WO2023272362A1 - Fish feed additives - Google Patents

Abstract

The present invention relates to the field of oral delivery of therapeutic compounds to fish through fish feed additives, including aquatic animal feed additive compositions and methods of treating or preventing diseases in aquatic animals.

Description

FISH FEED ADDITIVES

TECHNICAL FIELD

[0001] The present invention relates to the field of oral delivery of therapeutic compounds to fish through fish feed additives.

BACKGROUND ART

[0002] The commercial culture of fish is now a major industry in many countries around the world. However, commercially cultured fish can suffer from a variety of different diseases, the treatment of which can create significant challenges and cost burden on industry. For example, commercially cultured fish populations often suffer from parasitic infections which can be difficult to treat. Examples of such infections include polyopisthocotylean and monopisthocotylean monogenean flukes, a condition which is relatively common in commercially cultured fish populations such as the yellowtail kingfish ( Seriola lalandi).

[0003] Currently, the commercially used methods for treating parasites in fish populations involve bath treatments, where fish are bathed in solution containing medications, or oral administration. For example, the effective anthelmintic drug, praziquantel (2-(cyclohexylcarbonyl)- 1 ,2, 3, 6, 7,11 b-hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one), is used to treat S. lalandi suffering from polyopisthocotylean and monopisthocotylean monogenean flukes. However, due to the poor palatability of praziquantel, as it has a strong bitter taste, fish are bathed in praziquantel, rather than receiving the drug orally. Alternatively, fish are bathed in hydrogen peroxide.

[0004] Bathing can be labour intensive, time consuming, weather dependant and prolonged exposure can detrimentally impact the fish, causing reduced growth rates and in some cases mortalities (Gaikowski, M. P. et. a/., 1999, Acute toxicity of hydrogen peroxide treatments to selected life stages of cold, cool, and warm water fish Aquaculture, 178, 191-207). Bathing treatments can also be expensive. For example, in Japan, the cost of bathing to treat B. seriolae contributes up to 22% of the production costs of sea caged Seriola species (Ernst, I. et. at., 2002, Monogenean parasites in sea-cage aquaculture Austasia Aquaculture, 16, 46-48).

[0005] Constant handling, crowding, loss of feeding time and reductions in dissolved oxygen during bath treatments can cause mortalities as well as diminished appetite and loss of growth (Grant, A. N, 2002, Medicines for sea lice. Pest Management Science, 58, 521 -527).

[0006] Oral administration of medication to fish on the other hand has advantages over conventional bath treatments. For example, in-feed medications have wider safety margins and do not require crowding or increased handling of fish. Treatment efficiency is also increased, as all cages on the farm can be treated quickly, (Williams, R. et. a!., 2007, Efficacy of orally administered praziquantel against Zeuxapta seriolae and Benedenia seriolae (Monogenea) in yellowtail kingfish Seriola lalandi, Diseases of Aquatic Organisms, 77, 199-205.) reducing the chance of infection from nearby untreated fish. Through feeding medicated diets, the fish can also maintain their natural feeding regime, also reducing stress (Conte, F. S., 2004, Stress and the welfare of cultured fish. Applied Animal Behaviour Science, 86, 205-223.). The environmental impact of oral treatments is less than bathing treatments, as once the bathing process is finished, the chemicals are released into the surrounding environment, which may impact non-target organisms (Grant, A. N., 2002, Medicines for sea lice, Pest Management Science, 58, 521-527). Oral treatments are eventually released into the environment, but at much lower concentrations and at a much slower rate (Ramstad, A. et. al, 2002, Field trials in Norway with SLICE (0.2% emamectin benzoate) for the oral treatment of sea lice infestation in farmed Atlantic salmon Salmo salar, Diseases of Aquatic Organisms, 50, 29-33.).

[0007] Flowever, one major obstacle to orally administering medications to fish is that fish are often sensitive to the taste and smell of medications. Therefore, the inclusion of medications in fish feed often causes reduced palatability of the feed, resulting in reduced feeding and thereby reducing the dose of the medication that is received, meaning that the medication may ultimately be ineffective (Williams, R. et. al., 2007, Efficacy of orally administered praziquantel against Zeuxapta seriolae and Benedenia seriolae (Monogenea) in yellowtail kingfish Seriola lalandi, Diseases of Aquatic Organisms, 77, 199-205).

[0008] Some methods have been trialled in order to address the problems with oral administration of medications in fish populations. W02009/023013 describes multiparticulate in- feed additives that have a number of coatings applied to a core containing the medication (praziquantel and cimetidine). In particular, it describes a plurality of granular cores comprising a hydrogel-forming polymer, a second coating disposed on the first coating comprising a barrier material and a third coating disposed on the second coating comprising a taste-masking polymer being a Eudraguard^® polymer. This is however a complex structure from a manufacturing perspective, as the beads comprise several distinct coatings.

[0009] W01989/12442 describes a pharmaceutical dosage form for administration of medicaments to fish comprising an outer layer of animal or vegetable material, which is substantially impermeable to water, which surrounds an internal chamber which is filled with the active agent. AU2008100441 describes medicated fish feed containing therapeutic quantities of microencapsulated anthelmintic drugs (including praziquantel), as well as attractants such as amino acids, nucleotides and natural and synthetic extracts from various aquatic organisms, which trigger the olfactory and taste receptors of fish, eliciting feeding behaviour in the target fish species. Neither of these methods appear to have been used commercially. [0010] Partridge et. al. (2014) [Partridge, G.J., Michael, R.J. and Thuillier, L, 2014, Praziquantel form, dietary application method and dietary inclusion level affect palatability and efficacy against monogenean parasites in yellowtail kingfish. Diseases of Aquatic Organisms, 109 (2), 155-163.] assessed the palatability of fish feed either surface-coated with praziquantel powder or microencapsulated praziquantel, as well as fish feed where praziquantel powder or microencapsulated praziquantel had been incorporated within the pellet mash prior to extrusion. Fish feed containing microencapsulated praziquantel was more palatable than fish feed containing praziquantel powder. However, even with microencapsulation, the palatability of fish feed with high dietary inclusion levels of praziquantel was limited. Fish feed containing microencapsulated praziquantel also had lower bioavailability than fish feed containing praziquantel powder. Partridge et. al. (2019) [Partridge, G.J., Rao, S., Woolley, L., Pilmer, L., Lymbery, A.J. and Prestidge, C.A., 2018, Bioavailability and palatability of praziquantel incorporated into solid-lipid nanoparticles fed to yellowtail kingfish Seriola lalandi. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 218, 14-20.] examined the effect of incorporating praziquantel into solid-lipid nanoparticles which were then coated onto fish feed pellets but found that neither bioavailability nor palatability of the praziquantel was improved by incorporating it in the fish feed in this manner.

[0011] Therefore, there exists a need for alternative compositions that improve the palatability of therapeutically effective compounds for fish, including those with a particularly pungent taste or odour such as praziquantel, so that they can be delivered orally. Further, any effort to improve the palatability of medications such as praziquantel for fish should not compromise the bioavailability of the drug in vivo.

[0012] The previous discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

SUMMARY OF INVENTION

[0013] In a first aspect, the invention provides an aquatic animal feed additive composition capable of delivering a therapeutically effective amount of a therapeutic to an aquatic animal, said additive comprising a plurality of therapeutic beads, wherein each bead comprises:

(a) at least 45% w/w therapeutically effective compound;

(b) between 1 % and 10% w/w taste-masking agent;

(c) between 2% and 35% w/w carrier matrix; and wherein, the carrier matrix is:

(a) ingestible by the aquatic animal; and (b) is substantially stable for at least 60 minutes at room temperature in an aqueous environment in which the aquatic animal feed additive is used; and wherein, the taste-masking agent alone or in combination with the carrier matrix masks the taste of the therapeutically effective compound.

[0014] Preferably, the carrier matrix is digestible by the aquatic animal.

[0015] In an embodiment of the invention, the therapeutically effective compound is substantially stable in the carrier matrix when the amount of leaching of the therapeutically effective compound from the carrier matrix is less than 20% w/w over a 60 minute period in an aqueous environment, at room temperature, wherein the aqueous environment is an environment in which the aquatic animal feed additive is used (such as water or more preferably such as sea water).

[0016] In another embodiment of the invention, the additive is generally durable in the aqueous environment in which the aquatic animal lives, that is to be treated. For example, the carrier matrix is generally durable if it breaks down at room temperature by less than 1 , 1.1 , 1.2, 1.3, 1.4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10% w/w.

[0017] In yet another embodiment of the invention, the additive preferably disintegrates, at least in part, in the digestive tract of the aquatic animal that is treated by the feed additive. Preferably, the additive breaks down by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% w/w in the digestive tract of the aquatic animal.

[0018] Where the additive or carrier matrix is poorly soluble in an animal’s digestive tract (such as the gastric or intestinal tracts), the additive may also include a wetting agent or surfactants polymer or pharmaceutical disintegrant to assist in disintegration of the additive. Wetting agents or surfactants may include, for example and without limitation, anionic or cationic detergents or a non-ionic wetting agent. In a particular form of the invention, the wetting agent is Cremophor^® RH40 or Tween. Disintegrants can also be starches and modified starches, cellulose etc.

[0019] In an alternative embodiment, the additive or carrier matrix can include a wetting agent or surfactants polymer or pharmaceutical disintegrant, that may or may not breakdown in an animal’s digestive tract, which assists in the manufacture of the additive and or enhances the wetting of the additive in an aqueous environment. Wetting agents or surfactants suitable for this purpose include, for example and without limitation, anionic or cationic detergents or a non-ionic wetting agent. Disintegrants can also be starches and modified starches, cellulose etc. In a particular form of the invention, the wetting agent is Cremophor^® RH40 or Tween. [0020] In an embodiment of the invention the therapeutic is substantially stable in the additive for at least 6 months, more preferably 9, 12, 15, 18, 24 months.

[0021] Preferably, the carrier matrix is at least partially prepared from a blend of alginate and an acid soluble polymer (such as chitosan and/or Eudraguard^®) or alginate; or agar.

[0022] In a preferred embodiment, taste-masking agent is a garlic powder or a garlic derivative.

[0023] While it will be appreciated by a person skilled in the art that a range of therapeutic agents suitable for an aquatic environment can be delivered with this invention, preferably, the therapeutic is an anthelmintic drug, such as praziquantel, mebendazole and fenbendazole. Praziquantel is widely used to treat trematode, cestode and monogenean infestations in both freshwater and marine fish and elasmobranchs and therefore provides a model therapeutic.

[0024] In a preferred form of the invention the aquatic animal feed additive will include an appropriate amount of a therapeutic agent to achieve therapeutic efficacy. That amount of therapeutic agent will depend on, inter alia, the nature of the pathogen, the size of the aquatic animal and temperature of the water (amongst other matters). Where praziquantel is administered for blood feeding gill flukes the dose rate is approximately 50mg/kg. Where praziquantel is administered for mucus feeding gill flukes the dose rate is approximately 150mg/kg. The dietary inclusion level required to achieve these doses is something that a person skilled in the art can easily ascertain depending on water temperature (fish eat less in cold water) and the size of the fish. The bigger the fish the less they eat (on a %BW basis) therefore, a higher dietary inclusion rate of the drug (in g PZQ /kg of diet) is required to achieve the same dose (in mg PZQ/kg of body weight) as fish grow.

[0025] In a particularly preferred form of the invention, the effective dietary inclusion level, having regard to the average of all fish sizes and all water temperatures, could be achieved with a dietary inclusion level of between 10 g PZQ/kg to 20 g PZQ/kg of food. In many respects, the higher the dietary inclusion level the better.

[0026] In some embodiments, the invention provides that the therapeutically effective compound is present in an amount of at least about 30% to 70% w/w of the beads.

[0027] In some embodiments, the beads of the invention are about 0.1 to 5 millimetres in diameter.

[0028] In some embodiments, the composition comprises: (a) the therapeutically effective compound comprises praziquantel which is present in an amount of about 70-90% w/w of each bead; (b) the taste masking agent comprises a garlic derivative which is present in an amount of about 5% w/w of each bead; and (c) the carrier matrix comprises a combination of an acid soluble polymer and alginate, wherein the acid soluble polymer is present in an amount of 8-10% w/w and the alginate is present in an amount of 8-11% w/w of each bead.

[0029] In some embodiments, the composition comprises: (a) the therapeutically effective compound comprises praziquantel which is present in an amount of about 70-90% w/w of each bead; (b) the taste masking agent comprises a garlic derivative which is present in an amount of about 5% w/w of each bead; (c) the carrier matrix comprises alginate, wherein the alginate is present in an amount of 5-11 % w/w of each bead; and (d) the wetting agent is Cremophor^® RH40, wherein the Cremophor^® RH40 is present in an amount of 5-10% w/w of each bead.

[0030] In some embodiments, the composition comprises: (a) the therapeutically effective compound comprises praziquantel which is present in an amount of about 70-90% w/w of each bead; (b) the taste masking agent comprises a garlic derivative which is present in an amount of about 2-5 % w/w of each bead; and (c) the carrier matrix comprises a combination of agar, which is present in an amount of 18-20% w/w of each bead.

[0031] In some embodiments, the composition comprises: (a) the therapeutically effective compound comprises praziquantel which is present in an amount of about 70-90% w/w of each bead; (b) the taste masking agent comprises a garlic derivative which is present in an amount of about 5% w/w of each bead; (c) the carrier matrix comprises a combination of an acid soluble polymer and alginate, wherein the acid soluble polymer is present in an amount of 8-10% w/w and the alginate is present in an amount of 8-11% w/w of each bead; and (d) the wetting agent is Cremophor^® RH40 or Tween, wherein the RH40 or Tween is present in an amount of 5-10% w/w of each bead.

[0032] In some embodiments, the beads are further covered with a taste-masking agent.

[0033] In a second aspect, the invention provides a fish feed composition comprising the fish feed additive of the invention.

[0034] In a third aspect, the invention provides a method of treating or preventing a disease in a fish comprising administering to the fish the composition, fish feed or fish feed additive of the invention. Preferably, the disease is a parasitic infection, such as an infection caused by cestodes or trematodes, such as polyopisthocotylean and monopisthocotylean monogenean flukes. Preferably, the fish feed or fish additive is administered in a sufficient quantity to deliver to the fish a dose of the therapeutically effective compound in an amount of 50 mg/kg to 150 mg/kg.

[0035] In a fourth aspect, the invention provides a method for preparing the aquatic animal feed additive composition of the invention comprising the steps of: (a) preparing a dry mixture of the therapeutically effective compound and the taste-masking agent;

(b) adding a solution containing a carrier matrix to the dry mixture to form a homogenous suspension;

(c) dropping the homogenous suspension in a liquid medium to form beads;

(d) washing the beads with water; and

(e) drying the washed beads.

[0036] The dry mixture is dry because no water is added. The dry mixture is substantially or completely free of water.

[0037] In a fifth aspect, the invention provides a method for preparing the fish feed additive composition of the invention comprising the steps of: (a) mixing the aquatic animal feed additive with crushed fish feed to form a mixture; and (b) forming pellets from the mixture in (a).

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings.

[0039] Figure 1 A depicts the level of disintegration of beads comprising formulations A to E in seawater after 5 hours.

[0040] Figure 1 B depicts the level of disintegration of beads comprising formulations A to E in 0.1 M HCI after 5 hours.

[0041] Figure 2A depicts the cumulative percent dissolution of praziquantel (PZQ) from pure drug powder, Formulation B, and Formulation C after 180 min incubation in seawater.

[0042] Figure 2B depicts PZQ release profiles for Formulation B and Formulation C beads incubated sequentially in different media: SW = seawater, SGF = simulated gastric fluid, SIF = simulated intestinal fluid. Table shows cumulative PZQ release from pure drug powder under specified dissolution conditions. Data represent mean ± SD (n = 3).

[0043] Figure 3A depicts DSC thermograms of Formulation B in comparison with the DSC thermograms of PZQ powder and the corresponding blank beads.

[0044] Figure 3B depicts DSC thermograms of Formulation C in comparison with the DSC thermograms of PZQ powder and the corresponding blank beads. [0045] Figure 4A depicts the percentage of the total ration offered of fish feed comprising formulation A or E which was consumed by 175 gram (small) and 2000 gram (large) kingfish.

[0046] Figure 4B depicts the time taken to consume the total ration offered of fish feed comprising formulation A or E which was consumed by 175 gram (small) and 2000 gram (large) kingfish.

[0047] Figure 5A depicts the percentage of the total ration offered of fish feed comprising formulations B, C or D which was consumed by 260 gram kingfish.

[0048] Figure 5B depicts the time taken to consume the total ration offered of fish feed comprising formulations B, C or D which was consumed by 260 gram kingfish. [0049] Figure 6 depicts the dissected digestive tract showing undigested beads in the a) midgut and b) hindgut of a large kingfish fed fish feed comprising formulation E.

[0050] Figure 7 depicts the dissected digestive tract of fish 3 hours post-feeding, for fish offered fish feed comprising formulations B, C or D.

[0051] Figure 8A depicts the percentage of the total ration of 4 month old fish feed containing formulations B and C compared with fresh fish feed which was consumed by the fish.

[0052] Figure 8B depicts the average time taken to consume the total ration of 4 month old fish feed containing pure praziquantel, formulation B, or formulation C compared with fresh fish feed.

[0053] Figure 9A depicts the percentage of the total ration offered of fish feed comprising pure praziquantel, formulation B, or formulation C consumed by 1600 gram kingfish. [0054] Figure 9B depicts the percentage of the total ration offered of fish feed comprising praziquantel, formulation B, or formulation C consumed by 1600 gram kingfish over the 6 day experimental period.

[0055] Figure 9C depicts the average daily dose of praziquantel received by the fish that were offered fish feed comprising pure praziquantel, or formulation B, or formulation C. [0056] Figure 10 depicts the percentage reduction of Zeuxapta fluke in fish that were offered fish feed comprising pure praziquantel, formulation B, or formulation C.

DETAILED DESCRIPTION OF INVENTION

[0057] The present invention is based on the discovery that encapsulating a taste-masking agent together with a therapeutically effective compound in a carrier matrix that is ingestible for fish and which does not substantially dissolve or degrade in freshwater or seawater, can improve the palatability of therapeutically effective compounds that are otherwise difficult to administer to fish orally.

General

[0058] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0059] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0060] Any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0061] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0062] The invention described herein may include one or more range of values (e.g. size, percentage, concentration, etc.). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Hence “about 80%” means “about 80%” and also “80%”. At the very least, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0063] Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0064] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term "portion" can include part of a moiety or the entire moiety.

[0065] The term “fish feed” means any material such as plant, animal or other organic material that is intended for consumption by fish. Fish feed is used to provide nutrients for captive or farmed fish, and usually contains macronutrients, trace elements as well as vitamins. Sources of nutrients can be fishmeal and other marine sources, vegetable proteins as well as binding agents such as wheat. Fish feed is commonly provided in solid form, such as granules, pellets or flakes.

[0066] The term “aquatic animal” includes fish.

[0067] The term “aquatic animal feed additive” means a composition that can be added to aquatic animal feed, preferably fish feed. Aquatic animal feed additives can contain medications, or other biologically effective agents, taste-masking agents, including flavouring agents, carriers and other components that are desired to be ingested by an aquatic animal population. Aquatic animal feed additives can be incorporated within aquatic animal feed granules or pellets during the manufacturing of the aquatic animal feed. Alternatively, they can be mixed with manufactured aquatic animal feed granules or pellets prior to feeding, or otherwise coated on the surface of aquatic animal feed granules or pellets.

[0068] The term “beads” includes particles, granules and pellets. Beads can be any shape, or size, including spherical or flattened.

[0069] The term "therapeutically effective amount" or “therapeutically effective dose” refers to the amount or dose of the therapeutically effective compound determined to produce a therapeutic response in an aquatic animal. Such therapeutically effective amounts or doses are readily ascertained by one of ordinary skill in the art. [0070] The term "treat" and "treatment" includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.

[0071] The term “palatable” means that the taste and/or odour of a food or additive is sufficiently acceptable to the subject such that it is consumed by the subject. A food or additive does not need to be completely consumed in a given period (i.e. consumed 100%) in order to be considered palatable to the subject. A feed or additive containing a therapeutically effective compound is considered “palatable” if the subject finds it acceptable to consume it in a sufficient quantity to have a therapeutic effect. In some embodiments, a feed or additive containing a therapeutically effective compound is considered “palatable” if the presence of the therapeutically effective compound does not significantly affect the amount of feed consumed by an aquatic animal population. This can be measured by comparing the amount of medicated aquatic animal feed or aquatic animal feed additive consumed by the aquatic animal population to the amount of non-medicated aquatic animal feed or aquatic animal feed additive consumed by the aquatic animal population.

[0072] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

Aquatic Animal Feed Additive

[0073] In a first aspect, the invention provides an aquatic animal feed additive capable of delivering a therapeutically effective amount of a therapeutic to an aquatic animal, said additive comprising a plurality of therapeutic beads, wherein each bead comprises:

(a) at least 45% w/w therapeutically effective compound;

(b) between 1 % and 10% w/w taste-masking agent;

(c) between 2% and 35% w/w carrier matrix; and wherein, the carrier matrix is:

(a) ingestible by the aquatic animal; and

(b) is substantially stable for at least 60 minutes at room temperature in an aqueous environment in which the aquatic animal feed additive is used; and wherein, the taste-masking agent alone or in combination with the carrier matrix masks the taste of the therapeutically effective compound.

[0074] Preferably, the carrier matrix is digestible by the aquatic animal. [0075] The present invention provides a particulate aquatic animal feed additive. The beads of the invention can be of any shape or size that can be ingested by the target aquatic animal population, and which can be incorporated into aquatic animal feed.

[0076] In some embodiments, the beads are about 0.1 millimetres to 5 millimetres in diameter. Preferably the beads are 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 millimetres in diameter.

[0077] In one embodiment, the beads have a diameter selected from the group consisting of: 5000 pm, <5000 pm, 1000 pm, <1000 pm; 500 pm; <500 pm; 355-500pm; 212-355 pm; 150-355 pm; 150 pm; <150 pm; 100 pm and <100 pm.

[0078] Each bead comprises a therapeutically effective compound, a taste-masking agent and a carrier matrix. The relative amounts of each of these elements in a bead depends on the nature of the therapeutically effective compound, the taste-masking agent and the carrier matrix.

[0079] Preferably, the amount of therapeutically effective compound present in each bead will be at least 45% w/w, although a skilled reader will understand that the final amount will depend on the therapeutic being delivered by each bead and the number of beads in the additive. Preferably the amount of therapeutically effective compound present in each bead will be at least

45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70,

71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90 percent w/w.

[0080] Preferably the taste-masking agent is between 1% and 10% w/w such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 percent w/w. When multiple taste-masking agents are present each may be present in an amount of between 1% and 10% w/w such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 percent w/w.

[0081] Preferably the carrier matrix is between 2% and 35% w/w such as 1 , 2, 3, 4, 5, 6, 7, 8,

9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34,

35 percent w/w.

[0082] Each of the therapeutically effective compound, taste-masking agent and carrier matrix are preferably distributed throughout the beads of the invention, rather than coated in separate layers. Collectively, the percent w/w of each of the therapeutically effective compound, taste- masking agent and carrier matrix will amount to between 80 and 100 percent w/w such as 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 percent w/w.

[0083] In an embodiment of the invention, the therapeutically effective compound is substantially stable in the carrier matrix when the amount of leaching of the therapeutically effective compound from the carrier matrix is less than 20% w/w over a 60 minute period in an aqueous environment, at room temperature, wherein the aqueous environment is an environment in which the aquatic animal feed additive is used (such as water or more preferably such as sea water). Preferably, the amount of leaching is less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% w/w over a 60 minute period in the aqueous environment at room temperature. More preferably the amount of leaching is less than 5% w/w over a 60 minute period in an aqueous environment at room temperature.

[0084] Chemical stability of an additive preparation depends upon the amount of therapeutic leaching of the active ingredient in that preparation. Commonly, stability analysis of a preparation may be performed under accelerated temperature conditions, such as in an oven at temperatures higher than room temperature. The kinetic methods used in the accelerated stability analysis need not involve detailed studies of mechanism of degradation to be able to predict stability, but they are preferably based upon sound scientific principles and compliance with regulatory requirements.

[0085] In another embodiment of the invention, the additive is generally durable in the aqueous environment in which the aquatic animal lives, that is to be treated. For example, the carrier matrix is generally durable if it breaks down at room temperature by less than 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51 , 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61 , 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71 , 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81 , 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91 , 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1 , 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 .8, 1 .9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10% w/w.

[0086] In yet another embodiment of the invention, the additive preferably disintegrates, at least in part, in the digestive tract of the aquatic animal that is treated by the feed additive. Preferably, the additive breaks down by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% w/w in the digestive tract of the aquatic animal.

[0087] Where the additive or carrier matrix is poorly soluble in an animal’s digestive tract (such as the gastric or intestinal tracts), the additive may also include a wetting agent or surfactants polymer or pharmaceutical disintegrant to assist in disintegration of the additive. Wetting agents or surfactants may include, for example and without limitation, anionic or cationic detergents or a non-ionic wetting agent. In a particular form of the invention, the wetting agent is Cremophor^® RH40 or Tween. Disintegrants can also be starches and modified starches, cellulose etc. [0088] In an alternative embodiment, the additive or carrier matrix can include a wetting agent or surfactants polymer or pharmaceutical disintegrant, that may or may not breakdown in an animal’s digestive tract, which assists in the manufacture of the additive and or enhances the wetting of the additive in an aqueous environment.

[0089] Where the therapeutic is poorly wetted in the carrier matrix (e.g., the aqueous sodium alginate solution) during bead manufacture, a wetting agent can be added to help with the wetting of the drug powder so that it can be uniformly dispersed in the matrix. The wetting agent also assists in displacing entrapped air between the drug and the carrier matrix to give a stronger bind between the carrier matrix and the therapeutic- entrapped air gives rise to friable beads that are then easily crushed when incorporated into the fish feed, resulting in the fish being able to taste the exposed therapeutic.

[0090] Wetting agents or surfactants suitable for this purpose include, for example and without limitation, anionic or cationic detergents or a non-ionic wetting agent. Disintegrants can also be starches and modified starches, cellulose etc. In a particular form of the invention, the wetting agent is Cremophor^® RH40 or Tween.

[0091] In an embodiment of the invention the therapeutic is substantially stable in the additive for at least 6 months, more preferably 9, 12, 15, 18, 24 months.

[0092] Where the percent w/w of the beads is less than 100 percent w/w of the above components the beads may include other components or additional materials such as additional taste-masking agents may be deposited on (including as a coating or covering) or included in the beads.

[0093] Once the beads are prepared, they may be dried. Any suitable method can be used for drying the beads. For example, the bead may be air-dried using a fume-hood over a period of several days, as described below. The dried beads may then be incorporated into an aquatic animal feed.

Therapeutically effective compounds

[0094] Any therapeutically effective compound that can be administered orally to aquatic animals, preferably fish, may be used in the present invention. In some embodiments, there is more than one therapeutically effective compound in the bead. For example, the therapeutically effective compound can be an anthelmintic drug, a probiotic, a synbiotic or an antibiotic or a combination thereof. In a preferred embodiment, the therapeutically effective compound is an anthelmintic drug. [0095] Anthelmintic drugs that are used in the treatment and prevention of parasitic diseases in aquatic animals such as fish include trichlorfon, mebendazole, fenbendazole, praziquantel and 40% phoxim. In another embodiment, the anthelmintic drugs is selected from florfenicol, oxytetracycline dehydrate, sulfadimethoxine/ormetoprim, bioinsecticides based on B. thuringiensis and spores of the Gram-positive bacteria of the genus Bacillus. In another embodiment, the antibiotic is selected from a tetracycline, oxolinic acid and chloramphenicol.

[0096] Where a probiotic is used in the invention, the probiotic’s main purpose will be to establish or to maintain a relationship between beneficial and harmful bacteria, which is usually present in the intestine or gut of fish. Effective probiotics should possess certain qualities as specified below: a. The probiotics should have a beneficial effect on the growth, development and protection of fish against various pathogenic bacteria. b. The probiotic bacteria should not have any harmful effect on the host. c. The probiotics should not have the ability of drug resistance, and should have the ability to keep up hereditary traits. d. For the utilization of probiotics in an efficient feed, they should exhibit the following properties: i. acid and bile tolerance ii. resistance to gastric juices iii. adherence to digestive system surface iv. antagonism towards pathogens v. stimulation of immunity & increase in the gut motility & survival in mucous vi. production of enzymes and vitamins; and e. They should have good sensorial properties, fermentative action, tolerance towards freeze-drying and viability in feed during packaging and storing process.

[0097] By way of example only the most frequently used probiotic microorganisms belong to Bacillus, Lactobacillus and Bifidobacterium genus. Various species of Lactobacillus, Bifidobacterium and Streptococcus reported for use in aquaculture as probiotics, include L. acidophilus, L. casei, L fermentum, L. gasseri, L. plantarum, L. salivarius, L. rhamnosus, L. johnsonii, L. paracasei, L. reuteri, L helveticus, L. bugaricus, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Saccharromyces species, Saccharromyces boulardii, S. thermophiles and S. cremoris.

[0098] Many probiotics used in aquaculture are well-known for their antibacterial properties against known pathogens. Lactococcus lactis RQ516 probiotic shows inhibitory action against Aeromonas hydrophila when given to Tilapia (Oreochromis niloticus). Also L. lactis probiotic has anti-bacterial activity against two pathogens - Yersinia rukeri and Aeromonas salmonicida that can affect fish growth. Leuconostoc mesenteroides has the potential to inhibit the fish pathogens found in Nile tilapia ( O . niloticus). Bacillus subtilis considerably reduces the motile Aeromonads, total Conforms and Pseudomonads found in ornamental fishes. Lactic acid bacteria such as Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus fermentum, Lactococcus lactis, and Sterptococcus salivarius were isolated from Spanish mackerel {Scomberomorus commerson) intestine and were capable of inhibiting the Listeria innocua growth. Many Lactobacilli species isolated from the intestine of Anguilla species, Clarias orientalis, Labeo rohita, Oreochromis species and Puntius carnaticus showed significant anti-microbial activity against Aeromonas and Vibrio species.

[0099] The inhibition of viruses can also occur by secretion of extracellular enzymes produced by the bacteria. For example, Aeromonas species, Corynebacterium, Pseudomonas and Vibrio species show the antiviral activity against the IHNV (Infectious hematopoietic necrosis virus). Feeding of probiotic strain Bacillus megaterium has increased the resistance against WSSV (white-spot syndrome virus) in the shrimp, Litopenaeus vannamei. The probiotics strains Bacillus and Vibrio species are effective against WSSV and efficiently protect Litopenaeus vannamei. Application of Lactobacillus as a probiotic, either as a single strain or as a mixture with Sporolac resulted in better resistance against lymphocystis viral disease, which is found in Paralichthys olivaceus der).

[00100] Probiotics can also exhibit antifungal activity. For example, the Aeromonas strain A199 from Anguilla australis (eel) culture water, can inhibit the Saprolegnia species. Pseudomonas species M162, Pseudomonas species M174 and Janthinobacterium species M169 can increase the animal’s immunity against saprolegniasis, as has been demonstrated with Oncorhynchus mykiss (rainbow trout). Lactobacillus plantarum FNCC 226 has also shown inhibitory potential in catfish (Pangasius hypophthalamus) against Saprolegnia parasitica.

[00101] A major advantage of the present invention is that the aquatic animal feed additive provides a means for improving the palatability of therapeutically effective compounds. Therefore, in some embodiments of the invention, the therapeutically effective compound is a poorly palatable or pungent compound that is difficult to directly administer orally to aquatic animals.

[00102] In one embodiment, the therapeutically effective compound is selected from the group consisting of: a probiotic; a symbiotic; an anti-invertebrate compound; an anti-helminitic compound; an anti-viral compound; an antinematode compound; an antibiotic compound; an algaecide; an insecticide compound; an antifungal compound; an antiprotozoal compound; and a larvicide compound. [00103] In another embodiment, the therapeutically effective compound is selected from the group consisting of: a tetracycline; a sulfa-antibiotic; a diaminopyrimidine; a fluoroquinolone; a quinolone; a sulphonamide; a avermectin; a macrolide; a chlorinated bisphenol; a benzoylurea; a monochlorobenzene; an insect growth regulator; a sulfur compound; a salicylamide; a phosphorus compound; a benzimidazole; a pyrethrin; a triazine; a tetramisole; and an anticoccidal; a irreversible organophosphate acetylcholinesterase inhibitor; a organophosphate insecticide; a chloramphenicol; and a tetracycline;

[00104] In one preferred embodiment, the therapeutically effective compound is selected from the group consisting of: praziquantel; sulfadiazine; trimethoprim; flumequine; oxytetracycline; oxolinic acid (also known as terramycin); emamectin benzoate; trichlorfon; mebendazole; fenbendazole; 40% phoxim; florfenicol; oxytetracycline dehydrate; sulfadimethoxine/ormetoprim; sulfadimethoxine; ormetoprim; bioinsecticides based on B. thuringiensis and spores of the Gram positive bacteria of the genus Bacillus; tetracycline; and chloramphenicol.

[00105] In another preferred embodiment, the therapeutically effective compound is selected from the group consisting of: macrolide anthelmintic; bithionol; diflubenzuron; triflumuron; pyriproxyfen; sulfur powder; salicylanilide; organic phosphorus; benzimidazole; pyrethroid; triazine; tetramisole anthelmintic; levamisole anthelmintic; sulfonamide; and an anticoccidial drug.

[00106] In one preferred embodiment of the invention, the therapeutically effective compound is praziquantel. Praziquantel is an anthelmintic drug which is known to have a particularly bitter taste that is rejected by fish when administered directly orally to fish, simply mixed in with fish feed, or coated on fish feed. Praziquantel is used frequently in the treatment of flukes among certain commercially cultured fish species, such as the yellowtail kingfish, but is generally administered through bathing due to its bitter taste. The yellowtail kingfish is known to have a particularly sensitive palate.

[00107] The amount of the therapeutically effective compound in an aquatic animal feed additive bead of the invention is dependent on the nature of the therapeutically effective compound, and the amount required for achieving a therapeutic effect in the aquatic animal population. The amount of aquatic animal feed additive (or aquatic animal feed comprising the aquatic animal feed additive) consumed by the aquatic animal population influences the amount of therapeutically effective compound required in the beads. It is known to persons skilled in the art that the size of the aquatic animal, and the temperature of the water can also influence the amount of aquatic animal feed consumed by the aquatic animal population. For example, larger fish tend to eat a smaller percentage of their bodyweight in fish feed compared with smaller fish. Fish populations also tend to consume a smaller amount of feed in colder water conditions. This means that both for larger fish and in colder water conditions, a greater amount of the therapeutically effective compound must be present in the fish feed in order for it to be consumed in a therapeutically effective dose by the fish population.

[00108] A particular advantage of some embodiments of this invention is that very high drug loading can be achieved in the beads. In some embodiments, beads contain at least about 70% w/w of the therapeutically effective compound. This can be useful in delivering some therapeutically effective compounds in certain conditions and to some aquatic animal populations, as high drug loading is necessary in order to deliver the therapeutically effective compound in a sufficient quantity to have a therapeutic effect in the aquatic animal.

[00109] For example, depending on the nature of the disease to be treated, the therapeutically effective dose of praziquantel is between 50 mg/kg and 150 mg/kg. In a most preferred embodiment, drug loading of about 70% to 90% w/w praziquantel is present in the beads, which ensures that the aquatic animal population ingests enough aquatic animal feed such that a therapeutically effective dosage of praziquantel is delivered to the aquatic animal population.

Taste-masking agents

[00110] The present invention provides for a taste-masking agent that is incorporated within the beads of the invention.

[00111] The encapsulation of the taste-masking agent within the beads of the invention (as opposed to simply coated on or otherwise incorporated into the aquatic animal feed) provides the advantage of ensuring that the taste-masking agent remains proximal to the therapeutically effective compound and is not dispersed independently of the therapeutically effective compound. It is therefore better able to mask the taste of the therapeutically effective compound.

[00112] The taste-masking agent can be any agent that has the capacity to sufficiently conceal the flavour and/or smell of the therapeutically effective compound that is also comprised within the beads. Taste-masking agents can exert this effect in numerous ways. Some taste-masking agents do not have a taste or flavour of their own. Instead, these agents can influence the perception of the taste of the therapeutically effective compound, by for example, exerting an effect on the gustatory system of the aquatic animal, binding to the therapeutically effective compound and preventing its release in saliva, or reducing the dissolution of the therapeutically effective compound in the saliva. Other taste-masking agents are flavouring agents. Flavouring agents themselves have an odour and/or flavour that is more palatable to aquatic animals, which then have the capacity to mask the unpalatable flavour and/or odour of the therapeutically effective compound. In a preferred embodiment, the taste-masking agent is a flavouring agent. [00113] A large number of taste-masking agents for aquatic animals, particularly fish, are known in the art, including sucrose, free amino acids, nucleotides and nucleosides, organic acids, fish hydrolysates, and other compounds. The appropriate taste-masking agent can depend on the specific species of the aquatic animal population.

[00114] In a particularly preferred embodiment, the taste-masking agent is a derivative or extract of garlic (e.g. garlic powder, diluted or pure, synthetic garlic component (allicin) or garlic oil), including odorous compounds found in garlic. In another preferred embodiment, the taste- masking agent is an amino acid, such as betaine, L-alanine, L-glutamic acid, L-arginine, glycine and inosine. In another preferred embodiment, the taste-masking agent is natural fish oil (cod liver oil or tuna oil), anise oil, or castor oil.

[00115] The amount of the taste-masking agent required in the aquatic animal feed depends on the nature of the taste-masking agent, and is preferably in the range of 1 - 10% w/w. In a preferred embodiment, the taste-masking agent comprises about 2-8% w/w of the bead. In a particularly preferred embodiment, the flavouring or taste-masking agent comprises about 4-5% w/w of the bead.

Carrier Matrix

[00116] The carrier matrix provides a means of encapsulating the therapeutically effective compound and the taste masking agent together.

[00117] The carrier matrices used in the compositions of the invention are capable of forming beads incorporating the taste masking agent, and the therapeutically effective compound such that the beads are of a suitable size, shape and consistency for incorporation into aquatic animal feed and consumption by the target aquatic animal population. The carrier matrices also assist in maintaining the integrity of the beads when compressed into the aquatic animal feed.

[00118] Preferably, the beads are digestible by aquatic animals such that the therapeutically effective compound is released into the digestive tract of the animal once it is ingested, rather than regurgitated or excreted intact. The carrier matrices used in the present invention are preferably capable of at least partially breaking down in the aquatic animal digestive tract so that the therapeutically effective compound is able to be absorbed by the animal. This allows for targeted drug release in the gut.

[00119] Aquatic animal populations can find therapeutically effective compounds more palatable when provided in some carrier matrices rather than others. In some embodiments, the carrier matrix acts together with the taste-masking agent to improve the palatability of the therapeutically effective compound. [00120] Without being bound by theory, the therapeutically effective compound, when incorporated into a suitable carrier matrix, exhibits reduced leaching into the surrounding environment when it is delivered for consumption by a population of aquatic animals. The reduced leaching of the therapeutically effective compound reduces the detection of malodour/taste of the therapeutically effective compound by the aquatic animals, improving its palatability. This has the additional effect of reducing the amount of therapeutically active compound in the aquatic animal feed additive itself.

[00121 ] It is therefore desirable for the beads not to leach the therapeutically effective compound into the surrounding water when the aquatic animal feed additive (or aquatic animal feed comprising the aquatic animal feed additive) is introduced into the water for consumption by the aquatic animal population. Accordingly, the carrier matrix preferably does not substantially dissolve or disintegrate in freshwater or seawater. In a particularly preferred embodiment, the carrier matrix is substantially stable in sea water for at least 60 minutes, in that the beads will not disintegrate in sea water during this period.

[00122] In an embodiment of the invention, the rate of leaching of the therapeutic is less than 10% over a 60 minute period in sea water. More preferably, the rate of leaching is less than 5% over a 60 minute period in sea water.

[00123] In particularly preferred embodiments, suitable carrier matrices are at least partially prepared from hydrogel based compounds derived from marine environments, which do not substantially dissolve or disintegrate in seawater. Suitable marine based hydrogels include agar, agarose, carrageenan, fucoidan, chitosan, alginate or agar. In further preferred embodiments, suitable carrier matrices include a combination of alginate and an acid soluble polymer (such as chitosan and/or Eudraguard^®), alginate alone, or agar. In one embodiment, the suitable carrier matrix is a marine based collagen.

[00124] In a preferred embodiment, the carrier matrix is a combination of alginate and an acid soluble polymer (such as chitosan and/or Eudraguard^®). Preferably, the carrier matrix is comprised of about 5 - 15% w/w acid-soluble polymer and 5 - 15% w/w alginate. In a particularly preferred embodiment, the carrier matrix is comprised of about 8-10 w/w% acid-soluble polymer and about 8-11% w/w alginate. The beads are prepared by cross-linking a suspension containing the alginate/ acid-soluble polymer carrier matrix, therapeutically effective compound and taste masking agent in a solution of calcium chloride or zinc chloride.

[00125] In another preferred embodiment, the carrier matrix is comprised of alginate alone. Preferably, the carrier matrix is comprises 5 - 15% w/w alginate. In a particularly preferred embodiment, the carrier matrix is comprised of about 5-11% alginate. [00126] In another preferred embodiment, the carrier matrix is prepared from agar. Any type of agar can be used as the carrier matrix, including commercial food-grade agar, agarose and other pharmaceutical or microbiological grade agar. Preferably, the carrier matrix is comprised of about 15 - 25% agar. In a particularly preferred embodiment, the carrier matrix comprises food-grade agar and comprises about 18-20% w/w agar. These beads are prepared by setting the suspension containing the agar carrier matrix, therapeutically effective compound and taste-masking agent into an oil, emulsifier (e.g. Tween 80), or buffer. In a preferred embodiment, the oil, emulsifier or buffer used to set the agar carrier matrix has taste-masking properties as a flavouring agent, in that it has a flavour which is palatable to the aquatic animal. In an especially preferred embodiment, an oil is used to set the suspension containing the agar carrier matrix. Preferably, the oil used may be garlic oil (that is, oil containing a garlic derivative), and/or cod liver oil.

Wetting Agents / Surfactant / Disintegrant

[00127] In some preferred embodiments, the beads additionally comprise one or more wetting agents, surfactants or disintegrants.

[00128] Where the additive or carrier matrix is poorly soluble in an animal’s digestive tract (such as the gastric or intestinal tracts), the additive may include a wetting agent or surfactant polymer or pharmaceutical disintegrant to assist in disintegration of the additive.

[00129] In an alternative embodiment, the additive or carrier matrix can include a wetting agent or surfactants polymer or pharmaceutical disintegrant, that may or may not breakdown in an animal’s digestive tract, which assists in the manufacture of the additive and or enhances the wetting of the additive in an aqueous environment.

[00130] Where the therapeutic is poorly wetted in the carrier matrix (e.g., the aqueous sodium alginate solution) during bead manufacture, a wetting agent can be added to help with the wetting of the drug powder so that it can be uniformly dispersed in the matrix. The wetting agent also assists in displacing entrapped air between the drug and the carrier matrix to give a stronger bind between the carrier matrix and the therapeutic- entrapped air gives rise to friable beads that are then easily crushed when incorporated into the fish feed, resulting in the fish being able to taste the exposed therapeutic.

[00131] In some embodiments, the wetting agent has the effect of reducing the amount of air trapped in the suspension from which the beads are formed. The amount of air trapped in the suspension has the capacity to affect the brittleness of the beads. Preferably, a reduction of the air trapped in the suspension increases the compressibility of the bead, and therefore reduces the risk of crushing and consequent leaching of the therapeutic agent when the bead is incorporated into fish feed. For example, where the therapeutic is praziquantel, the wetting agent can be added to help with the wetting of the drug powder so that it can be uniformly dispersed in the alginate solution. In addition, the wetting agent also helps to displace the entrapped air between the drug and the sodium alginate solution (a result of poor wetting) to give strong beads - entrapped air in the final beads gave rise to friable beads that were then easily crushed when incorporated into the fish feed, resulting in the fish being able to taste the exposed praziquantel.

[00132] Wetting agents or surfactants include, for example, and without limitation, anionic or cationic detergents or a non-ionic wetting agent. Preferably, wetting agents or surfactants include, for example and without limitation, anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be optionally used and could include, without limitation, benzalkonium chloride or benzethomium chloride. The list of potential nonionic wetting agents that could be included in the formulation include Cremophor^® RH40, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80 (Tween), sucrose fatty acid ester, methyl cellulose and carboxymethylcellulose. When used, these wetting agents can be present in the dosage forms of the present invention either alone or as a mixture in different ratios.

[00133] Preferably, the wetting agent is Cremophor^® RH40 or polysorbate (Tween). For example, the wetting agent is 21 , 21 , 40, 60, 61 , 65, 80, 81 and/or 85 Tween. In an embodiment, the bead is comprised of about 5 - 15% Cremophor^® RH40.

[00134] Disintegrants can be, for example, starches and modified starches, cellulose, etc.

[00135] In particularly preferred embodiments, suitable carrier matrices include a combination of alginate and an acid soluble polymer (such as chitosan and/or Eudraguard^®) and a wetting agent (such as Cremophor^® RH40 or Tween).

[00136] In a preferred embodiment, the carrier matrix is comprised of about 5 - 15% w/w acid- soluble polymer and 5 - 15% w/w alginate and a wetting agent. Preferably, the wetting agent is selected from Cremophor^® RH40 and/or Tween. Most preferably, the wetting agent is present at about 5 - 10% w/w. Most preferably, the wetting agent is Cremophor^® RH40 and is present at about 5 - 10% w/w.

[00137] In another preferred embodiment, the carrier matrix is comprised of alginate and a wetting agent. Preferably, the carrier matrix is comprised 5 - 15% w/w alginate and a wetting agent. In a particularly preferred embodiment, the carrier matrix is comprised of about 5-11% w/w alginate and a wetting agent. Preferably, the bead additionally comprises a wetting agent selected from Cremophor^® RH40 and/or Tween. Most preferably, the wetting agent is Cremophor^® RH40 and is present at about 5 - 10% w/w. Aquatic Animal Feed comprising the Aquatic Animal Feed Additive

[00138] In an embodiment, the invention provides an aquatic animal feed comprising the aquatic animal feed additive of the invention in a therapeutically effective amount.

[00139] The aquatic animal feed additive beads of the invention can be incorporated into aquatic animal feed, which can then be delivered to the target aquatic animal population. Preferably, the aquatic animal is a fish. Any aquatic animal feed that can safely be offered to the target aquatic animal population can be used to prepare the aquatic animal feed of the invention.

[00140] In a preferred embodiment, the beads of the invention are mixed with crushed aquatic animal feed during the manufacturing process, compressed and then cut into pellets or granules of an appropriate size for the target aquatic animal population. In this embodiment, the beads of the invention are dispersed throughout the aquatic animal feed, and not merely coated on the surface of the aquatic animal feed.

[00141] A person skilled in the art can readily determine the amount of aquatic animal feed additive that must be added to the aquatic animal feed in a given situation in order to constitute a therapeutically effective amount. This will depend on a range of factors including the nature of the therapeutically effective compound, the aquatic animal population, and the percentage drug loading in the beads.

Methods of Treatment

[00142] In a further aspect, this invention provides a method of treating a disease in an aquatic animal comprising administering an aquatic animal feed additive or aquatic animal feed of the invention to the aquatic animal population. Preferably, the aquatic animal is a fish.

[00143] The compositions of the invention can be used to treat a disease that an aquatic animal population is suffering. Alternatively, the compositions of the invention can be used prophylactically to prevent an aquatic animal population from developing a disease.

[00144] Many different therapeutically effective compounds can be used in the beads of the invention. In a particularly preferred embodiment, the therapeutically effective compound is an anthelmintic drug, such as praziquantel. In a particularly preferred embodiment, the aquatic animal feed additive or aquatic animal feed of the invention comprising praziquantel is administered to a fish population such that the fish population receives a dose of between 50mg/kg and 150mg/kg of praziquantel.

[00145] A range of different diseases can be treated or prevented using the compositions of the invention including parasitic diseases such as those caused by cestodes or trematodes including monogenean flukes, and blood flukes. Families of monogenean parasites that causes disease in aquatic animals include Capsalidae, Dactylogyrus vastator, Centrocestus formosanus and Digenea Heterophydae.

[00146] In a preferred embodiment, the disease is a parasitic infection caused by monogenean flukes. In a further preferred embodiment, the disease is a parasitic infection caused by Zeuxapta flukes.

[00147] The compositions of the inventions can be used to treat or prevent diseases in an aquatic animal. Preferably, the aquatic animal is any species of fish, including yellowtail kingfish or Pacific Bluefin Tuna.

[00148] In a preferred embodiment, the fish is of a species that is known to be particularly taste and/or odour sensitive. In a further preferred embodiment, the fish is of the species Seriola lalandi.

[00149] The skilled person will know the amount of the aquatic animal feed or aquatic animal feed additive to deliver to the aquatic animal population, and the dosage schedule required in order to achieve a therapeutic effect in the aquatic animal population.

Methods of Preparing the Aquatic Animal Feed Additive

[00150] In a further aspect, the aquatic animal feed additives of the invention are prepared using a method comprising the following steps:

(a) preparing a dry mixture of the therapeutically effective compound and the taste masking agent, and optionally a part of the carrier matrix and/or wetting agent;

(b) adding a solution containing a carrier matrix and optionally a wetting agent to the dry mixture to form a homogenous suspension;

(c) dropping the homogenous suspension in a liquid medium to form beads;

(d) washing the beads with water; and

(e) drying the washed beads.

[00151 ] In a particularly preferred embodiment, the dry mixture in step (a) is prepared by mixing an effective amount of a therapeutically effective compound such as praziquantel with a taste-masking agent such as garlic powder. This step can also optionally include mixing a portion of the carrier matrix (such as chitosan) and/or wetting agent.

[00152] In step (b), a solution containing a carrier matrix is added to the dry mixture to form a homogenous suspension. In a preferred embodiment, the carrier matrix is an acid soluble polymer (such as chitosan or Eudraguard^®), and the solution is made by mixing acid-soluble polymer powder with acetic acid. In a particularly preferred embodiment, chitosan powder is dissolved in 0.2M acetic acid to prepare a 1% chitosan solution. In another preferred embodiment, the carrier matrix is alginate, preferably in a 1% w/v solution. In another preferred embodiment, the carrier matrix is agar, and is added in a 2% w/v solution. In another embodiment, the carrier matrix is alginate. In another embodiment, the carrier matrix is chitosan dispersed in alginate, where chitosan is included as part of the dry mixture in step (a) and alginate solution is then added to the dry mixture, stirred and sonicated in a sonicating bath to form a homogenous suspension (stirring time may range from 30 minutes to overnight). In a further embodiment, there is also provided a wetting agent. Preferably, the wetting agent is Cremophor^® RH40 and/or Tween. Preferably, Cremophor^® RH40 is mixed together with the therapeutically effective compound and taste-masking agent (and optionally a portion of the carrier matrix) in step (a), and a solution of alginate preferably 1% w/v is added to the dry mixture.

[00153] In step (c), the homogenous suspension is dropped in a liquid medium to form beads. The appropriate liquid medium is selected based on carrier matrix used, and will be known to persons in the art. The beads may be required to sit in the liquid medium for some time in order to form properly. In a preferred embodiment, the carrier matrix comprises alginate, and the liquid medium is a calcium chloride solution. Preferably, the calcium chloride solution is at a concentration of 1% w/v. In another preferred embodiment, the carrier matrix comprises agar, and the liquid medium is a cold oil or mixture of oils, emulsifier or buffer. In a preferred embodiment, the oil is selected from one or more of garlic oil, cod liver oil and soybean oil. In a particularly preferred embodiment, the oil contains garlic oil, which can have an additional taste-masking effect.

[00154] In step (d), the beads are taken out of the liquid medium and washed with water, preferably deionised water.

[00155] In step (e), the beads are dried. The beads may be dried through any of the methods known in the art. For example, the beads may be dried through air drying, spray drying, convective drying, vacuum drying, microwave drying, lyophilisation and combinations thereof. Preferably, the beads are air dried, for example, in a fume hood. Most preferably, the beads are dried for at least 7 days.

[00156] In a preferred embodiment, the dried beads are additionally mixed with a taste-masking agent in order to coat the beads with this agent. In a particularly preferred embodiment, this taste- masking agent is garlic powder.

[00157] Once the aquatic animal feed additive has been prepared in accordance with the above method, it can be incorporated into aquatic animal feed. Preferably, the fish feed is prepared by mixing the aquatic animal feed additive prepared by the above process with a commercial fish feed, and then pelleting the mixture. The mixture may be pelleting using any machine or device suitable for that process. The commercial fish feed used in the process, and the size and shape of the pellets can be chosen based on the requirements of the target aquatic animal population. [00158] In one embodiment, the aquatic animal feed additive composition is prepared using an extruder. In a further embodiment, the extruder is subjected to the following heat and pressure parameters: >90 °C pre-conditioner for approximately 30 seconds, followed by extruder temperature of 90 °C to 120 °C at >20 bar cylinder pressure in the zone of high pressure, followed by drying at 90 °C to 110 °C for 44 minutes. The clearance between the screw and barrel within an extruder is less than 1 mm. In this embodiment, the aquatic animal feed additive composition is not substantially disrupted or degraded by these parameters. In this embodiment, the bead size is kept to a diameter of 500 pm (0.5 mm) and preferably under 1 mm.

[00159] In another embodiment, the extruder is subjected to the following heat and pressure parameters: 70 °C and pressure at 20 - 30 bar, with a extruder retention time (mixing through the extruder) of 25 - 30 seconds. In this embodiment, the aquatic animal feed additive composition is not substantially disrupted or degraded by these parameters.

EXAMPLES [00160] Further features of the present invention are more fully described in the following non limiting Examples. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad description of the invention as set out above.

Example 1 - Preparation of beads. [00161] In order to test the palatability and digestibility of fish feed additive compositions comprising praziquantel (PZQ), five different bead formulations were prepared, with the following compositions:

Table 1 - Composition of Bead Formulations

[00162] Formulations A and E (the chitosan beads) were prepared by dissolving the chitosan powder in 0.2M acetic acid to prepare a 1% chitosan solution. The praziquantel and garlic derivative was then mixed with the chitosan solution. The resulting suspension was then dropped in a bath of 2% w/v sodium triphosphate solution in order to form beads. Formulation A was allowed to complex for 3 hours, whereas formulation E was allowed to complex overnight. The beads were then air dried in a fume hood for at least 7 days. Once dried, garlic powder equating to 1% of the total bead weight was mixed with the dry beads.

[00163] Formulation B was prepared by preparing a dry mixture of the praziquantel, chitosan and garlic derivative. The 1% (w/v) alginate solution was then added to the dry mixture, and stirred to form a homogenous suspension. The suspension was then dropped into a 1% (w/v) calcium chloride solution in order to form the beads. The beads were allowed to sit in the calcium chloride solution for another 10-15 minutes and washed with deionised water. The beads were then air dried in a fume hood for at least 7 days. Once dried, garlic powder equating to 1% of the total bead weight was mixed with the dry beads. [00164] Formulation B^* was prepared by preparing a dry mixture of the praziquantel, chitosan, garlic derivative and Cremophor^® RH40. The 1% (w/v) alginate solution was then added to the dry mixture, and stirred to form a homogenous suspension. The suspension was then dropped into a 1% (w/v) calcium chloride solution in order to form the beads. The beads was then allowed to sit in the calcium chloride solution for another 10-15 minutes and washed with deionised water. The beads was then air dried in a fume hood for at least 7 days. Once dried, garlic powder equating to 1% of the total bead weight was mixed with the dry beads.

[00165] Formulation C was prepared by preparing a dry mixture of the praziquantel and garlic derivative. The 2% (w/v) agar solution (when hot and runny) was then added to the dry mixture and stirred to form a homogenous suspension. The suspension was then dropped into cold garlic oil (which was comprised of garlic oil: soybean oil: cod liver oil in a ratio of 5:3:1 ) to form the beads. The beads were then washed with deionised water and then air dried in a fume hood for at least 7 days. Once dried, garlic powder equating to 1% of the total bead weight was mixed with the dry beads.

[00166] Formulation D was prepared by preparing a dry mixture of the praziquantel, Cremophor® RFI40 and garlic derivative. The 1% (w/v) alginate solution was then added to the dry mixture and stirred to form a homogenous suspension. The suspension was then dropped into a 1 % (w/v) calcium chloride solution in order to form the beads. The beads were allowed to sit in the calcium chloride solution for another 10-15 minutes and washed with deionised water. The beads were then air dried in a fume hood for at least 7 days. Once dried, garlic powder equating to 1% of the total bead weight was mixed with the dry beads.

Example 2- Disintegration Trials.

[00167] The beads of Formulation A, B, C, D and E were tested for their disintegration capacity in 0.1 M HCI.

[00168] Beads were placed in vials containing 10ml_ 0.1 M HCI or seawater and were observed over a period of 5 hours they were stirred using a magnetic stirrer at 10Orpm.

[00169] The results of the disintegration experiment are set out in Figure 1 . The results showed that the beads of all Formulations, A, B, C, D and E, did not disintegrate in the seawater (Figure 1 A), but they did at least expand and soften in the HCI solution (Figure 1 B).

Example 3- In Vitro Dissolution Profile of Formulation B and Formulation C.

[00170] Approximately 10 mg of dry beads of Formulation B and Formulation C were separately weighed and transferred to USP dissolution baskets (708-DS model, Agilent Technologies, Mulgrave, VIC, Australia), each of which was then attached to a USP dissolution paddle shaft. The set-up ensured the beads were able to be submerged in a dissolution medium of low volume; beads were not removed during media sampling, medium could be agitated during dissolution, and beads could be quickly and collectively transferred from one medium to the next. Dissolution profiles were performed in triplicate at stirring speed of 100 rpm and ambient temperature (22-25 °C). The beads were incubated for 5 min in 500 ml. of seawater, followed by 60 min in 100 ml. of simulated fish gastric fluid (SGF, seawater adjusted to pH 2.0 ± 0.05 with HCI, with 0.1% w/v Polysorbate 80 and 0.8 mg/ml_ pepsin) and finally 120 min in 100 ml. of simulated fish intestinal fluid (SIF, consisting of PBS 137 mM NaCI, 2.7 mM KCI, 10 mM Na₂HP0₄, and 1 .8 mM KH₂P0₄, pH 7.8 ± 0.05 with 0.1% w/v Polysorbate 80 and 0.4 mg/ml_ trypsin). The respective dissolution medium was sampled (1 ml.) at 0 and 5 min (seawater), 35 and 65 min (SGF), and 125 and 185 min (SIF). Withdrawn samples were filtered (0.45 _m) and analysed for praziquantel content using the HPLC assay. Control experiments were performed using pure PZQ powder (7-13 mg per basket). However, as the basket could not be transferred from one dissolution medium to the next without loss of powder, the experiments for the PZQ powder were performed separately in the three dissolution media as follows: 5 min in 500 ml. of seawater, 60 min in 100 ml. of SGF, and 120 min in 100 ml. of SIF. Additionally, the dissolution profiles of Formulation B and Formulation C beads were determined over 3 h in seawater using the same equipment set-up.

[00171] Dissolution of PZQ was not detectable after 5 min in seawater; however, 56.7 ± 4.1% of the drug powder had dissolved after 3 h in 500 ml. of seawater (Figure 2a), which was comparable to the 53.7 ± 0.9% dissolution obtained after 1 h in 100 mL of SGF (Figure 2b). Incubation for 2 h in SIF led to the dissolution of 78.2 ± 4.8% of the drug powder (Figure 2b). There was incomplete dissolution of the PZQ powder under the simulated GIT conditions, although the drug:media ratio (7-13 mg in 100 mL) was below the solubility of PZQ in water (approximately 36 mg/100 mL).

[00172] Formulation B and Formulation C beads did not disintegrate even after 3 h incubation in 500 mL of seawater, and only 6.51 ± 0.59% and 1.36 ± 0.71% of the drug loads from the respective beads were leached into the seawater at 3 h (Figure 2a). Simulation of the bead passage from seawater into the fish GIT showed undetectable drug release after 5 min in seawater from both formulations. The Formulation B beads remained intact after a further 60 min incubation in SGF, releasing only 2.3 ± 0.4% of the drug load; however, bead disintegration was noted in the SIF accompanied by the release of 84.7 ± 2.9% of the drug load at 185 min (Figure 2b). In contrast, the beads of Formulation C remained intact not only in seawater, but also in the SGF and SIF, with low levels of drug release measured over the entire dissolution period. Cumulative percent drug release from the Formulation C beads was only 3.87 ± 0.24% at 185 min.

Example 4 - Analysis by Differential Scanning Calorimetry (DSC). [00173] Formulation B, Formulation C, blank agar, alginate and alginate-chitosan beads, and PZQ alone were analysed in a differential scanning calorimeter (Discovery DSC25 System, TA Instruments, Newcastle, DE, USA). Bead samples were analysed within 14 days of manufacture. Samples (~3 mg) were analysed in standard aluminium pans (DSC Consumables Incorporated, Austin, MN, USA) over 0 to 250°C at a heating rate of 10°C/min with empty aluminium pans as reference. DSC thermograms were analysed using the TRIOS Software (TA Instruments, New Castle, DE, USA.

[00174] DSC analysis was conducted for Formulations B and C, and the thermograms were compared with the DSC thermograms of PZQ and the corresponding blank beads in Figure 3. PZQ (3.4 mg) exhibited a sharp melting endotherm with onset at 138.9°C and peak temperature of 141.99°C. The enthalpy for melting was 99.870 J/g. PZQ melting peak for Formulation B (3.8 mg) had onset at 138.35°C and peak temperature at 141.50°C. Its enthalpy of 72.607 J/g was 72.70% that of pure PZQ, and corresponded to the drug loading of Formulation B. PZQ peak in Formulation C (3.4 mg) had onset at 138.03°C and peak temperature at 141.93°C. Its enthalpy of 71 .645 J/g was 71 .74% that of pure PZQ, which again corresponded closely to the drug loading of Formulation C. Thus, it may be concluded from the respective DSC thermograms that PZQ retained its crystalline characteristics, and did not interact with the matrix materials in Formulations B and C.

Example 5 - Fish palatability trials.

[00175] Each of the dried beads of formulations A to E were mixed with crushed commercial yellowtail kingfish fish feed, and pelleted. The feed contained the equivalent of 10 grams of pure praziquantel per kilogram of feed. Healthy yellowtail kingfish were offered a fixed ration based on their weight and water temperature of the fish feed pellets containing the formulations. The fish were fed for a maximum duration of 3 minutes in a single morning feed over a 5 day period.

[00176] Fish feed containing formulations A and E was delivered to large healthy fish with an average weight of 2000 grams and to healthy small fish with an average weight of 175 grams. Fish feed containing formulations B, C and D was delivered to healthy small fish with an average weight of 263 grams.

[00177] Healthy yellowtail kingfish fish were offered a fixed ration per tank per day in a single morning feed over a day period, calculated on the basis of their size, and the water temperature.

[00178] The average time for the fish population to consume the full ration of fish feed was measured. The average percentage of fish feed consumed was also recorded. The data was analysed with each of the 5 days as replicates. This was compared with a control group which was fed fish feed that did not contain a fish feed additive. [00179] Figure 5 presents the results for fish feed containing formulations A and E (the chitosan beads). The large fish fed the formulation E fish feed never ate a full ration of feed during the 5- day trial. As shown in Figure 5A, two-way ANOVA revealed there to be no effect of fish size (P = 0.77) or diet (P = 0.07) on ingestion. Fish fed formulation E ate on average 75% of their offered ration compared to fish fed formulation A, which only ate on average 68% of the ration. Fish in the unmedicated control treatment consumed 90% of the ration (pooled across small and large fish). These results were contrary to the hypothesis that larger fish are more susceptible to the taste and/or smell of praziquantel. The small control fish ate their entire ration of unmedicated feed, but only ate 60% and 70% of formulation A and formulation E treatment rations, respectively. As shown in Figure 5B, time taken to consume a full ration was significantly affected by treatment (P < 0.0001) but not fish size (P = 0.27). On those occasions when the fish ate the entire medicated ration, the time to do so was ca. 3 minutes. Fish offered the unmedicated control treatment consumed their ration in ca. 1 .5 minutes.

[00180] Figure 3 presents the results for fish feed containing formulations B, C and D. As there was no evidence of larger fish being more susceptible to the taste/small of praziquantel in the formulation, only small fish (260 gram) were used for assessing the palatability of formulations B, C and D. Fish ate ³99% of all treatment diets (Figure 5A), with no difference between treatments (P = 0.28). Whilst the fish ate their full ration at each feeding, the time taken to consume the ration was significantly different between treatments (P= 0.02). There was no difference in the amount of time required for fish fed diets containing Formulation B (1.15 ± 0.14 minutes) and Formulation C (1.16 ± 0.14 minutes) to eat their ration compared with the control (0.64 ± 0.07 minutes), but those fed the diets containing Formulation D took significantly longer than the control to consume the ration (1.42 ± 0.04 minutes) (Figure 5B). This presents acceptable palatability for each of formulations B, C and D. On the basis that both a greater percentage of feed containing formulations B, C and D was consumed and that these feeds were also consumed more quickly in comparison to feed containing formulations A and E, formulations B, C and D were more palatable to the fish population.

Example 6 - Digestibility trials.

[00181] In respect of formulations A and E, after the final feed on day 5, fish were dissected to determine the digestibility of the beads at approximately 3 hours post feeding.

[00182] There was no evidence of beads found in the digestive tract in either small or large fish fed the Formulation A treatment. However, undigested and partly digested beads were found throughout the entire length of the tract of the Formulation E treatment in the large fish, as can be seen in Figure 6. [00183] In respect of formulations B, C and D, prior to feeding on Day 5, one fish per treatment was dissected to determine if there were beads remaining in the digestive tract after the previous days feeding. Apart from a single Formulation D bead in the stomach, there was no digesta or evidence of beads in the digestive tracts of fish from all treatments. [00184] On day 5, all fish were fed their respective treatment diets and two fish per treatment were dissected 3 and 6 hours post-feeding. The results are presented in Figure 7.

[00185] By 3 hours post feeding, the feed had moved through the entire length of the fish gut. There were whole beads in the stomach but no evidence of the beads in any section of the tract for all treatments. Similar findings were made 6 hours post feeding. [00186] These results indicate that Formulations A, B, C and D were digested by the fish, as the beads did not pass through the gut intact.

Example 7 - Drug Loading Determination.

[00187] The drug loading of the beads of each of formulations A to E was determined by dissolving the beads in 0.4M NaCI solution, and then adding methanol to make a 50% methanol solution. FIPLC analysis was then performed on the solution to determine the drug loading percentage. The results are presented in the table below:

Table 2

Example 8 - Drug Loading Stability. [00188] The drug loading capacity (or more specifically residual intact drug content) of the beads of formulations B, C and D was determined 18 months after the beads were initially prepared according to the method described in Example 7. The results are set out in Table 3 below: Table 3

Example 9 - Palatability of fresh beads compared with stored beads.

[00189] Fish feed comprising formulations B and C were tested to compare the palatability of freshly prepared beads with beads that were approximately 4 months old. Healthy yellowtail kingfish fish were offered a fixed ration of fish feed containing Formulations B and C per tank per day in a single morning feed over a 5 day period. The percentage of feed consumed per ration was measured, as well as the average time to consume the ration in the last 2 days. The results are presented in Figure 8. The data indicates that there were no significant difference in the palatability of the 4 month old beads and the fresh beads.

Example 10- Second Palatability Trial.

[00190] A second palatability trial was conducted to compare formulation B and C, with fish feed incorporating pure praziquantel (which had not been incorporated within bead, or with garlic extract). Beads of formulation B or C were mixed with ground 3mm commercial yellowtail kingfish feed, and were reconstituted into pellets using a Dolly pasta maker. Pellets were also made with pure praziquantel powder using the same process. All pellets contained the equivalent of 10 grams/kg of pure praziquantel. The control diet was also made using the same process, which contained no praziquantel. Yellowtail kingfish fish with an average weight of 1600 grams, infected with Zeuxapta flukes were offered a fixed ration of 155 grams per tank per day in a single morning feed over a 6 day period.

[00191] If the fish consumed the entire feed ration within 3 minutes, the time was recorded. The weight of any dry, uneaten feed was also recorded. Any pellets in the tank uneaten or regurgitated were also collected, weighed and converted to the dry weight. Both weights were combined to measure the percentage of feed consumed per ration, as well as the actual praziquantel dose received by the fish (in mg/kg) calculated on the basis of the amount of feed consumed. The results are presented in Figure 9.

[00192] The data shows that food intake was significantly lower in fish fed the pure praziquantel fish feed (17 ± 4%), compared to plain fish feed (with no additives). However, there was no significant difference between the food intake of fish fed the plain fish feed and the fish feed incorporating formulations B or C (Figure 9A). Fish fed the non-medicated control diet consumed 79 ± 6% of the ration and fish fed the Formulation B and C treatments consumed ca. 62%. None of the diets were consumed at 100%, and this may be because these fish were still acclimating to the tank conditions after having been moved from the seacage. Figure 9B shows feed intake by day. This graph demonstrates that all tanks fed poorly on the second day and that feed intake subsequently increased in all treatments over the following days, with the exception of the pure praziquantel which remained low. The fact that the intake of fish feed containing formulations B and C treatment diets increased over time, similarly to the control diet, is further evidence that the diets are highly palatable, as intake in pure PZQ diets typically tends to decrease over time.

[00193] Figure 9C presents the average daily dose of praziquantel ingested by the fish receiving the different treatments in mg/kg. The actual praziquantel dose ingested by fish fed the pure praziquantel fish feed composition was 21 mg/kg. On the other hand, the fish populations that were fed the fish feed containing formulations B or C ingested approximately 75mg/kg of praziquantel (P < 0.001). The results therefore demonstrate that formulations B and C clearly allow the oral delivery of greatly increased quantities of praziquantel to the fish. It is generally considered that fish must receive approximately 50 mg/kg for three consecutive days to eliminate Zeuxapta. This dose was easily achieved in the fish feed containing formulations B and C but not in the pure praziquantel treatment.

Example 11 - Treatment of fish populations suffering from fluke.

[00194] Following the palatability trial, the fish population from example 10 was bathed using praziquantel to assess fluke infection. The fluke count in each treatment group was measured.

[00195] The results are set out in Figure 10. Fish fed the unmedicated control diet had an average of 55 ± 13 Zeuxapta flukes per fish at the end of the trial. There was a significant effect of diet on the percentage reduction in fluke numbers in the three medicated treatments relative to the control (P < 0.001). Fish fed the formulation B and formulation C treatments had reductions of 93 ± 2% and 94 ± 3%, respectively, both significantly higher than the pure praziquantel control, where a 73 ± 4% reduction was observed. These data demonstrate that formulations B and C are both palatable, digestible and efficacious against Zeuxapta infections.

Example 12- Methods to prepare beads of different sizes. [00196] Beads of different sizes (diameters) in the range 0.4 to 2.5 mm were prepared using methods described in the following paragraph. The bead sizes were regulated by choosing the appropriate chitosan particle size, extrusion needle and stirring speed. The chitosan size ranges were: (1) 355-500pm; (2) 212-355 pm; (3) 150-355 pm; and (4) <150 pm. [00197] Firstly, a mixture of the praziquantel, chitosan and garlic derivative (with and without

Cremophor^® RH40) was prepared. The 1% (w/v) alginate solution was then added to the mixture, stirred and sonicated to form a homogenous suspension. Stirring of the suspension ranged from 30 minutes to overnight. The suspension was then dropped into a 1% (w/v) calcium chloride solution in order to form the beads. Syringe needles of different sizes were used to drop the suspension, e.g. 18G or 21 G needles were used to form smaller beads with the smaller chitosan particles. The beads were allowed to sit in the calcium chloride solution for another 10-15 minutes and washed with deionised water. The beads were then air dried in a fume hood for at least 7 days. Once dried, garlic powder equating to 1% of the total bead weight was mixed with the dry beads.

Claims

1 . An aquatic animal feed additive composition capable of delivering a therapeutically effective amount of a therapeutic to an aquatic animal, said additive comprising a plurality of therapeutic beads, wherein each bead comprises:

(a) at least 45% w/w therapeutically effective compound;

(b) between 1% and 10% w/w taste-masking agent;

(c) between 2% and 35% w/w carrier matrix; and wherein, the carrier matrix is:

(a) ingestible by the aquatic animal; and

(b) is substantially stable for at least 60 minutes at room temperature in an aqueous environment in which the aquatic animal feed additive is used; and wherein, the taste-masking agent alone or in combination with the carrier matrix masks the taste of the therapeutically effective compound.

2. The composition of claim 1 , wherein the carrier matrix is digestible by an aquatic animal.

3. The composition of claims 1 or 2, wherein the carrier matrix comprises a marine based hydrogel.

4. The composition of claim 3, wherein the carrier matrix is selected from a combination of alginate and an acid-soluble polymer; alginate; or agar.

5. The composition of any of the above claims, wherein the composition additionally comprises a wetting agent.

6. The composition of claim 5, wherein the wetting agent is Cremophor^® RH40 and/or Tween.

7. The composition of any of the above claims, wherein the taste masking agent is a flavouring agent.

8. The composition of claim 7, wherein the taste-masking agent is garlic or a derivative thereof.

9. The composition of claim 8, wherein the taste-masking agent is garlic.

10. The composition of any of the above claims, wherein the therapeutically effective compound is an anthelmintic compound.

11. The composition of claim 10, wherein the anthelmintic compound is praziquantel.

12. The composition of any of the above claims, wherein the therapeutically effective compound is present in an amount of at least about 70% w/w of each bead.

13. The composition of any of the above claims, wherein the carrier matrix comprises about 10 - 25% w/w of each bead.

14. The composition of any of the above claims, wherein the taste-masking agent comprises 2- 8% w/w of each bead.

15. The composition of any of the above claims, wherein the wetting agent comprises 5-10% w/w of each bead.

16. The composition of any of the above claims 1 to 15 wherein:

(a) the therapeutically effective compound comprises praziquantel which is present in an amount of about 70-90% w/w of each bead;

(b) the taste masking agent comprises a garlic derivative which is present in an amount of about 5% w/w of each bead; and

(c) the carrier matrix comprises a combination of an acid soluble polymer and alginate, wherein the acid soluble polymer is present in an amount of 8-10% w/w and the alginate is present in an amount of 8-11% w/w of each bead.

17. The composition of any of the above claims 1 to 15 wherein :

(a) the therapeutically effective compound comprises praziquantel which is present in an amount of about 70-90% w/w of each bead;

(b) the taste masking agent comprises a garlic derivative which is present in an amount of about 5% w/w of each bead;

(c) the carrier matrix comprises alginate, wherein the alginate is present in an amount of 5-11 % w/w of each bead; and

(d) the wetting agent is Cremophor^® RH40, wherein the Cremophor^® RH40RH40 is present in an amount of 5-10% w/w of each bead.

18. The composition of any of the above claims 1 to 15 wherein :

(a) the therapeutically effective compound comprises praziquantel which is present in an amount of about 70-90% w/w of each bead;

(b) the taste masking agent comprises a garlic derivative which is present in an amount of about 2-5 % w/w of each bead; and

(c) the carrier matrix comprises a combination of agar, which is present in an amount of 18-20% w/w of each bead.

19. The composition of any of the above claims 1 to 15 wherein:

(a) the therapeutically effective compound comprises praziquantel which is present in an amount of about 70-90% w/w of each bead; (b) the taste masking agent comprises a garlic derivative which is present in an amount of about 5% w/w of each bead;

(c) the carrier matrix comprises a combination of an acid soluble polymer and alginate, wherein the acid soluble polymer is present in an amount of 8-10% w/w and the alginate is present in an amount of 8-11% w/w of each bead; and

(d) the wetting agent is Cremophor^®RH40 or Tween, wherein the Cremophor^® RH40RH40 or Tween is present in an amount of 5-10% w/w of each bead.

20. The composition of any of the above claims, wherein the beads have a diameter selected from the group consisting of: 5000 pm, <5000 pm, 1000 pm, <1000 pm; 500 pm; <500 pm; 355-500pm; 212-355 pm; 150-355 pm; 150 pm; <150 pm; 100 pm and <100 pm.

21. The composition of any of the above claims, wherein the beads are further covered with a taste-masking agent.

22. A fish feed composition comprising the composition of any of the above claims.

23. A method of treating or preventing a disease in a fish comprising administering to a fish a composition of any of the above claims 1 to 22.

24. The method of claim 23, wherein the disease is a parasitic infection.

25. The method of claim 24, wherein the therapeutically effective compound is praziquantel.

26. The method of any of claims 23 to 25, wherein the fish population receives a dose of the therapeutically effective compound of about 50 mg/kg to 150 mg/kg.

27. A method of preparing an aquatic animal feed additive composition of any of claims 1 to 21, comprising the steps of:

(a) preparing a dry mixture of the therapeutically effective compound and the taste-masking agent and optionally a portion of the carrier matrix and/or wetting agent;

(b) adding a solution containing a carrier matrix and/or wetting agent to the dry mixture to form a homogenous suspension;

(c) dropping the homogenous suspension in a liquid medium to form beads;

(d) washing the beads with water; and

(e) drying the washed beads.

28. The method of claim 27, wherein the dried beads are additionally mixed with a taste-masking agent.

29. A method of preparing a fish feed composition of claim 22, comprising the steps of:

(a) mixing the aquatic animal feed additive prepared by the method in claims 27 or 28 with crushed fish feed to form a mixture; and

(b) forming pellets from the mixture in (a).