellulose;

and/or 25-80% ethyl cellulose and chitosan; and/or 25-80% Acetic acid ester of monoglycerides from vegetable oil; and/or 25-80% Vegetable oil; and/or 25-80% Vegetable hydrogenated oil; and/or 5-10% Amylopectin, pregelatinized or not pregelatinized; <20% Saccharose <20% Lactose <20% Free flow auxiliary (e. g Silicium dioxide) <1% Synthetic sweetener Water 100% Total Example 2 Typical compositions of the invention include: 1) A composition essentially consisting of an inner core of sugar sphere coated with a first layer of zinc sulphate and thereafter coated with one or more layer of ethyl cellulose and finally coated with a layer of sucrose, either sprayed on or as a powder.

2) A composition essentially consisting of an inner core of sugar sphere coated with a first layer of zinc sulphate and thereafter one layer of chitosan (deacetylised) and thereafter one or more layers of ethyl cellulose and finally coated with a layer of sucrose, either sprayed on or as a powder.

3) A composition essentially consisting of a mixture of zinc sulphate and a vegetable fat (hydrogenated or not), which is spray cooled into solid particles. Optionally, further coated with ethyl cellulose. Optionally, wherein chitosan (deacetylated) is added to the mixture before the spray-cooling process.

4) A composition essentially consisting of zinc sulphate and an acetic acid ester of a monoglyceride from a vegetable oil sprayed in a spray cooler into particles. Optionally,

further coated with ethyl cellulose. Optionally, wherein chitosan (deacetylated) is added to the mixture before the spray-cooling process.

5) A composition essentially consisting of pelletised or extruded zinc sulphate and chitosan micro particles coated with one or more outer layers of ethyl cellulose in a rotation drum.

6) A composition essentially consisting of zinc sulphate coated with one or more layers of ethyl cellulose in a fluid bed.

All of the above mentioned compositions optionally further comprise binders or fats/oils or starch as pre-conditioners and are optionally covered with sugar film or sugar powder.

Example 3 Investigation of local bioavailability of controlled release formulation of zinc in comparison with conventional formulations.

For the proof of principles and in vivo testing of the dissolution profile of the modified release compound, an animal experiment is performed with 36 piglets at weaning. The piglets are divided into three groups and feed daily for a period of 14 days after weaning with either: i) A composition according to the present invention comprising an amount of Zn equivalent to 250 ppm of Zn element ii) Zinc sulphate monohydrate in an amount equivalent to 250 ppm of Zn element (easy soluble zinc salt) iii) ZnO in an amount equivalent to 3000 ppm of Zn element Piglets from each trial group are investigated as follows: At day 14 after weaning a number of piglets from each group are euthanasized and an autopsy is performed. Samples of

mucosal intestinal tissue are taken from different compartments of the small intestinal canal, the duodenum, the jejunum and the ileum and immediately preserved for the purpose of determination of the intracellular content of both metallothionein (MT) and Zinc.

By measuring the induction of intracellular metallothionein in enterocytes of different compartments of the small intestine it is shown that the invention induces high intracellular concentration of metallothionein which can support proliferation and differentiation of enterocytes in the different compartments of the small intestinal canal.

Intestinal metallothionein is determined by a Cadmium saturation assay.

By measuring the induction of intracellular Zn in enterocytes of different compartments of the small intestine it is shown that the invention induces high intracellular concentration of Zn which can support proliferation and differentiation of enterocytes in the different compartments of the small intestinal canal.

Zinc content of the different intestinal segments is determined by a spectrophotometrical analysis.

Example 4 The histological investigation of intestinal mucosa and the histological measurement of villus height and crypt depth from different compartments of the small intestinal canal show the positive impact of the compositions according to the invention on intestinal morphology.

The trial is conducted as described above in example 3and piglets from each trial group are investigated as follows: At day 5 after weaning a number of piglets from each group are euthanasized and an autopsy is performed. Samples of intestinal tissue are taken from different compartments

of the small intestinal canal, the duodenum, the jejunum and the ileum and preserved for histological examination and determination of villus height and crypt depth.

The mean differences of the different parameters between the treatments of the experiments are tested for statistically significant by standard statistical methods.

By registration of feed intake and daily growth in the piglets used for this trial, the influence of the composition according to the invention on production parameters is measured.

By registration of incidence of diarrhoea in the piglets in this trial, the influence of the compositions according to the invention on weaning diarrhoea is measured.