EP0923312A1 - Oral delivery form having a high absorption efficiency and method for making same - Google Patents

Abstract

The present invention relates to a premix material for the production of an oral delivery form for animals, in particular fish, which premix is obtainable by preparing a first emulsion of at least one oily substance and optionally at least one watery substance; optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; adding the preparation obtained so far to a solution of a colloid binding agent to obtain a liquid mixture; absorbing this mixture to a carrier mixture of hydrophobic and hydrophilic silica particles to obtain a cream or viscous liquid; gelling this scream or viscous liquid to obtain a premix gel; optionally drying the gel and grinding the dry substance obtained hereof to a particulate premix material. As an alternative the silica may be added before the binding agent. The invention further relates to a series of oral delivery forms per se, to methods for making the premix material and the delivery forms, and to the use of the premix material and the delivery forms in the treatment of animals. The invention also relates to the use of the premix material, the delivery forms or at least some of the components for increasing the uptake of a bioactive agent in the intestinal tract.

Description

ORAL DELIVERY FORM HAVING A HIGH ABSORPTION EFFICIENCY AND METHOD FOR MAKING SAME

The present invention relates to a premix material for the production of an oral delivery form for animals, in particular fish, to a series of oral delivery forms per se, to methods for making the premix material and the delivery forms, and to the use of the premix material and the delivery forms in the treatment of animals. The invention further relates to the use of the premix material, the delivery forms or at least some of their components for increasing the uptake of a bioactive agent in the intestinal tract.

When administering bioactive agents to animals, including humans, oral administration is usually preferred as compared to injections, intubation, suppositories and the like. Oral administration forms may be simply ingested, optionally after having been added to the food, whereas the other types of administration require handling of the animals. This generally causes stress which is not beneficial for the animals, and requires a high amount of work. It is for example known to administer bioactive agents, like hormones, to fish in order to produce a desired physiological effect, such as spawning. In practice, fish are individually treated by first catching them, followed by injection or parenteral administration of the bioactive agent. Being caught and handled is particularly stressful for the fish. Furthermore, this procedure is very labour intensive and thus not cost- effective.

Although an oral administration or delivery form would seem the solution to the above stated problem, these types of delivery forms are not suitable for administering substances that are not or not very well absorbed in the intestine or which are degraded in the digestive tract. Substances presenting these problems are for example peptides, polypeptides and proteins. In order to avoid degradation in the acid environment of the stomach coatings may be applied to protect the bioactive agent from being degraded. However, even if the bioactive agent reaches the intestine intact, it has been described in various articles that the uptake of peptides and proteins therein is extremely low (Zhou, X.H. & Po, A.L.W., International Journal of Pharmaceutics 75, 117-130 (1991)) . To increase the uptake suggestions have been made to incorporate absorption enhancers in the delivery form (Swenson, E.S. & Curatolo, W.J. (1991) Means to enhance penetrations. Advanced Drug Delivery Reviews 8, 39-92) .

Previous manuscripts of experiments on oral delivery to estimate the bioavailability in fish have only been giving evidence of very limited uptake after delivery of an high dose (in the mg/kg body weight range) . The studies of McLean and Ash (Comp. Biochem. Physiol., Vol. 84A, No. 4, pp 687-690 (1986) and J. Fish Biol. 31 (Supplement A) , 219-223, (1987)) reported the time-course of appearance of horseradish peroxidase (HRP) after oral presentation of a very high dose (20 mg per fish) to common carp of approximately 95 g and to rainbow trout of approximately 200 g. By means of the peak plasma concentration the authors deduced that the maximum intact HRP blood content was 41.435 μg/ml (1/482 of dose of delivery (20 mg) ) and 52,03 ng/ml (1/384394 of dose of delivery (20 mg) ) in carp and rainbow trout, respectively.

Furthermore, Suzuki et al. , (J. Comp. Physiol. B 157:753-758 (1988a)) proved that salmon-gonadotropin (sGtH) can be transported intactly from the intestinal lumen to blood circulation of male goldfish, Carassius auratus. He delivered orally sGtH in crude pituitary extract at 1.382 mg per kg body weight. The remaining bioactivity of absorbed sGtH was proved by increased testosterone, 17α, 20S-dihydoxy-4-pregnen-3-one levels and augmentation of milt production. Induction of ovulation in goldfish was reported, as well (Suzuki, Y., Kobayashi M. , Nakamura, 0., Aida, K. , and Hanyu, I., Aquaculture, 74, 379-384 (1988b)) after oral delivery of sGtH in a crude pituitary extract at 2 x 2.62 mg/kg body weight. Peak values of 1.2 μg/ml plasma were reached in the study on male goldfish. Since the blood volume in goldfish is around 3% percent, at the moment of the peak levels only a maximal sGtH content of 3.72 μg in the systemic circulation could have been available for delivery at the pituitary receptors. This equals 0.26% (1/384) of the delivered dosage. In this study the biological activity of plasma exogenous sGtH was estimated from the elevated 17α, 20β-dihydoxy-4-pregnen-3-one and testosterone and increased milt production. In fact the exogenic sGtH from the crude salmon-pituitaries could have been inactive while other pituitary factors were exerting the endocrine and testicular response.

Furthermore the publication of P. Thomas and N.W. Boyd (Aquaculture, 80 (1989) 363-370) , described an experiment carried out on 5 spotted sea trout (Cynoscion nebulosus) , and suggests evidence that low dosage delivery of sGnRHa is ineffective to induce spawning in fish. In this experiment a liquid formulation of sGnRHa had been injected in dead shrimp which were fed to five fish. The dosage of 200 μg sGnRH/kg body weight delivered to 2 of the 5 fish did not induce spawning. Only in the range of 1000 μg/kg body weight - 2500 μg/kg body weight (100 to 250 times the dosage of injection) spawning could be observed.

Le Bail et al . , (J. Exp. Zool. 31 (Supplement A) , 219-223 (1989)) clearly demonstrated, that even after direct delivery of the bioactive protein, growth hormone (GH) , into the hind gut of fish, the uptake is very limited. Rectal intubation of 25 mg GH/kg body weight gives an absorption rate of 0.04% of the delivery dose. Data of Moriyama et al. (Proceedings of the first international marine biotechnology conference, Miyachi, S., Karube, I., Ishida, Y. , eds:299-302 (1989) The Japanese Society for Marine Biotechnology, Tokyo) demonstrate that from a oral dosage of 10 mg/kg, only 0.024% is absorbed. It can be concluded that although macromolecules such as GH might slip through the wall of the intestine this intestinal absorption of GH is of a very limited nature. Thus, it is clear that the present state of the art does not present any efficient or cost effective oral delivery for protein of peptide macromolecules in fish resulting in an acceptable absorption of the macromolecules. In the research that led to the present invention it was found that a combination of a tensio- active substance and a fatty acid, such as polyoxyethylene sorbitan surfactant, in combination with oleic acid, forms an excellent absorption enhancer. It is possible to add the bioactive agent to an emulsion of a tensio-active substance and a fatty acid to produce a liquid delivery form. It was found that this liquid delivery form when administered by intubation already increased the amount of bioactive agent absorbed in the intestine. However, intubation is not a very practical administration method, especially not in large scale fish farms. For obvious reasons a liquid delivery form is not suitable for simply adding it to the water as is done with solid fish feed for example. Therefore, and to further increase the absorption efficiency of the bioactive agent, it is the object of the invention to provide means for obtaining an oral delivery form that leads to a high absorption efficiency of the active substance. According to the invention it was thus found that a suitable delivery form may be prepared from a premix material, which is obtainable by a) preparing a first emulsion of at least one oily substance and optionally at least one watery substance; b) optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; c) adding the preparation obtained so far to a solution of a colloid binding agent to obtain a liquid mixture; d) absorbing this mixture to a carrier mixture of hydrophobic and hydrophilic silica particles to obtain a cream or viscous liquid; e) gelling this cream or viscous liquid to obtain a premix gel; f) optionally drying the gel and grinding the dry substance obtained hereof to a particulate premix material .

In an alternative embodiment of the invention the premix material is obtainable by a) preparing a first emulsion of at least one oily substance and optionally at least one watery substance; b) optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; c) absorbing either of the emulsions to a carrier mixture of hydrophobic and hydrophilic silica particles to obtain a powder, a cream or a viscous liquid depending on the proportion of the ingredients; d) adding the preparation obtained so far to a solution of a colloid binding agent to obtain a mixture; e) gelling the mixture to obtain a premix gel; f) optionally drying the gel and grinding the dry substance obtained hereof to a particulate premix material .

The particulate premix material may then be used in combination with known granulation additives, such as Avicel™ (microcrystalline cellulose) or lactose to obtain granules. Optionally the bioactive agent can be added to the other ingredients during the granulation process instead of during the preparation of the premix. The granules may be added to the feed or be brought in a separate dosage form such as capsules.

In the process of finding a way to transform a mixture of an absorption enhancer and a bioactive agent into granules various problems had to be solved. Since the preferred absorption enhancer consists of an oily substances it appeared impossible to combine these with watery bioactive substances into one delivery form. However, after extensive research it was found that absorption of an emulsion of the oily and watery substance on a carrier mixture of hydrophilic and hydrophobic silica resulted in a dry powder that could be used for further processing. However, the powder proved to be unsuited for direct granulation, probably because of the water-soluble tensio-active substance used. In granulation the initial formation and subsequent growth of granules depends to some extent on liquid bridges that hold the primary particles together. Therefore it is important that the liquid (mostly water) wets the particles effectively. To improve wettability the addition of tensio-active substances is a suitable means. However, tensio-active substances are known to lower the interfacial tension between the liquid and the particles and thus have a negative impact on the strength between the primary particles. That is, by lowering the surface tension of the binding liquid, tensio-active substances make the formation of difficult. For granulation the use of tensio-active substances is therefore to be avoided

("Pharmaceutical Pelletization Technology", Ed. : Isaac Ghebre-Sellassie, Marcel Dekker Inc., New York (1989))

However, the absorption enhancer of the invention comprises a tensio-active substance. Therefore, again after extensive research it was found that when the silica powders having the oily and tensio-active substance absorbed thereon were incorporated in a colloid binding agent, after gelling, drying and milling, small particles were obtained that could be used for standard (micro)granulation.

The invention in a first aspect thus resides in the provision of this unique combination of process steps that surprisingly led to a particulate premix material that could be used in standard (micro)granulation techniques.

Although this a-spect of the invention was made starting with the specific absorption enhancer that forms another aspect of the invention it will be clear for the skilled person that at least some steps of this particular set of steps may also be used for preparing (micro)granulation premixes from other starting materials, which do not have absorption enhancing effects but need to be (micro)granulated for some other reason. Therefore, the invention in another aspect relates to the use of carrier mixtures of hydrophilic and hydrophobic silica as an additive for (micro)granulating oils, fats, fatty acids, tensio-active substances, or mixtures thereof, either with or without hydrophilic substances. Thus, the first and second emulsion in the above described method may also be substituted by fats, oils, fatty acids or tensio-active substances per se.

The silica is preferably a mixture of Sipernat D17™ and/or Sipernat 22™ from the Degussa company.

Although the (bio)active substance might be a separate component that is added to the emulsion, fat, oil or fatty acid, the bioactive substance might as well be the oily or watery component itself. Step b) is therefore optional.

Absorption of the emulsion, oil, fat or fatty acid to the silica mixture results in a composition referred to in this application as "powder" . This powder per se is also encompassed by the present invention. The colloid binding agent is preferably a hydrocolloid material, such as those originating from terrestrial plant (galactomannans, pectins) , seaweed (carrageenans, alginate) , animal (gelatin) or microorganism (xantan gum) , and is most preferably, for optimum results, an alcohol extracted kappa-carrageenan. The dissolution of the binding agent is in the case of kappa-carrageenan preferably effected by heating or by removing K⁺ from the kappa-carrageenan composition by alcohol extraction. Gelling is then effected by cooling or the addition of K⁺.

The final delivery form can be the premix gel before drying and grinding, but may also be the particulate premix material obtained after drying and grinding. However, in a preferred embodiment the particulate premix material is further processed into granules which are preferably substantially spherical, and have a diameter of between 150 μm and 10 mm, preferably between 250 μm and 1 mm, the optimal size of the granules being dependent on the application of the granules, such as the size of the animal to be treated. The invention thus provides for four types of delivery forms, namely the powder of silica with the oil, fat, fatty acid or emulsion absorbed thereon, the premix gel, the particulate premix material and the granules.

For use in the treatment of animals with a stomach, the different delivery forms may optionally be encapsulated within a protective layer suitable for providing protection against breakdown thereof in the stomach or for targeting to particular areas in the intestine. The protective layer comprises at least one enteric polymer, such as cellulose acetate trimellitate (CAT) , polyvinyl acetate phthalate (PVAP) , hydroxypropyl methylcellulose phthalate (HPMCP) , shellac or any other suitable polymer. The enteric polymer is most preferably cellulose acetate phthalate (CAP) which was found to be very effective.

To target the bioactive agents to particular areas in the intestine, the core may be coated with other controlled release polymers. For instance, it can be a hydrophilic swellable polymer responsible for a longer "lag phase" period by a delayed disintegration of the core over the intestinal transit time. It can also be chemically modified glycosidase-sensitive carbohydrates. Optionally the delivery forms may further comprise degradation decreasing agents for inhibiting degradation of bioactive agents in the digestive tract, such as enzyme inhibitors and/or chelating agents.

Optionally the delivery forms may comprise several other bioactive substances acting in combination, for example anti-dopaminergic compounds to enhance in fish the effect of GnRH.

The delivery forms according to the present invention can be simply fed to animals as part of a diet . For example, concerning fish, the granules after incorporation into the feed for example can be thrown onto the water whereby the need to individually treat fish is obviated.

"Bioactive agents" to be incorporated in one of the delivery forms of the invention are chemical compounds or compositions that have an effect on or create a response in living organisms, cells or tissues. The bioactive agents can, for example, be polypeptides, hormonally active substances, antibodies, enzymes, oligo- or polynucleotides, like RNA, immuno-stimulators, (essential) nutrients, vitamins, carotenoids, therapeutics, and viral and bacterial antigens. The bioactive agent may also be (modified) micro-organisms, for example for use in vaccines. All of these are biodegradable, which means that they are susceptible of degradation by biological processes, such as bacterial or other enzymatic action. The bioactive agents may be naturally occurring, be synthetic or be derived from recombinant DNA technology.

Compounds which have a specific regulatory effect on the activity of certain body organs are hormonally active. Mostly they are secreted by an endocrine gland. Compounds, which are not secreted by an endocrine gland, but exhibit a specific regulatory affect on a body organ are also classified as hormonally active compounds. According this definition synthetically prepared analogues of naturally occurring hormonally active compounds are also to be considered as hormonally active compounds. Pharmaceutical acceptable salts of the naturally occurring hormones and their synthetic analogues, which retain the same type of activity as their parent compound, also are to be understood as "hormonally active compounds" . Hormonally active substances comprise a diverse group of chemical and biophysical identity but because of their functional specificity, they are conveniently grouped into discrete classifications by physiological effect. Each group generally regulates one specific physiological function by interacting only with the organ or organs directly affecting that function. For example GnRH-active polypeptides act through their receptors on the gonadotrops of the anterior pituitary gland to affect release of gonadotropic hormones, which affect the activity of reproductive organs. In most fish species this GnRH(-a) action is inhibited by dopamine. Simultaneously delivering anti-dopaminergic compounds can thus enhance the gonadotropin release stimulating effect of GnRH(-a) . It has been proved that the delivery form of the invention may contain for example a gonadotropin hormone releasing hormone (GnRH) or analogue (GnRH-a) thereof which affect fertility and the physiological effects related thereto.

Growth hormones (GH) , on the other hand, act on receptors in various tissues causing the release of somatomedines, the peptide factors responsible for skeletal growth and various other somatic and osmoregulatory processes. Several studies have, also, provided evidence for a direct gonadal site of action for GH. In teleost fish GH and prolactines affect calcium metabolism and osmoregulation in specific ways.

Oligonucleotides are biodegradable compounds of a few nucleotides.

Antigens are substances which are capable, under appropriate conditions, of inducing the formation of antibodies and reacting specifically in some detectable manner with the antibodies produced. Antigens may be soluble substances, such as toxins and foreign proteins, or particulate, such as bacteria and tissue cells. They may be lipids, proteins, carbohydrates, proteoglycans, glycolipids, glycoproteins or lipoproteins such as the numerous lipopolysaccharides isolated from bacteria which function as specific haptens or as complete antigens in another individual or by cell culture.

Antibodies are immunoglobulin molecules that have a specific amino acid sequence by virtue of which they interact only with the antigen that induces its synthesis in lymphoid tissue, or with antigen closely related to it. The meaning of entrapment of antibodies in the proposed delivery system of the invention is its usage a an immunotherapeutic for the modulation of the passive immunity of an individual, which passive immunity is conferred by administration of pre-formed antibodies (serum or gamma globulin) actively produced in another individual. They can also be used as vaccine in immuno- prophylaxis for prevention of disease and for any process of rendering a subject immune.

Enzymes are proteins capable of accelerating or producing by catalytic action some change in a substrate for which they are often specific.

Therapeutics are compounds that will heal and which will be used for treatment of diseases.

Prophylactic compounds will prevent disease, while immunostimulants are substances that activate the cellular or humoral immune system or possibly result in enhanced formation of antibodies and the specific reaction of the antibodies produced.

Essential nutrients are nutrients an animal requires for proper functioning.

This list is not intended to be exhaustive of bioactive, biodegradable compounds for administration of which this invention is intended, but merely sets out examples to illustrate the type of biodegradable macromolecules that may be used. All these bioactive, biodegradable substances have to be targeted from the intestinal lumen into the blood circulation. The invention makes this possible by providing a delivery form that combines the bioactive agent with an absorption enhancer - these enhancers improve the uptake of the bioactive agent from the intestinal lumen into the blood - in a form which is suitable for oral administration. The delivery form resists breakdown in the stomach if necessary. The invention further relates to a method for producing the particulate premix material of the invention, comprising a) preparing a first emulsion of at least one oily substance and optionally at least one watery substance; b) optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; c) adding the preparation obtained so far to a solution of a colloid binding agent to obtain a liquid mixture; d) absorbing this mixture to a carrier mixture of hydrophobic and hydrophilic silica particles to obtain a cream or viscous liquid; e) gelling this cream or viscous liquid to obtain a premix gel; f) optionally drying the gel and grinding the dry substance obtained hereof to a particulate premix material.

In an alternative embodiment of the invention the method comprises the steps of a) preparing a first emulsion of at least one oily substance and optionally at least one watery substance; b) optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; c) absorbing either of the emulsions to a carrier mixture of hydrophobic and hydrophilic silica particles to obtain a powder, a cream or a viscous liquid depending on the proportion of the ingredients; d) adding the preparation obtained so far to a solution of a colloid binding agent to obtain a mixture; e) gelling the mixture to obtain a premix gel; f) optionally drying the gel and grinding the dry substance obtained hereof to a particulate premix material. To the method the same principles apply as have been explained for the premix material which was defined by its method of preparation.

The invention further relates to the use of the premix material in the preparation of granules. To achieve granulation various standard techniques may be used such as "Pharmaceutics: The Science of Dosage Form Design", Ed. Michael E. Aulton, Churchill Livingstone, Edinburgh London Melbourne and New York (1988) , pages 616-628, and "Pharmaceutical Pelletization Technology", Ed. Isaac Ghebre-Sellassie, Marcel Dekker, Inc., New York and Basel (1989) , pages 101-122 and 187-216. The invention further relates to method for producing the various delivery forms and premixes. Sometimes, a premix may at the same time be classified as a delivery form. However, a premix is also always a starting or intermediate material for the preparation of the delivery form.

The invention in another aspect relates to the use of the delivery forms and premixes in various (therapeutical) applications.

In this application "delivery form(s) " and "administration form(s)" are used interchangeably. The same applies to "bioactive agent" and " (bio) active substance" .

"Oily substances" encompass a single substance or mixtures of substances having a hydrophobic character, e.g. oils, fats, fatty acids, tensio-active substances etc.

A "watery substance" is any watery compound or compound with a hydrophilic character. "Powder" is used for the mixture of hydrophilic and hydrophobic silica having an oil, fat, fatty acid, tensio-active substance, emulsion or any combination of these absorbed thereon. "Binding agent" encompasses every agent encapsulating the powder. The powder is added to a solution of the binding agent after which the solution is gelled. Thus, the "premix gel" or "gel" is obtained. After drying the gel and comminuting the dried gel in any way (grinding, milling, pulverizing etc.) a "particulate premix material" is obtained. "Granules" are obtained after (micro)granulation of the particulate premix material .

"Absorption enhancer" is used to indicate any substance or combination of substances that somehow stimulates the absorption of a bioactive agent by the intestine.

"Oral delivery form" is used to encompass any combination of a bioactive agent with one or more excipients. Excipients as used herein are both the silica, binding agent, emulsion, tensio-active substance, fatty acid, fats, oils etc. and any other additive necessary for preparing the composition.

The invention will now be further described by way of the following examples and research results. The inventors have used LHRHa or GnRHa as a model substance to show that absorption thereof by fish can be readily stimulated. GnRHa or LHRHa absorption was estimated from its ability to stimulate ovulation and spermiation in African catfish, rainbow trout and carp and by the induced gonadotropin (GtH) release in these animals.

Although most of the work concerned with the present invention has been focused on these molecules and fish in order to develop rapidly a method to induce spawning after oral treatment, the invention is not limited thereto. It will be clear to the skilled person that analogous to these examples treatments may be developed with other molecules for different animals (including humans) .

EXAMPLES

EXAMPLE 1

Preparation of the delivery forms

1.1. First method for preparing a particulate premix material Delivery forms were prepared that allowed the inventors to entrap bioactive (poly)peptides and a dosage of Tween/oleic acid, necessary to enhance intestinal uptake, into a rigid gel of K-carrageenan. After producing the gel it was further treated to obtain a particulate premix material suitable for (micro) granulation.

The gel was formed as follows, K-carrageenan was added to water (up to 5 gram per 100 ml) , mixed vigorously, whereafter the mixture was warmed to 85°C to obtain a K-carrageenan solution in water.

Commercial K-carrageenan contains potassium salts. Potassium ions prevents κ-carrageenan to solve in cold water. One has to heat such κ-carrageenan up to 85°C in water to make a solution. Fast cooling results in immediate gelling.

All other premix ingredients (polyoxyethylene sorbitan surfactant, oleic acid, hydrophobic silica, hydrophilic silica and occasionally EDTA or other substances) were added and vigorously mixed. This mixture was gradually cooled to 50°C.

Subsequently, the bioactive compounds were added and mixed intensively by a (ultraturax) mixer. This mixture was cooled suddenly to obtain a rigid gel. Occasionally a KCl solution (2M) was added just prior to cooling to improve gelling.

From this gel the particulate premix material was made as follows. The gel was cut in small pieces which were dried by freeze drying, fluidized bed drying or tray drying. This product was ground to a powder consisting of particles of less than 70 μm diameter.

1.2. Second method for preparing a particulate premix material

Because commercially available K-carrageenan contains potassium ions it is not soluble in cold water. In the above procedure we therefore used an elevated temperature to effect dissolution. However when using alcohol extracted K-carrageenan it was possible by a cold procedure to make a solution which gelled immediately on addition of a KCl solution.

Hence an alternative procedure was also carried out for producing the particulate premix material, as follows. Potassium chloride was shown to be removed from K-carrageenan by three alternative methods:

1) 40 gram K-carrageenan was put into 1400 ml of a hot medium of NaOH (pH 8-9) by adding 2% NaOH. This solution filtered under in a buchner filter and was treated with 10% sodium chloride to salt out the K- carrageenan upon precipitation with 1.5 volumes of 85% isopropanol . The extract was washed twice with 85% isopropanol. This was dried at 50-55°C and ground to a fine powder. 2) Alternatively, 150 ml 40-60% ethanol was used per 10 gram K-carrageenan at 5°C overnight. This fluid was filtered. Then the filtrate was salted out in a saturated NaCl solution, washed, dried and ground to a powder. 3) In a water bath (50°C) 10 gram K-carrageenan per 400 ml and 2 gram NaCl while stirring for 3 hours. 1000 ml ethyl alcohol was added and this was stirred at 4°C for 16 hours. The liquid was filtered. The filtrate was dried and milled to a fine powder. Subsequently the treated K-carrageenan was added to water (up to 5%) , mixed with an ultraturax at room temperature to obtain complete dissolution. All other premix ingredients (polyoxyethylene sorbitan surfactant, oleic acid, hydrophobic silica, hydrophilic silica and occasionally EDTA or other substances) were added and vigorously mixed.

Also the bioactive agents like GnRH analogues and/or Domperidone were added and mixed intensively.

To obtain a rigid gel, a KCl solution (2M) was mixed in this liquid. The gel was cut into small pieces which were dried by freeze drying, fluidized bed drying or tray drying. This procedure was repeated to yield 7 particulate premixes (A-F and X) wherein the basic components were carrageenan, Tween + oleic acid, silica and bioactive agents.

Car = carrageenan enhanc = Tween 80/oleic acid (4/0.6] hyfeel = hydrophilic silica hyfoob = hydrophobic silica CAP = cellulose acetate phthalate

These premixes were ground to a powder consisting of microbeads of diameter of less than 70 μm. Subsequently these premixes were subjected to granulation. The particulars of granulation are set out under 1.3. hereinbelow. The above described particulate premixes were put into a spheronizer with the aim of producing spheres with a diameter of around- 250-750 μm. For granulation 200 gram of each premix was mixed (during 10 minutes) with 175 gram microcrystalline cellulose (Avicel™) and 125 lactose. The mixtures A, B, C, D, E and F gave in the granulating machine a low yield of only some spheres (10%) . Phase separation of Tween (polyoxyethylene sorbitan surfactant) and the oleic acid (fatty acid) occurred.

For this reason further mixtures were developed by the inventors, keeping in mind that mixtures with improved granulating properties could have been correlated with diminished absorption enhancement capacities.

In premix X the inventors replaced a fraction of hydrophilic silica by hydrophobic silica, to yield a hydrophobic versus hydrophilic silica ratio of 1:1. Premix X was then used by the inventors to develop enteric microgranules. The formulation of the premix for encapsulation was shown in the above table.

1.3. Granulation

For optimalisation purposes various granulation techniques were tested. These techniques are known per se and are for example described in "Pharmaceutics: The Science of Dosage Form Design", Ed. Michael E. Aulton, Churchill Livingstone, Edinburgh London Melbourne and New York (1988) , pages 616-628, and "Pharmaceutical Pelletization Technology", Ed. Isaac Ghebre-Sellassie, Marcel Dekker, Inc., New York and Basel (1989), pages 101-122 and 187-216.

Two processes of granulation, the pressure pellet and agitation method, were carried out to shape the irregular particles of the premix into granules of a regular shape. Main carrier materials currently used in granulation processes are monohydrate lactose, micro crystalline cellulose and potato starch or lactose (Microencapsulation and related drug processes, Patrick B. Deasy, Marcel Dekker, Inc., New York and Basel, p 282) . These carrier materials, contribute to processes of attractive force between solid particles. These attractive forces may be molecular (Valence and Van der Waals) , electrostatic or magnetic in nature (Drug and the Pharmaceutical Science, Vol. 37, Marcel Dekker Inc, 1989, p.124) .

1.3.1. Pressure pellet method

Extrusion is a process that initially mixes powders by forcing them through a mesh screen or plate through action of wiper mechanism. Carrier materials such as lactose, micro crystalline cellulose and potato starch improve this process. By an extruder (Fitzpatrick Company, Europe) the irregular shaped granules (microbeads) of premix X were shaped, after addition of monohydrate lactose (FEDERA) and of micro crystalline cellulose (AVICEL, FEDERA) , into cylindrical extruded granules. After extrusion they were spheronized (300 μm diameter) in a marmurizer, from the Fuji Paudal Co. Ltd.

1.3.2. Agitation method

The Aeromatic or the Freund type spheronizers, like high speed mixer/granulators and also fluidized bed granulators, use wet granulation in which the powder microbeads are made to adhere and form larger particles.

The premix X and monohydrate lactose and microcrystalline cellulose were put into a centrifugal force granulating machine (Aeromatic rotorprocessor) to produce spherical pellets of 250 μm - 750 μm.

1.4. Enteric coating of the pellets

Coating was done in a fluidized bed with a 10% CAP salt solution and 25% (to CAP w/w) triacetin as plasticizer. The CAP salts were produced from CAP (Eastman Kodak) . Coating condition in the fluidized bed were: - Income temperature: 50°C

- The sprayer pressure: 1 atm.

- the spraying speed: 3.5 ml/min

An advantage of coating under these conditions is that no environmentally unfriendly organic solvents were used either for granulation or for enteric coating, furthermore coating with cellulose acetate phthalate in a fluidized bed has a large flexibility and the size of the coating layer can be easily controlled.

EXAMPLE 2

Effect of nutrients on the intestinal uptake of sGnRH-a from the microcapsules in three fish species

1. The common carp (Cyprinus carpio) Materials and methods

The capsules X were mixed together with different ratios of a mixture containing fat, hydrolysable sugar and proteins. The overall mixture was introduced by forced feeding. The treatment protocol is explained in table 1.

Table 1

^* control is blank microcapsules/no food

Results

There was no significant effect of a mixture of nutrients (up to 66,6% of the weight of the microcapsules) on the sGnRH-a induced GtH secretion. This indicates a similar bioavailability of GnRH for all dietary GnRH delivery forms (fig. 1) . With and without food, after intestinal delivery, of the enteric sGnRH-a loaded microcapsules were highly efficient in stimulating the GtH2 plasma levels in carp. In contrast, in the control groups the circulating GtH2 levels tended to decrease slightly (P<0.05 at 24h, 48h and 60h) , which may be due to a handling stress effect.

Within groups T-test (dependent samples) revealed that except for the control groups all post treatment values were significantly higher than the 0 hour values (★ = P<0.05, ★* = P<0.005, ★★★ = P<0.0005). In the control group GtH2 levels tended to decrease (■ = significantly lower than 0 hour (P<0.05). Bars underlined by the same line represent values that are not significantly different (Fig. 1) .

Table 2 shows the statistical analysis of data of the common carp experiment explained in table 1 and demonstrated in fig. 1.

Table 2

NS = not significant S = significant

Table 2 shows that there is no significant effect of diet (0 - 66,6%) on sGnRH-a induced GtH2 release. The sGnRH-a loaded microcapsules were under the various nutritional conditions highly efficient in stimulating GtH2 release.

2. The rainbow trout (Onchorynchus mykiss)

Lipids are certainly the nutrients which could interfere the most with enhancers. These enhancers, are more often tensio-active hydrophobic substances which can either be solubilised in lipids or form emulsions, thus reducing or inhibiting their action on the intestinal border. For that reason, special attention must be paid to the effects of lipids on the absorption of bioactive material from food entrapped microcapsules. Materials and methods

This work has been done in two years old maturing rainbow trout weighing between 1 and 2 kg. In this experiment, two concentrations of lipids were studied together with other nutrients (sucrose and gluten) . The enteric microcapsules made out of premix X were mixed with particular food ingredients and introduced by forced feeding to the fish. Table 3 shows the outline.

Table 3

Results

In general the data of fig. 2 prove that the sGnRH-a loaded enteric microcapsules after oral delivery are highly efficient in inducing GtH release in rainbow trout . This can be revealed by Manova as well as by a within groups T-test (dependent samples) .

Micro-encapsulated sGnRH-a, when delivered alone, did not stimulate significantly more the GtH secretion than when ingested together with low un-saturated fat concentrations of 1% to 5%. On the contrary in the presence of fat there was a tendency (but not significant) to induce a better stimulation. This indicated comparable bioavailabilities .

Other nutrients together with lipids (1 and 5%) did not significantly modify the response to encapsulated GnRH (fig. 2) .

In fig. 2, bar codes of the same sampling time underlined by the same line are not significantly different (T-test for independent samples) . ★ = significantly (P<0.05) different from zero hour control & ★★ = significantly (P<0.005) different from zero hour control (T-test dependent samples) .

Table 4 shows the statistical analysis of this experiment on rainbow trout .

Table 4

No significant nutrient effect on sGnRH-a induced GtH2 release. The effect of sGnRH-a from microcapsules is highly significant.

3. African catfish (Clarias gariepinus) Materials and methods

Female mature catfish weighing 662 ± 143 g (mean ± std) and kept at 26°C were used. Enteric microcapsules (X) were loaded with 20μg/g of GnRH, they were mixed with a commercial catfish food. In group 2 the food was sprayed on top of the microcapsules . Ovulation rates were checked after 24 hours.

Table 5

food sprayed on the capsules

Results

In general the sGnRH-a loaded microcapsules were highly efficient after oral delivery to increase the GtH2 levels.

Eight hours after treatment, only the group that received 20% commercial diet, "Biomeerval" , had significantly lower GtH2 plasma levels than the group which received the GnRH loaded microcapsules without feed. There was on the other hand no significant difference in GtH2 levels between the group that received the microcapsules with 20% spray coated feed and the group that received the microcapsules without feed Between all sGnRH-a treatment groups there were also no significant differences in the rate of ovulation in catfish, which were about 70%.

In contrast the highest commercial feed (Biomeerval) levels (40 & 60%) tended to result in higher GtH2 levels than the 20% commercial feed level, which was confirmed by statistics for the 20h samplings. This shows that, in these species, in the perspective of a nutritional delivery of the microcapsules, that they can be incorporated in a pellet containing up to 70% of nutrients. In fig. 3 the following symbols are used: it P < 0.05 significant different from the zero hour control levels ick p < 0.005 significant different from the zero hour control levels jckit P < 0.0005 significant different from the zero hour control levels

(within groups, T-test for dependent samples, n=8) □ significant different from 20% Biomeerval at 20 h ♦ significant different from 20% Biomeerval at 8 h (between groups, T test for independent samples, n=8) The effect of nutrient on the GtH2 release effect of sGnRH-a from enteric microcapsules is shown in table 6.

Table 6

No significant Biomeerval diet (0-60%) effect on sGnRH-a induced GtH2 release. The groups with spray coated microcapsules was more effective than the groups with Biomeerval nutrient .

EXAMPLE 3

Voluntary uptake of the microcapsules by African catfish (Clarias gariepinus) , goldfish (Carrasius auratus) & barbell (Barbus conchonius)

1. African catfish (Clarias gariepinus)

Experiments were also carried out to determine the voluntary uptake of microcapsules (X) loaded with sGnRH-a and incorporated in food pellets.

Based on previous results of nutriment/sGnRH-a uptake competition, a test diet (A) was formulated as oral delivery medium for enteric microcapsules with the mixed micelle (polyoxyethylene sorbitan surfactant/oleic acid) and sGnRH analogue (sGnRH-a) 20 μg/g capsule. This diet was moistened (50 ml water per 100 gram dry materials) and pelletised with CMC and Satagel (Sanofi Bioindustries) as binder and by the procedure of extrusion and of freeze drying. The pellets were floating which had the advantage that the food uptake could be checked. The food composition is described in Table 7. Table 7 Formulation of the test diet (A) gluten - 5 casein 25.5 yeast 20 silica 2 alpha starch 10 sucrose 15

CMC 1 carra Sanofi 2.5 fat 15 vitamin premix 2 mineral premix 2

TOTAL 100

The catfish had been raised on a commercial diet, "Biomeerval" (B) , they had to be weaned to the new dietary formulation. Biomeerval pellets were milled to a fine powder and after vitamin/mineral enrichment it was re-pelletized (formulation B) in a same procedure with the same binders.

Test diet (A) and a commercial diet (B) had been mixed in the following proportions and had been pelletized on the same way by moistening, extrusion and freeze drying with CMC and Satagel as binder and had been fed according following protocol: day 1-2: A/B = 0/1, day 3-4 : A/B = l/l; day 5-6: A/B - 4/1, day 7-8: A/B = 9/1, day 9-10: A/B = 1/0.

The feeding rate was 1% of the body weight. To induce spawning microcapsules loaded with 20 μg s-GnRH-a per gram and coated 10% w/w CAP, were introduced into the food pellets. 104 gram of enteric microcapsules had been mixed into 520 gram of nutrients. Fourteen broodstock fish (11 females and 3 males) were kept in a 1000 litre flow-through holding facility at 27°C. On day eleven they were fed a diet enriched with s-GnRH-a loaded microcapsules.

During this experiment, the fish being around 2 kg were fed per fish 20 g dietary pellets containing 4 gram enteric capsules loaded with sGnRH-a (80 μg) . Four out of 11 fish (36.36%) ovulated the fecundity and the relative fecundity being respectively 292, 138 eggs per fish and 65 ± 28 gram of eggs per kg. In a control group of 11 female and 3 male fish which were fed a control diet containing no sGnRH-a loaded microcapsules non of these fish ovulated, which is obvious since spontaneous ovulation has not been recorded for Clarias gariepinus in captivity. The Fisher Exact Test confirmed the ovulation inducing effect of the dietary delivery form, since both treatments gave significant different (P < 0.05) ovulation rates.

The delivery dose for sGnRH-a was only 40 μg/kg body weight . This is only twice more than what is currently administered by intraperitoneal injection to obtain spawning in catfish. These results are very promising. Firstly, because they made prove that the formulation, which had been changed to make granulating of the premix possible was still an efficient absorption enhancement medium. And secondly, because this is also the first record of an efficient sGnRH-a enriched spawning diet .

To validate the dietary delivery system, other feeding experiments have been carried out on goldfish, Carassius auratus and the barbell, Barbus conchonius.

2. Goldfish (Carassius auratus) Materials and Methods

The sGnRH-a loaded enteric microcapsules were incorporated into food pellets and were fed to the goldfish. For this, 40 goldfish (mean body weight around 48 gram) were separated into 2 groups over 4 aquaria of 300 litre water at 24°C to these conditions during 2 weeks. No male goldfish were present. During 10 days before experiment the fish were adapted to the experimental food which consisted of a sticky past from boiled fine wheat grains. After cooling little pellets of a diameter of approximately 1 mm were hand made and fed to the fish. The experiments started after two days of starvation. The microcapsules coated with 10% CAP were mixed with the paste in a 1/1 ratio dry weight and the pellets were formed as described. This preparation was given to the fish in the morning. The food uptake was confirmed and after 12 hours ovulation was checked.

Group 1 was fed a diet containing blank control microcapsules. Groups 2 was fed at 40 μg sGnRH-a per kg fish by microcapsules (containing 20 μg sGnRH-a/g) in the food pellets .

Results

After 12 hours 1 out of 20 goldfish ovulated spontaneously in the control group, while 10 out of 20 goldfish ovulated in the experimental groups. These ovulation rates between control and the group receiving dietary sGnRH-a delivery system were highly significantly different (P < 0.005) as revealed by the Fisher Exact Test.

3. Barbell (Barbus conchonius) Materials and Methods

Fifty six female Barbus conchonius (mean body weight 2.92 g) were separated into 4 groups (14 fish per group) in four 80 litre aquaria. No male barbells were present .

As for goldfish, during 10 days before experiment the fish were adapted to the experimental food which consisted of a sticky paste from boiled fine wheat grains. After cooling little pellets of a diameter of approximately 1 mm were hand made and fed to the fish. The experiments started after two days of starvation. The microcapsules coated with 10% CAP were mixed with the paste in a l/l ratio dry weight and the pellets were formed as described. This preparation was given to the fish in the morning. The food uptake was confirmed and after 10 hours ovulation was checked. Group 1 was fed a diet containing blank control microcapsules. Groups 2, 3 and 4 were fed with the sGnRH-a loaded microcapsules (containing 20 μg sGnRH-a/g) in the food pellets. They obtain an average delivery dosage of sGnRH-a of respectively 50 μg/kg b.w., 100 μg/kg b.w. and 150 μg/kg b.w..

Results

Ovulation rates were 1/14, 3/14, 10/14 and 8/14 in the groups that were respectively fed control capsules, 50 μg sGnRH-a/kg, 100 μg sGnRH-a/kg, 150 μg sGnRH-a/kg.

The Fisher exact test revealed that a dietary delivery dosage of 50 μg sGnRH-a /kg b.w. was not enough to affect significantly spawning in mature barbells (P > 0.05) . However a dietary delivery system of 100 μg sGnRH- a/kg b.w. (P < 0.005) or 150 sGnRH-a/kg b.w. (P < 0.007) highly significantly induced ovulations as compared to the control group. A dietary delivery of 100 μg sGnRH- a/kg b.w. was significantly more affective than the 50 μg sGnRH-a/kg dietary delivery form (P < 0.05) . There were no significant differences between the highest dose delivery forms.

EXAMPLE 4 Comparison between the liquid, powdered premix and microencapsulate delivery form, for the premix X

1. Common carp (Cyprinus carpio)

Different delivery forms (liquid, powdered premix & enteric microcapsules) containing the same absorption enhancer and s-GnRH-a were administrated orally to groups of 10 carps at the dosage of 20 μg sGnRH-a/kg b.w. (experiment 1) and at the dosage of 40 μg sGnRH-a /kg b.w. (experiment 2) . Plasma samples collected at 0, 1, 3, 6, 12, 32 and 48 hours after delivery were analysed for GtH2. And the AUC (area under the curve) was calculated to estimate the bioavailability of GtH2 in the different conditions.

For the 20 μg sGnRH-a dosage forms, there was no significant difference in GtH2 release after sGnRH-a delivery by intraperitoneal injection or by forced feeding of the enteric microcapsules. Both these treatments were, however, more efficient that the liquid delivery form.

The 40 μg sGnRH-a delivery experiment revealed that there was no significant difference in efficiency between the powdered premix , the microcapsules and intraperitoneal injection (figures 4 and 5) .

All the delivery forms significantly stimulated the levels of circulating GtH2.

2. Rainbow trout (Qnchorvnchus mvkiss) In three experiments on a dosage of 40 μg/kg sGnRH-a the efficiency of respectively the liquid, the powdered premix and the microencapsulate delivery forms have been compared every time with intraperitoneal injection of the same dosage and the control GtH2 levels. All three delivery forms significantly stimulated GtH2 release as compared to the control groups which got the same delivery form but without sGnRH-a. Intraperitoneal injection was, however, significantly more efficient than the liquid and the powdered premix delivery form. Oral delivery of the enteric microcapsules did not result in significantly different GtH2 levels than intraperitoneal injection (figures 6 and 7) .

3. African catfish (Clarias gariepinus) The efficiency of the liquid, powdered premix and enteric microencapsulate delivery form was evaluated by a Fisher exact test in two experiment on basis of the ovulation rates. In the first experiment the delivery dosage was 30 μg sGnRH-a per kg. The powdered premix significantly induced ovulation in the catfish and was as efficient as intraperitoneal injection.

The second experiment with delivery dosages of 40 μg sGnRH-a/kg b.w. demonstrated that the enteric microcapsules are a more efficient medium to transfer bioactive sGnRH-a into catfish blood circulation than the liquid delivery form (figures 8 and 9) .

EXAMPLE 5

Action of the microencapsulate delivery form, in which the bioactive agent had been incorporated during and not before the granulation process

1. Materials en methods

1.1. Preparation of the microencapsulate delivery form

The premix X, containing no bioactive agent, was ground to a powder of diameter less than 90 μm. This powder was subsequently subjected to granulation, using the agitation method (Aeromatic rotorprocessor; see example 1, 1.3.2.) . Monohydrate lactose (FEDERA) and micro crystalline cellulose were used as granulating additives. The bioactive agent, the GnRH-analogue Azagly- nafarelin, was dissolved in the water, serving as binder liquid in the granulation process. Thereafter, the obtained granules were dried and coated (see example 1, 1.4.) . The final concentration of Azagly-nafarelin in the capsules was 40 μg beadlets.

1.2. Administration of the capsules to Rainbow trout

Blank capsules and GnRH-a loaded capsule were respectively administered to two groups (n=12) of rainbow trout by forced feeding (1 g capsules per kg of body weight) . Plasma samples were collected immediately before administration and 6, 12, 24, 36, 48 and 72 hours later. The plasma GtH II levels, as a response to the GnRH-a were determined in both groups. 2 . Results

In the control group plasma GtH II levels remained stable around 1 ng/ml throughout the whole sampling period, except in 2 fish which had higher basal GtH levels than average at time 0. In these fish a slow increase of plasma GtH levels was noticed until 36 hours after the last sampling. These 2 fish were the first to ovulate in this group. It can be suggested that the natural process leading to ovulation had already started in these fish.

In the Azagly-nafarelin treated fish a sharp increase of the mean plasma GtH II levels was noticed 6 hours after the start of the experiment . In this group plasma GtH II levels were significantly higher compared to the control group from 12 hours till the end of the sampling period. Accordingly, oral administration of Azagly-nafarelin resulted in higher ovulation rates as compared to the control group.

Claims

1. Premix material for the preparation of a delivery form for oral ingestion by animals, in particular fish, obtainable by a) preparing a first emulsion of at least one oily substance and optionally at least one watery substance; b) optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; c) adding the preparation obtained so far to a solution of a colloid binding agent to obtain a liquid mixture; d) absorbing this mixture to a carrier mixture of hydrophobic and hydrophilic silica particles to obtain a cream or viscous liquid; e) gelling this cream or viscous liquid to obtain a premix gel; f) optionally drying the gel and grinding the dry substance obtained hereof to a particulate premix material .

2. Premix as claimed in claim 1, obtainable by a) preparing a first emulsion of at least one oily substance and optionally at least one watery substance; b) optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; c) absorbing either of the emulsions to a carrier mixture of hydrophobic and hydrophilic silica particles to obtain a powder, a cream or a viscous liquid depending on the proportion of the ingredients; d) adding the preparation obtained so far to a solution of a colloid binding agent to obtain a mixture; e) gelling the mixture to obtain a premix gel; f) optionally drying the gel and grinding the dry substance obtained hereof to a particulate premix material.

3. Premix material as claimed in claim 1 or 2, wherein the first emulsion is an absorption enhancer capable of enhancing uptake of a bioactive agent in the digestive tract, which absorption enhancer comprises a tensio-active substance and a fatty acid.

4. Premix material as claimed in claim 3, wherein the tensio-active substance is selected from the group consisting of the polyoxyethylene sorbitan surfactants of the Tween™ series, such as Tween 20™ and Tween 80™, and the fatty acid is oleic acid.

5. Premix material as claimed in claims 1-4, wherein the binding agent is a hydrocolloid, such as kappa-carrageenan.

6. Premix material as claimed in claims 1-5, wherein the carrier mixture of hydrophilic and hydrophobic silica is a mixture of Sipernat D17™ and Sipernat 22™.

7. Premix material as claimed in claims 1-6, wherein the carrier mixture of hydrophilic and hydrophobic silica is substituted with one or more silicates.

8. Delivery form for oral administration to animals, comprising a suitable form of a premix material, as claimed in claims 1-7, which premix material comprises a bioactive agent.

9. Delivery form as claimed in claim 8, wherein the suitable form is the premix gel.

10. Delivery form as claimed in claim 8, wherein the suitable form is the particulate premix material.

11. Delivery form as claimed in claim 10, wherein the particulate premix material consists of microbeads with a diameter of about 50-100 μm, preferably less than 70 μm.

12. Delivery form as claimed in claim 8, wherein the suitable form is a granulate obtainable by (micro)granulating the particulate premix material in the presence of the usual (micro)granulation additives.

13. Delivery form as claimed in claim 12, wherein granules of the granulate are substantially spherical and have a diameter between 150 μm and 10 mm, preferably between 250 μm and 1 mm.

14. Delivery form as claimed in any one of the claims 8-13, wherein the premix material or the suitable form prepared therefrom are encapsulated within a protective layer suitable for providing protection against breakdown thereof in the stomach.

15. Delivery form as claimed in claim 14, wherein the protective layer comprises at least one enteric polymer, preferably being CAP.

16. Delivery form as claimed in any one of the claims 8-15, wherein the premix material or the suitable form prepared therefrom are encapsulated within a polymer that controls release in the intestine, such as a hydrophilic swellable polymer or chemically modified glycosidase-sensitive carbohydrates.

17. Delivery form as claimed in any one of the claims 8-16, wherein the bioactive agent is selected from the group consisting of hormonally active substances, antibodies, enzymes, oligonucleotides, immunostimulators, (essential) nutrients, vitamins, carotenoids, therapeutics, antigens.

18. Delivery form according to claim 17, wherein the hormonally active substance is selected form the group consisting of GnRH or GnRH-a, LHRH or LHRH-a.

19. Powder for use in the preparation of a delivery form as claimed in claims 8-18, comprising a mixture of hydrophilic and hydrophobic silica having an absorption enhancer absorbed thereon.

20. Powder for use in the preparation of (micro)granules, comprising a mixture of hydrophilic and hydrophobic silica having oils, fats, fatty acids or combinations thereof with each other and/or with an aqueous phase, absorbed thereon.

21. Use of a mixture of hydrophilic and hydrophobic silica as an additive in the preparation of (micro) granules, which comprise oils, fats, fatty acids or combinations thereof with each other and/or with an aqueous phase.

22. Method of preparing (micro) granules, comprising: a) preparing a powder as claimed in claim 19 or 20; b) adding the powder to a solution of a colloid binding agent; c) gelling the solution to obtain a gel containing the powder; d) drying the gel and comminuting the dried gel to obtain a particulate material; e) (micro) granulating the particulate material in the presence of the usual (micro)granulation additives .

23. (Micro) granules, obtainable by the method as claimed in claim 22.

24. Method of producing a delivery form or premix therefore having the form of a gel, comprising: a) preparing a first emulsion of at least one oily substance and at least one watery substance; b) optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; c) absorbing either of the emulsions to a mixture of hydrophilic ana hydrophobic silica particles to obtain a powder; d) adding the powder to a solution of a colloid binding agent to obtain a mixture; e) gelling the mixture to obtain a premix gel.

25. Premix having the form of a gel obtainable by the method as claimed in claim 24.

26. Delivery form having the form of a gel obtainable by the method as claimed in claim 24.

27. Method for producing a delivery form or premix therefore having the form of a particulate material, comprising: a) preparing a first emulsion of at least one oily substance and at least one watery substance; b) optionally adding a bioactive agent to the first emulsion to obtain a second emulsion; c) absorbing either of the emulsions to a mixture of hydrophilic and hydrophobic silica particles to obtain a powder; d) adding the powder to a solution of a colloid binding agent to obtain a mixture; e) gelling the mixture to obtain a premix gel; f) optionally drying and grinding the gel to obtain a particulate premix material.

28. Premix having the form of a particulate material obtainable by the method as claimed in claim 27.

29. Delivery form having the form of a particulate material obtainable by the method as claimed in claim 27.

30. Use of the premix as claimed in any one of the claims 1-7, 25 and 28 and/or a powder as claimed in claim 19 or 20 for the preparation of an oral delivery form.

31. Delivery form according to any of the claims 8-18, 26 or 29 for use in delivering a bioactive agent to an animal .

32. Delivery form according to any of the claims 8-18, 26 or 29 for use in inducing spawning in fish.

33. Delivery form according to any of the claims 8-18, 26 or 29 for use as a therapeutical or prophylactic composition.

34. Liquid delivery form, comprising a bioactive agent incorporated in an emulsion of a tensio- active substance and a fatty acid, in particular polyoxyethylene sorbitan surfactant and oleic acid.

35. Method as described in claims 1-8 or claimed in claims 24 or 27, wherein the bioactive agent is added during one or more of the other steps of the preparation method of the premix and/or during granulation.

36. Absorption enhancer consisting of one or more tensio-active substances, for example selected from surfactants of the Tween™ series (in particular sorbitan polyoxyethylene surfactants) and one or more oily substances, in particular a fatty acid, such as oleic acid for use in a premix or (micro)granules .

37. Method for preparing (micro)granules, comprising the usual (micro) granulation steps, for example selected from the extrusion/spheronisation and agitation method, and wherein the material to be (micro)granulated comprises one or more oily substances and/or one or more tensio-active substances.

38. Mixture of hydrophobic and hydrophilic silica for use in the preparation of (micro) granules .

39. Method of preparing an oral delivery form, comprising the steps of: a) preparing an emulsion of at least one oily substance and optionally at least one watery substance; b) adding the emulsion to a solution of a colloid binding agent to obtain a liquid mixture; c) absorbing this mixture to a carrier mixture of hydrophobic and hydrophilic silica particles to obtain a cream or viscous liquid; d) gelling this cream or viscous liquid to obtain a premix gel; e) drying the gel and grinding the dry substance obtained hereof to a particulate premix material; f) granulating the particulate premix material in the presence of the usual (micro)granulation additives and a bioactive agent dissolved in water; and g) drying and optionally coating the thus obtained granules.