Fish feed additives

Abstract

The present invention relates to the field of oral delivery of therapeutic compounds to fish via fish feed additives, including aquatic animal feed additive compositions and methods for treating or preventing diseases in aquatic animals. In a first aspect, the present invention provides an aquatic animal feed additive composition capable of delivering a therapeutically effective amount of a therapeutic agent to an aquatic animal, the additive comprising a plurality of therapeutic beads, each bead comprising (a) at least 45% w / w of a therapeutically active compound, (b) 1%-10% w / w of a flavoring agent, and (c) 2%-35% w / w of a carrier matrix, the carrier matrix being (a) ingestible by the aquatic animal, and (b) substantially stable at room temperature for at least 60 minutes in an aqueous environment in which the aquatic animal feed additive is used, and the flavoring agent, alone or in combination with the carrier matrix, masks the taste of the therapeutically active compound.

Description

[Technical field]

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

[0002] Commercial fish farming is now a major industry in many countries around the world. However, commercially farmed fish can suffer from a variety of different diseases, the treatment of which can pose significant challenges and costs to the industry. For example, commercially farmed fish populations are often affected by parasitic infections that can be difficult to treat. Examples of such infections include polyopisthocotylean and monopisthocotylean monogenean flukes, which are relatively common conditions in commercially farmed fish populations, such as yellowtail amberjack (Seriola lalandi).

[0003] Currently, commercially used methods to treat parasites in fish populations include bathing, where fish are immersed in a solution containing a drug, or oral administration. For example, the effective anthelmintic drug praziquantel (2-(cyclohexylcarbonyl)-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]isoquinolin-4-one) is used to treat S. lalandi, which is infected with polyopisthocotylean and monopisthocotylean monogenean trematodes. However, praziquantel has a bitter taste and is poorly palatable, so fish are bathed in praziquantel rather than administered orally. Alternatively, fish are bathed in hydrogen peroxide.

[0004] Bathing is laborious, time-consuming, and weather-dependent, and long-term exposure can have adverse effects on fish, causing reduced growth rates and even large-scale mortality (Gaikowski, MP et al., 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 accounts for up to 22% of the production cost of sea-caged Seriola species (Ernst, I. et al., 2002, Monogenean parasites in sea-cage aquaculture Austasia Aquaculture, 16, 46-48).

[0005] Constant handling, overcrowding, loss of feeding time and reduced dissolved oxygen during bathing treatments can cause massive mortality as well as loss of appetite and loss of growth (Grant, AN, 2002, Medicines for sea lice. Pest Management Science, 58, 521-527).

[0006] On the other hand, oral administration of drugs to fish has advantages over traditional bathing treatments. For example, in-feed drugs have a wider safety margin and do not require increased crowding or handling of fish. Treatment efficiency is also increased, as all cages on a farm can be treated quickly (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.), reducing the possibility of infection from nearby untreated fish. Feeding medicated diets also allows fish to maintain their natural feeding regime, reducing stress (Conte, FS, 2004, Stress and the welfare of cultured fish. Applied Animal Behaviour Science, 86, 205-223.). The environmental impact of oral treatments is less than that of bathing treatments because once the bathing process is completed, the chemicals are released into the surrounding environment and may affect non-target organisms (Grant, AN, 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] However, one major obstacle to administering drugs orally to fish is that fish are often sensitive to the taste and smell of drugs, therefore including drugs in fish feed often reduces the palatability of the feed, which in turn means reduced intake, which in turn means reduced doses of drugs administered, and ultimately potentially no effect of the drug (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] To address the problem of oral administration of drugs in fish populations, several methods have been tested. WO 2009 / 023013 describes a multiparticulate feed additive with multiple coatings applied to a core containing drugs (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, which is a Eudraguard® polymer. However, this is a complex structure from a manufacturing point of view, since the beads comprise several separate coatings.

[0009] WO 1989 / 12442 describes a pharmaceutical dosage form for administering drugs to fish, comprising an outer layer of animal or plant material that is substantially impermeable to water surrounding an inner chamber filled with an active agent. AU2008100441 describes a medicated fish feed containing therapeutic amounts of microencapsulated anthelmintics (including praziquantel) and attractants such as amino acids, nucleotides and natural and synthetic extracts from various aquatic organisms that trigger olfactory and gustatory receptors in fish and induce feeding behavior in the target fish species. None of these methods appear to be in commercial use.

[0010] Partridge et al. (2014) [Partridge, GJ, Michael, RJ 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.] evaluated the palatability of fish feeds that were surface coated with praziquantel powder or microencapsulated praziquantel, as well as fish feeds in which praziquantel powder or microencapsulated praziquantel was incorporated into the pellet mash prior to extrusion. Fish feeds containing microencapsulated praziquantel were more palatable than fish feeds containing praziquantel powder. However, even with microencapsulation, the palatability of fish feeds 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, GJ, Rao, S., Woolley, L., Pilmer, L., Lymbery, AJ and Prestidge, CA, 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.] tested the effect of incorporating praziquantel into solid lipid nanoparticles, which were then coated onto fish feed pellets, but found that incorporating praziquantel into fish feed in this way did not improve either the bioavailability or the palatability of praziquantel. Thus, there is a need for alternative compositions that improve the palatability of therapeutically active compounds for fish, including those with particularly pungent tastes or odors such as praziquantel, and thus enable oral delivery. Furthermore, any efforts to improve the palatability of drugs such as praziquantel to fish should not compromise the bioavailability of the drug in vivo. The foregoing discussion of the background art is intended only to facilitate an understanding of the present invention. The discussion is not an admission or acknowledgement that any of the material referred to was or was part of the common general knowledge at the priority date of the application. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2009 / 023013 [Patent Document 2] International Publication No. 1989 / 12442 [Patent Document 3] Australian Patent Application Publication No. 2008100441 [Non-patent literature]

[0012] [Non-Patent Document 1] Gaikowski, MP et al., 1999, Acute toxicity of hydrogen peroxide treatments to selected life stages of cold, cool, and warm water fish Aquaculture, 178, 191-207 [Non-Patent Document 2] Ernst, I. et al., 2002, Monogenean parasites in sea-cage aquaculture Austasia Aquaculture, 16, 46-48 [Non-Patent Document 3] Grant,AN,2002,Medicines for sea lice.Pest Management Science,58,521-527 [Non-Patent Document 4] 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. [Non-Patent Document 5] Conte, FS, 2004, Stress and the welfare of cultured fish. Applied Animal Behavior Science, 86, 205-223. [Non-Patent Document 6] 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. [Non-Patent Document 7] Partridge et al., (2014) [Partridge, GJ, Michael, RJand 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.] [Non-Patent Document 8] Partridge et al. (2019) C:Toxicology&Pharmacology,218,14-20.] Summary of the Invention

[0013] In a first aspect, the present invention provides an aquatic animal feed additive composition capable of delivering a therapeutically effective amount of a therapeutic agent to an aquatic animal, the additive comprising a plurality of therapeutic beads, each bead comprising: (a) at least 45% w / w of a therapeutically active compound; (b) 1% to 10% w / w of a flavoring agent; (c) 2% to 35% w / w of a carrier matrix; Including, The carrier matrix is (a) be ingestible by aquatic animals; (b) is substantially stable for at least 60 minutes at room temperature in the aqueous environment in which the aquatic animal feed additive is used; An aquatic animal feed additive composition is provided in which a flavoring agent, alone or in combination with a carrier matrix, masks the taste of a therapeutically active compound.

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

[0015] In one embodiment of the invention, the therapeutically active compound is substantially stable in the carrier matrix if the amount of leaching of the therapeutically active compound from the carrier matrix is ​​less than 20% w / w over a period of 60 minutes at room temperature in an aqueous environment, where the aqueous environment is the environment in which the aquatic animal feed additive is used (such as water, more preferably seawater).

[0016] In another embodiment of the invention, the additive is generally durable in the aqueous environment in which the aquatic animal lives, i.e., the aqueous environment in which it is treated. For example, a carrier matrix is ​​generally durable if it degrades 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 at room temperature.

[0017] In yet another embodiment of the invention, the additive is preferably at least partially degraded in the digestive tract of an aquatic animal treated with the feed additive. Preferably, the additive is 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) degraded in the digestive tract of the aquatic animal.

[0018] If the additive or carrier matrix is ​​poorly soluble in the digestive tract (such as the stomach or intestinal tract) of an animal, the additive may also include a wetting agent or surfactant polymer or pharmaceutical disintegrant to aid in disintegration of the additive. The wetting agent or surfactant may include, for example, but is not limited to, anionic or cationic surfactants or non-ionic wetting agents. In a particular embodiment of the present invention, the wetting agent is Cremophor® RH40 or Tween®. The disintegrant may also be starch and modified starch, cellulose, and the like.

[0019] In alternative embodiments, the additive or carrier matrix may include a wetting agent or surfactant polymer or pharmaceutical disintegrant that may or may not degrade in the animal's digestive tract, which aids in the manufacture of the additive or promotes wetting of the additive in an aqueous environment. Wetting agents or surfactants suitable for this purpose include, for example, but are not limited to, anionic or cationic surfactants or non-ionic wetting agents. Disintegrants may also be starches and modified starches, celluloses, and the like. In a particular form of the invention, the wetting agent is Cremophor® RH40 or Tween®.

[0020] In one embodiment of the invention, the therapeutic agent 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 ​​prepared at least in part from a blend of alginate and an acid soluble polymer (eg, chitosan and / or Eudraguard®) or alginate; or agar.

[0022] In a preferred embodiment, the flavouring agent is garlic powder or a garlic derivative.

[0023] It will be appreciated by those skilled in the art that a range of therapeutics suitable for aqueous environments can be delivered with the present invention, but 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 cartilaginous fish, and thus provides a model therapeutic.

[0024] In a preferred form of the invention, the aquatic animal feed additive contains an appropriate amount of therapeutic agent to achieve therapeutic efficacy. The amount of therapeutic agent depends, among other things, on the nature of the pathogen, the size of the aquatic animal, and (among other things) the temperature of the water. When praziquantel is administered to blood feeding gill flukes, the dosage rate is about 50 mg / kg. When praziquantel is administered to mucus feeding gill flukes, the dosage rate is about 150 mg / kg. The dietary inclusion levels required to achieve these doses are easily ascertainable by those skilled in the art depending on the water temperature (fish do not eat as much in cold water) and the size of the fish. Thus, the larger the fish, the less they eat (on a % BW basis), so a higher dietary inclusion rate of drug (g PZQ / kg diet) is required to achieve the same dose (mg PZQ / kg body weight) as the fish grow.

[0025] In a particularly preferred form of the invention, taking into account the average for all fish sizes and all water temperatures, effective dietary inclusion levels can be achieved at dietary inclusion levels of 10 g PZQ / kg to 20 g PZQ / kg food. In many respects the higher the dietary inclusion level the better.

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

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

[0028] In some embodiments, the composition comprises: (a) the therapeutically active compound comprises praziquantel present in an amount of about 70-90% w / w of each bead; (b) the flavoring agent comprises a garlic derivative 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 an alginate, the acid soluble polymer being present in an amount of 8-10% w / w of each bead and the alginate being present in an amount of 8-11% w / w of each bead.

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

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

[0031] In some embodiments, the composition comprises: (a) the therapeutically active compound comprises praziquantel present in an amount of about 70-90% w / w of each bead; (b) the flavoring agent comprises a garlic derivative 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 an alginate, the acid soluble polymer being present in an amount of 8-10% w / w of each bead and the alginate being present in an amount of 8-11% w / w of each bead; (d) the wetting agent is Cremophor® RH40 or Tween®, the RH40 or Tween® being present in an amount of 5-10% w / w of each bead;

[0032] In some embodiments, the beads are further coated with a flavoring agent.

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

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

[0035] In a fourth aspect, the present invention provides a method for preparing the aquatic animal feed additive composition of the present invention, comprising the steps of: (a) preparing a dry mix of a therapeutically active compound and a flavoring agent; (b) adding a solution containing a carrier matrix to the dry mixture to form a uniform suspension; (c) dropping the homogenous suspension into a liquid medium to form beads; (d) washing the beads with water; (e) drying the washed beads; The present invention provides a method comprising:

[0036] A dry mix is ​​dry because no water has been added. A dry mix is ​​substantially or completely free of water.

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

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

[0039] [Figure 1A] FIG. 1A shows the level of disintegration of beads containing formulations A through E in seawater after 5 hours.

[0040] [Figure 1B] FIG. 1B shows the disintegration levels of beads containing formulations A through E in 0.1 M HCl after 5 hours.

[0041] [Figure 2A] FIG. 2A shows the cumulative percent dissolution of praziquantel (PZQ) from pure drug powder, formulation B and formulation C after incubation in seawater for 180 minutes.

[0042] [Figure 2B] Figure 2B shows the PZQ release profile of formulation B and formulation C beads incubated successively in different media: SW = seawater, SGF = simulated gastric fluid, SIF = simulated intestinal fluid. The table shows the cumulative PZQ release from pure drug powder under specific dissolution conditions. Data represent the mean ± SD (n = 3).

[0043] [Figure 3A] FIG. 3A shows the DSC thermogram of Formulation B compared to that of the PZQ powder and the corresponding blank beads.

[0044] [Figure 3B] FIG. 3B shows the DSC thermogram of Formulation C compared to that of the PZQ powder and the corresponding blank beads.

[0045] [Figure 4A] FIG. 4A shows the percentage of the total provided supply of fish feed containing Formulations A or E consumed by 175 gram (small) and 2000 gram (large) kingfish.

[0046] [Figure 4B] FIG. 4B shows the time it takes to consume the entire offered supply of fish feed containing Formulation A or E consumed by 175 gram (small) and 2000 gram (large) kingfish.

[0047] [Figure 5A] FIG. 5A shows the percentage of the total provided feed of fish feed containing Formulations B, C or D consumed by a 260 gram kingfish.

[0048] [Figure 5B] FIG. 5B shows the time it takes to consume the entire provided supply of fish feed containing Formulations B, C or D consumed by a 260 gram kingfish.

[0049] [Figure 6] FIG. 6 shows the dissected gut of fish feed containing Formulation E fed to large kingfish showing undigested beads in a) midgut and b) hindgut.

[0050] [Figure 7] FIG. 7 shows the dissected digestive tract of fish fed fish feed containing formulations B, C or D 3 hours after feeding.

[0051] [Figure 8A]FIG. 8A shows the percentage of the total feeding of 4-month old fish food containing Formulations B and C compared to fresh fish food consumed by the fish.

[0052] [Figure 8B] FIG. 8B shows the average time it takes to consume an entire supply of 4-month-old fish food containing pure praziquantel, Formulation B or Formulation C compared to fresh fish food.

[0053] [Figure 9A] FIG. 9A shows the percentage of the total provided supply of fish feed containing pure praziquantel, Formulation B or Formulation C consumed by a 1600 gram kingfish.

[0054] [Figure 9B] FIG. 9B shows the percentage of the total provided supply of fish feed containing praziquantel, Formulation B or Formulation C consumed by 1600 gram kingfish over the 6 day experimental period.

[0055] [Figure 9C] FIG. 9C shows the average daily dose of praziquantel received by fish fed fish feed containing pure praziquantel or formulation B or formulation C.

[0056] [Figure 10] FIG. 10 shows the percentage reduction of Zeuxapta trematodes in fish fed fish feed containing pure praziquantel, formulation B or formulation C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description of the Invention The present invention is based on the discovery that the palatability of therapeutically active compounds that are otherwise difficult to administer orally to fish can be improved by encapsulating a flavoring agent along with the therapeutically active compound in a carrier matrix that is ingestible by fish and that does not substantially dissolve or degrade in fresh or salt water.

[0058] General Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described.The present invention includes all such variations and modifications.The present invention also includes all of the steps, features, preparations and compounds mentioned or shown in this specification, individually or collectively, and any and all combinations of steps or features or any two or more.

[0059] Each document, reference, patent application or patent cited herein is expressly incorporated herein in its entirety by reference, meaning that it should be read and considered by the reader as part of this document. It is for the sake of brevity only that documents, references, patent applications or patents cited herein are not repeated herein.

[0060] Any manufacturer's instructions, descriptions, product specifications, and product sheets for any product described in this specification, or in any document incorporated by reference herein, are incorporated by reference herein and may be used in the practice of this invention.

[0061] The scope of the present invention is not limited by any of the specific embodiments described herein. These embodiments are for illustrative purposes 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 ranges of values ​​(e.g., size, percentage, concentration, etc.). A range of values ​​is understood to include all values ​​within the range, including the values ​​defining the range and adjacent values ​​in the range that give the same or substantially the same result as the values ​​immediately adjacent to the values ​​defining the boundaries of the range. Thus, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. Thus, "about 80%" means "about 80%" and also means "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" are understood to mean the inclusion of a recited integer or group of integers but not the exclusion of any other integer or group of integers. Also, in this disclosure, particularly in the claims and / or paragraphs, it is noted that terms such as "comprises," "comprised," "comprising," and the like can have the meaning ascribed to them in U.S. patent law. For example, they can mean "includes," "included," "including," and the like. Terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, for example, they allow for elements not expressly recited but exclude elements found in the prior art or that affect the basic or novel characteristics 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 limiting of the invention as claimed. In this application, the use of the singular includes the plural unless otherwise stated. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limiting. Also, terms such as "element" or "component" encompass both elements and components that include one unit and elements and components that include two or more subunits unless otherwise stated. Also, the use of the term "moiety" can include a portion of a moiety or the entire moiety.

[0065] The term "fish feed" means any material, such as a plant, animal, or other organic material intended for consumption by fish. Fish feed is used to provide nutrients to captured or farmed fish and typically contains macronutrients, trace elements, and vitamins. The source of nutrients can be fishmeal and other marine sources, vegetable proteins, and binders such as wheat. Fish feed is generally provided in solid forms such as granules, pellets, or flakes.

[0066] The term "aquatic animals" includes fish.

[0067] The term "aquatic animal feed additive" refers to a composition that can be added to an aquatic animal feed, preferably a fish feed. The aquatic animal feed additive can contain flavorings, including drugs or other biologically active agents, flavorings, carriers, and other ingredients that are desirable for the aquatic animal population to ingest. The aquatic animal feed additive can be incorporated into the aquatic animal feed granules or pellets during the manufacture of the aquatic animal feed. Alternatively, they can be mixed with the manufactured aquatic animal feed granules or pellets before feeding, or else coated on the surface of the aquatic animal feed granules or pellets.

[0068] The term "beads" includes particles, granules, and pellets. Beads may be of any shape or size, including spherical or flat.

[0069] The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount or dose of a 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 terms "treat" and "treatment" include therapeutic treatments, prophylactic treatments, and applications in which a subject reduces the risk of developing a disorder or other risk factors. Treatment does not require a complete cure of the disorder, but encompasses embodiments in which symptoms or underlying risk factors are alleviated.

[0071] The term "palatable" means that the taste and / or smell of a food or additive is sufficiently acceptable to a subject to be consumed by the subject. A food or additive does not need to be completely consumed in a given period of time (i.e., 100% consumption) to be considered palatable by a subject. A feed or additive containing a therapeutically effective compound is considered "palatable" if a subject finds it acceptable to consume in an amount sufficient 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 the 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 with the amount of non-medicated aquatic animal feed or aquatic animal feed additive consumed by the aquatic animal population.

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

[0073] Feed additives for aquatic animals In a first aspect, the present invention provides an aquatic animal feed additive capable of delivering a therapeutically effective amount of a therapeutic agent to an aquatic animal, the additive comprising a plurality of therapeutic beads, each bead comprising: (a) at least 45% w / w of a therapeutically active compound; (b) 1% to 10% w / w of a flavoring agent; (c) 2% to 35% w / w of a carrier matrix; Including, The carrier matrix is (a) be ingestible by aquatic animals; (b) is substantially stable for at least 60 minutes at room temperature in the aqueous environment in which the aquatic animal feed additive is used; To provide an aquatic animal feed additive in which a flavoring agent, alone or in combination with a carrier matrix, masks the taste of a therapeutically active compound.

[0074] Preferably, the carrier matrix is ​​digestible by aquatic animals.

[0075] The present invention provides a granular aquatic animal feed additive. The beads of the present invention can be of any shape or size that can be ingested by the target aquatic animal population and incorporated into the aquatic animal feed.

[0076] In some embodiments, the beads are between about 0.1 millimeters and 5 millimeters 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 millimeters in diameter.

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

[0078] Each bead contains a therapeutically active compound, a flavoring agent, and a carrier matrix. The relative amounts of each of these components in the bead will depend on the nature of the therapeutically active compound, the flavoring agent, and the carrier matrix.

[0079] Preferably, the amount of therapeutically effective compound present in each bead is at least 45% w / w, although one of skill in the art will appreciate that the final amount will depend on the number of beads in the therapeutic agent and additive delivered by each bead. Preferably, the amount of therapeutically effective compound present in each bead is 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 flavoring agent is from 1% to 10% w / w, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 percent (w / w). When multiple flavoring agents are present, each may be present in an amount of from 1% to 10% w / w, for example, 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, for example 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 active compound, flavoring agent, and carrier matrix is ​​preferably distributed throughout the beads of the invention, rather than being coated in a separate layer. Taken together, the percent (w / w) of each of the therapeutically active compound, flavoring agent, and carrier matrix is ​​between 80 and 100 percent (w / w), e.g., 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 one embodiment of the present 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 60 minutes in an aqueous environment at room temperature, where the aqueous environment is the environment in which the aquatic animal feed additive is used (such as water, more preferably seawater). 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 60 minutes in an aqueous environment at room temperature. More preferably, the amount of leaching is less than 5% w / w over 60 minutes in an aqueous environment at room temperature.

[0084] The chemical stability of an excipient formulation depends on the therapeutic leaching of the active ingredient in the formulation. In general, the stability analysis of the formulation may be performed under accelerated temperature conditions, such as in an oven at a temperature higher than room temperature. The kinetic methods used in the accelerated stability analysis do not need to study the degradation mechanisms in detail to be able to predict stability, but should be based on sound scientific principles and comply with regulatory requirements.

[0085] In another embodiment of the present invention, the additive is generally durable in the aqueous environment in which the aquatic animal lives, i.e., the aqueous environment in which it is treated. For example, the carrier matrix may be 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, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0. 7, 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 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 decomposition is generally durable.

[0086] In yet another embodiment of the invention, the additive is preferably at least partially degraded in the digestive tract of an aquatic animal treated with the feed additive. Preferably, the additive is 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) degraded in the digestive tract of the aquatic animal.

[0087] If the additive or carrier matrix is ​​poorly soluble in the digestive tract (such as the stomach or intestinal tract) of an animal, the additive may also include a wetting agent or surfactant polymer or pharmaceutical disintegrant to aid in disintegration of the additive. The wetting agent or surfactant may include, for example, but is not limited to, anionic or cationic surfactants or non-ionic wetting agents. In a particular embodiment of the present invention, the wetting agent is Cremophor® RH40 or Tween®. The disintegrant may also be starch and modified starch, cellulose, and the like.

[0088] In alternative embodiments, the additive or carrier matrix may include a wetting agent or surfactant polymer or pharmaceutical disintegrant that may or may not degrade in the animal's digestive tract, which aids in the manufacture of the additive or promotes wetting of the additive in an aqueous environment.

[0089] If the therapeutic agent is not well wetted into the carrier matrix (e.g., aqueous sodium alginate solution) during bead manufacture, a wetting agent can be added to help wet the drug powder and disperse it evenly throughout the matrix. The wetting agent also assists in moving trapped air between the drug and carrier matrix to provide a strong bond between the carrier matrix and the therapeutic agent - trapped air results in friable beads that are easily broken up when incorporated into fish feed, allowing the fish to taste the exposed therapeutic agent.

[0090] Wetting agents or surfactants suitable for this purpose include, for example, but are not limited to, anionic or cationic surfactants or non-ionic wetting agents. Disintegrants can also be starches and modified starches, celluloses, etc. In a particular form of the invention, the wetting agent is Cremophor® RH40 or Tween®.

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

[0092] Where the percentage (w / w) of the beads is less than 100% w / w of the above ingredients, the beads may contain other ingredients or additional materials, such as additional flavorings, which may be deposited on the beads (including as a coating or covering) or included in the beads.

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

[0094] Therapeutically Active Compounds Any therapeutically effective compound that can be orally administered to aquatic animals, preferably fish, can be used in the present invention.In some embodiments, there are two or more therapeutically effective compounds in the beads.For example, the therapeutically effective compound can be an anthelmintic, a probiotic, a synbiotic, or an antibiotic, or a combination thereof.In a preferred embodiment, the therapeutically effective compound is an anthelmintic.

[0095] Anthelmintics used for 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 is selected from florfenicol, oxytetracycline dihydrate, sulfadimethoxine / ormetoprim, B.thuringiensis and bioinsecticides based on gram-positive spores of the Bacillus genus. In another embodiment, the antibiotic is selected from tetracycline, oxolinic acid and chloramphenicol.

[0096] When probiotics are used in the present invention, the main purpose of the probiotics is to establish or maintain the relationship between beneficial and harmful bacteria that are normally present in the intestine or gut of fish. Effective probiotics should have certain qualities as specified below. a. Probiotics should have beneficial effects on the growth, development and protection of fish against various pathogenic bacteria. b. Probiotic bacteria should not have adverse effects on the host. c. Probiotics should not have the ability of drug resistance, but should have the ability to maintain genetic traits. d. For efficient use of probiotics in feed, they should exhibit the following characteristics: i. Acid and bile resistance ii. Gastric juice resistance iii. Adhesion to surfaces of the digestive system iv. Antagonism against pathogens v. Immune stimulation & increased intestinal motility & mucosal survival vi. Production of enzymes and vitamins; and e. They should have good sensory properties, fermentation action, resistance to freeze-drying, and viability in the feed during the packaging and storage process.

[0097] For example, the only probiotic microorganisms most frequently used belong to the genera Bacillus, Lactobacillus and Bifidobacterium.Various species of Lactobacillus, Bifidobacterium and Streptococcus that have been 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. bugarius, 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 fed to Tilapia (Oreochromis niloticus). L.lactis probiotic also has antibacterial activity against two pathogens that can affect fish growth, namely Yersinia rukeri and Aeromonas salmonicida. Leuconostoc mesenteroides has the potential to inhibit fish pathogens found in Nile tilapia (O.niloticus). Bacillus subtilis significantly reduces motile Aeromonads, total Coliforms and Pseudomonads found in ornamental fish. Lactic acid bacteria such as Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus fermentum, Lactococcus lactis, and Sterptococcus salivarius were isolated from the intestine of Spanish mackerel (Scomberomorus commerson) and were able to inhibit the growth of Listeria innocua. Many Lactobacilli species isolated from the intestine of Anguilla species, Clarias orientalis, Labeo rohita, Oreochromis species, and Puntius carnaticus showed significant antibacterial activity against Aeromonas and Vibrio species.

[0099] Inhibition of viruses can also occur through the secretion of extracellular enzymes produced by bacteria. For example, Aeromonas species, Corynebacterium species, Pseudomonas species and Vibrio species show antiviral activity against IHNV (infectious hematopoietic necrosis virus). Feeding of the probiotic strain Bacillus megaterium enhanced resistance to WSSV (white spot syndrome virus) in shrimp, Litopenaeus vannamei. Probiotic 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 in a mixture with Sporolac, resulted in better resistance to lymphocystis viral disease, which is found in Paralichthys olivaceus der.

[0100] Probiotics may also exhibit antifungal activity. For example, Aeromonas strain A199 from Anguilla australis (eel) culture water can inhibit Saprolegnia species. Pseudomonas species M162, Pseudomonas species M174 and Janthinobacterium species M169 can increase animal immunity against saprolegniasis, as demonstrated in Oncorhynchus mykiss (rainbow trout). Lactobacillus plantarum FNCC 226 also showed inhibitory ability in catfish (Pangasius hypophthalamus) against Saprolegnia parasitica.

[0101] A major advantage of the present invention is that an aquatic animal feed additive provides a means for improving the palatability of therapeutically active compounds. Thus, in some embodiments of the present invention, the therapeutically active compounds are poorly tasting or harsh compounds that are difficult to administer orally directly to aquatic animals.

[0102] In one embodiment, the therapeutically effective compound is selected from the group consisting of probiotics; synbiotics; anti-invertebrate compounds; anthelmintic compounds; antiviral compounds; anti-nematode compounds; antibiotic compounds; algicides; insecticides; antifungal compounds; antiprotozoal compounds; and larvicide compounds.

[0103] In another embodiment, the therapeutically effective compound is selected from the group consisting of tetracyclines; sulfa antibiotics; diaminopyrimidines; fluoroquinolones; quinolones; sulfonamides; avermectins; macrolides; chlorinated bisphenols; benzoyl ureas; monochlorobenzenes; insect growth regulators; sulfur compounds; salicylamides; phosphorus compounds; benzimidazoles; pyrethrins; triazines; tetramizoles; and anticoccidals; irreversible organophosphate acetylcholinesterase inhibitors; organophosphate insecticides; chloramphenicol; and tetracyclines.

[0104] In a preferred embodiment, the therapeutically active 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 spores of gram-positive bacteria of the genus Bacillus and B. thuringiensis; tetracycline; and chloramphenicol.

[0105] In another preferred embodiment, the therapeutically active compound is selected from the group consisting of macrolide anthelmintics; bithionol; diflubenzuron; triflumuron; pyriproxyfen; sulfur powder; salicylanilide; organophosphates; benzimidazoles; pyrethroids; triazines; tetramisole anthelmintics; levamisole anthelmintics; sulfonamides; and anticoccidials.

[0106] In a preferred embodiment of the present invention, the therapeutically active compound is praziquantel.Praziquantel is an anthelmintic drug known to have a particularly bitter taste that is rejected by fish when administered orally directly to fish, or when simply mixed with fish feed, or coated on fish feed.Praziquantel is frequently used to treat trematodes among certain commercially farmed fish species, such as yellowfin amberjack, but is generally administered by bathing due to its bitter taste.Yellowfin amberjack is known to have a particularly sensitive sense of taste.

[0107] The amount of therapeutically effective compound in the aquatic animal feed additive beads of the present invention depends on the nature of the therapeutically effective compound and the amount required to achieve a therapeutic effect in the aquatic animal population. The amount of aquatic animal feed additive (or aquatic animal feed containing the aquatic animal feed additive) consumed by the aquatic animal population will affect the amount of therapeutically effective compound required in the beads. It is known to those skilled in the art that the size of the aquatic animal and the temperature of the water can also affect the amount of aquatic animal feed consumed by the aquatic animal population. For example, larger fish tend to eat a smaller percentage of fish feed relative to their body weight compared to smaller fish. Fish populations also tend to consume less feed in colder water conditions. This means that a greater amount of 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 in both larger fish and colder water conditions.

[0108] A particular advantage of some embodiments of the present invention is that very high drug loading can be achieved in the beads. In some embodiments, the beads contain at least about 70% w / w of the therapeutically effective compound. This can be useful for delivering some therapeutically effective compounds to some aquatic animal populations in certain conditions, since high drug loading is necessary to deliver the therapeutically effective compound in sufficient amounts to have a therapeutic effect in the aquatic animal.

[0109] For example, depending on the nature of the disease being treated, a therapeutically effective dose of praziquantel is between 50 mg / kg and 150 mg / kg. In a most preferred embodiment, a drug loading of about 70% to 90% w / w praziquantel is present in the beads, which ensures that the aquatic animal population ingests sufficient aquatic animal feed such that a therapeutically effective dose of praziquantel is delivered to the aquatic animal population.

[0110] Flavoring agents The present invention provides flavoring agents that are incorporated within the beads of the present invention.

[0111] Encapsulation of the flavoring agent within the beads of the present invention (as opposed to simply being coated or otherwise incorporated into the aquatic animal feed) provides the advantage of ensuring that the flavoring agent remains in proximity to the therapeutically active compound and does not disperse independently of the therapeutically active compound, and therefore can better mask the taste of the therapeutically active compound.

[0112] The taste masking agent may be any agent that has the ability to adequately mask the taste and / or odor of the therapeutically effective compound that is also contained within the beads. The taste masking agent may exert this effect in many ways. Some taste masking agents have no taste or flavor of their own. Instead, these agents may affect the taste perception of the therapeutically effective compound, for example, by exerting an effect on the taste system of the aquatic animal, by binding to the therapeutically effective compound and preventing its release in saliva, or by reducing the dissolution of the therapeutically effective compound in saliva. Other taste masking agents are flavoring agents. The flavoring agent itself has an odor and / or flavor that is more pleasant to the aquatic animal, having the ability to mask the unpleasant taste and / or odor of the therapeutically effective compound. In a preferred embodiment, the taste masking agent is a flavoring agent.

[0113] Numerous flavoring 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 flavoring agent may depend on the particular species of the aquatic animal population.

[0114] In a particularly preferred embodiment, the flavoring agent is a derivative or extract of garlic (e.g., garlic powder, diluted or pure, synthetic garlic component (allicin) or garlic oil) and contains the odor compounds found in garlic. In another preferred embodiment, the flavoring agent is an amino acid such as betaine, L-alanine, L-glutamic acid, L-arginine, glycine and inosine. In another preferred embodiment, the flavoring agent is natural fish oil (cod liver oil or tuna oil), aniseed oil, or castor oil.

[0115] The amount of flavouring required in the aquatic animal feed depends on the nature of the flavouring and is preferably in the range of 1-10% w / w. In a preferred embodiment, the flavouring comprises about 2-8% w / w of beads. In a particularly preferred embodiment, the flavouring or flavouring comprises about 4-5% w / w of beads. Support matrix

[0116] The carrier matrix provides a means for encapsulating the therapeutically active compound and the flavoring agent together.

[0117] The carrier matrix used in the compositions of the present invention is capable of forming beads incorporating the flavoring agent and therapeutically active compound such that the beads have a size, shape and consistency suitable for incorporation into an aquatic animal feed and consumption by the target aquatic animal population. The carrier matrix also helps to maintain the integrity of the beads when compressed into the aquatic animal feed.

[0118] Preferably, the beads are digestible by the aquatic animal, so that the therapeutically active compound, when ingested, is released into the animal's digestive tract, rather than being regurgitated or excreted intact. The carrier matrix used in the present invention is preferably capable of at least partially degrading in the aquatic animal digestive tract, so that the therapeutically active compound can be absorbed by the animal, allowing targeted drug release in the intestine.

[0119] Aquatic animal populations may perceive a therapeutically effective compound as tastier when presented in one carrier matrix than in another, hi some embodiments, the carrier matrix works in conjunction with a flavoring agent to improve the palatability of the therapeutically effective compound.

[0120] Without wishing to be bound by theory, therapeutically active compounds, when incorporated into a suitable carrier matrix, exhibit reduced leaching into the surrounding environment when delivered for consumption by a population of aquatic animals. Reduced leaching of the therapeutically active compound reduces the detection of the bad odor / taste of the therapeutically active compound by the aquatic animals and improves their palatability. This has the added effect of reducing the amount of therapeutically active compound in the aquatic animal feed additive itself.

[0121] Thus, when the aquatic animal feed additive (or aquatic animal feed containing the aquatic animal feed additive) is introduced into water for consumption by an aquatic animal population, it is desirable that the beads do not leach the therapeutically active compound into the surrounding water. Thus, the carrier matrix preferably does not substantially dissolve or disintegrate in fresh water or seawater. In a particularly preferred embodiment, the carrier matrix is ​​substantially stable in seawater for at least 60 minutes, in that the beads do not disintegrate in seawater during this period.

[0122] In one embodiment of the invention, the leaching rate of the therapeutic agent is less than 10% over 60 minutes in seawater. More preferably, the leaching rate is less than 5% over 60 minutes in seawater.

[0123] In a particularly preferred embodiment, the suitable carrier matrix is ​​at least partially prepared from a hydrogel-based compound derived from a marine environment that does not substantially dissolve or disintegrate in seawater. Suitable marine-based hydrogels include agar, agarose, carrageenan, fucoidan, chitosan, alginate, or agar. In a further preferred embodiment, the suitable carrier matrix comprises a combination of alginate and an acid-soluble polymer (e.g., chitosan and / or Eudraguard®), alginate alone, or agar. In one embodiment, the suitable carrier matrix is ​​a marine-based collagen.

[0124] In a preferred embodiment, the carrier matrix is ​​a combination of alginate and an acid soluble polymer (e.g., 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 crosslinking a suspension containing the alginate / acid soluble polymer carrier matrix, the therapeutically active compound and the flavoring agent in a solution of calcium chloride or zinc chloride.

[0125] In another preferred embodiment, the carrier matrix is ​​composed solely of alginate. Preferably, the carrier matrix comprises 5-15% w / w alginate. In a particularly preferred embodiment, the carrier matrix is ​​composed of about 5-11% alginate.

[0126] In another preferred embodiment, the carrier matrix is ​​prepared from agar. Any type of agar can be used as the carrier matrix, including commercially available food grade agar, agarose and other pharmaceutical or microbiological grade agar. Preferably, the carrier matrix is ​​composed of about 15-25% agar. In a particularly preferred embodiment, the carrier matrix comprises food grade agar, and contains about 18-20% w / w agar. These beads are prepared by solidifying a suspension containing the agar carrier matrix, the therapeutically active compound and a flavoring agent in an oil, an emulsifier (e.g., Tween® 80), or a buffer. In a preferred embodiment, the oil, emulsifier or buffer used to solidify the agar carrier matrix has taste-masking properties as a flavoring agent, in that it has a pleasant flavor to aquatic animals. In a particularly preferred embodiment, an oil is used to solidify the suspension containing the agar carrier matrix. Preferably, the oil used can be garlic oil (i.e., an oil containing a garlic derivative), and / or cod liver oil.

[0127] Wetting agents / surfactants / disintegrants In some preferred embodiments, the beads further comprise one or more wetting agents, surfactants or disintegrants.

[0128] If the excipient or carrier matrix is ​​poorly soluble in the animal's digestive tract (such as the stomach or intestinal tract), the excipient may include a wetting agent or surfactant polymer or pharmaceutical disintegrant to aid in disintegration of the excipient.

[0129] In alternative embodiments, the additive or carrier matrix may include a wetting agent or surfactant polymer or pharmaceutical disintegrant that may or may not degrade in the animal's digestive tract, which aids in the manufacture of the additive or promotes wetting of the additive in an aqueous environment.

[0130] If the therapeutic agent is not well wetted into the carrier matrix (e.g., aqueous sodium alginate solution) during bead manufacture, a wetting agent can be added to help wet the drug powder and disperse it evenly throughout the matrix. The wetting agent also assists in moving trapped air between the drug and carrier matrix to provide a strong bond between the carrier matrix and the therapeutic agent - trapped air results in friable beads that are easily broken up when incorporated into fish feed, allowing the fish to taste the exposed therapeutic agent.

[0131] 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 ability to affect the fragility of the beads. Preferably, the reduction in air trapped in the suspension increases the compressibility of the beads, thus reducing the risk of crushing and resulting leaching of the therapeutic agent when the beads are incorporated into fish feed. For example, if the therapeutic agent is praziquantel, a wetting agent can be added to help wet the drug powder so that it can be uniformly dispersed in the alginate solution. In addition, the wetting agent also helps to displace air trapped between the drug and the sodium alginate solution to give stronger beads (as a result of insufficient wetting), while air trapped in the final beads results in fragile beads that are then easily crushed when incorporated into fish feed, so that the fish can eat the exposed praziquantel.

[0132] Wetting agents or surfactants include, for example, but are not limited to, anionic or cationic surfactants or non-ionic wetting agents. Preferably, wetting agents or surfactants include, for example, but are not limited to, anionic surfactants such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic surfactants can be used as needed, including, but are not limited to, benzalkonium chloride or benzethomium chloride. A list of potential non-ionic wetting agents that can be included in the formulation includes 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 esters, methylcellulose and carboxymethylcellulose. When used, these wetting agents can be present in the dosage form of the present invention alone or as a mixture in different ratios.

[0133] Preferably, the humectant is Cremophor® RH40 or polysorbate (Tween®). For example, the humectant is 21, 21, 40, 60, 61, 65, 80, 81 and / or 85 Tween®. In one embodiment, the beads are comprised of about 5-15% Cremophor® RH40.

[0134] The disintegrants can be, for example, starches and modified starches, celluloses, and the like.

[0135] In a particularly preferred embodiment, a suitable carrier matrix comprises alginate in combination with an acid soluble polymer (eg, chitosan and / or Eudraguard®) and a humectant (eg, Cremophor® RH40 or Tween®).

[0136] In a preferred embodiment, the carrier matrix is ​​comprised of about 5-15% w / w of an acid soluble polymer and 5-15% w / w of an alginate and a humectant. Preferably, the humectant is selected from Cremophor® RH40 and / or Tween®. Most preferably, the humectant is present at about 5-10% w / w. Most preferably, the humectant is Cremophor® RH40 and is present at about 5-10% w / w.

[0137] In another preferred embodiment, the carrier matrix is ​​comprised of an alginate and a humectant. Preferably, the carrier matrix is ​​comprised of 5-15% w / w alginate and humectant. In a particularly preferred embodiment, the carrier matrix is ​​comprised of about 5-11% w / w alginate and humectant. Preferably, the beads further comprise a humectant selected from Cremophor® RH40 and / or Tween®. Most preferably, the humectant is Cremophor® RH40 and is present at about 5-10% w / w.

[0138] Feed for aquatic animals, including feed additives for aquatic animals In one embodiment, the present invention provides an aquatic animal feed comprising a therapeutically effective amount of the aquatic animal feed additive of the present invention.

[0139] The aquatic animal feed additive beads of the present 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 be safely provided to the target aquatic animal population can be used to prepare the aquatic animal feed of the present invention.

[0140] In a preferred embodiment, the beads of the present invention are mixed with the ground aquatic animal feed during the manufacturing process, compressed, and then cut into pellets or granules of a size suitable for the target aquatic animal population. In this embodiment, the beads of the present invention are dispersed throughout the aquatic animal feed and are not merely coated on the surface of the aquatic animal feed.

[0141] Those 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 to constitute a therapeutically effective amount, which depends on a variety of factors including the nature of the therapeutically active compound, the aquatic animal population, and the drug loading rate in the beads.

[0142] Treatment In a further aspect, the present invention provides a method for treating a disease in an aquatic animal comprising administering to a population of aquatic animals an aquatic animal feed additive or an aquatic animal feed of the present invention. Preferably, the aquatic animal is a fish.

[0143] The compositions of the present invention can be used to treat diseases suffered by aquatic animal populations. Alternatively, the compositions of the present invention can be used prophylactically to prevent aquatic animal populations from developing diseases.

[0144] Many different therapeutically active compounds can be used in the beads of the invention. In a particularly preferred embodiment, the therapeutically active 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 50 mg / kg to 150 mg / kg of praziquantel.

[0145] A range of different diseases can be treated or prevented using the compositions of the present invention, including parasitic diseases such as those caused by tapeworms or trematodes, including monogenean trematodes and schistosomes. Families of monogenean parasites that cause disease in aquatic animals include Capsalidae, Dactylogyrus vastator, Centrocestus formosanus, and Digenea Heterophydae.

[0146] In a preferred embodiment, the disease is a parasitic infection caused by a monogenean trematode. In a further preferred embodiment, the disease is a parasitic infection caused by a Zeuxapta trematode.

[0147] The composition of the present invention can be used to treat or prevent diseases in aquatic animals.Preferably, the aquatic animals can be any kind of fish, such as yellowtail amberjack, bluefin tuna, etc.

[0148] In a preferred embodiment, the fish is of a species known to be particularly sensitive to taste and / or smell, in a further preferred embodiment, the fish is of the species Seriola lalandi.

[0149] Those skilled in the art will know the amount of aquatic animal feed or aquatic animal feed additive to deliver to the aquatic animal population and the dosing schedule necessary to achieve a therapeutic effect in the aquatic animal population.

[0150] Method for preparing aquatic animal feed additive In a further aspect, the aquatic animal feed additive of the present invention is prepared using a method comprising the steps of: (a) preparing a dry mix of a therapeutically active compound, a flavoring agent, and optionally a portion of a carrier matrix and / or a wetting agent; (b) adding a solution containing a carrier matrix and optionally a wetting agent to the dry mixture to form a uniform suspension; (c) dropping the homogenous suspension into a liquid medium to form beads; (d) washing the beads with water; (e) Drying the washed beads.

[0151] In a particularly preferred embodiment, the dry mixture of step (a) is prepared by mixing an effective amount of a therapeutically active compound, such as praziquantel, with a flavoring agent, such as garlic powder. This step may also include mixing a portion of a carrier matrix (e.g., chitosan) and / or a humectant, if desired.

[0152] In step (b), a solution containing a carrier matrix is ​​added to the dry mix to form a uniform suspension. In a preferred embodiment, the carrier matrix is ​​an acid soluble polymer (e.g., chitosan or Eudraguard®) and the solution is made by mixing an 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, the chitosan being included as part of the dry mix in step (a), and then the alginate solution is added to the dry mix, stirred and sonicated in a sonication bath to form a uniform suspension (stirring times can range from 30 minutes to overnight). In a further embodiment, a wetting agent is also provided. Preferably, the humectant is Cremophor® RH40 and / or Tween®. Preferably, Cremophor® RH40 is mixed together with the therapeutically active compound and the flavouring agent (and optionally part of the carrier matrix) in step (a) and a solution of alginate, preferably 1% (w / v), is added to the dry mix.

[0153] In step (c), the homogenous suspension is dropped into a liquid medium to form beads. The appropriate liquid medium is selected based on the carrier matrix used and is known to those skilled in the art. The beads may need to be placed in the liquid medium for a period of time 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 a mixture of oil, 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 may have an additional taste masking effect.

[0154] In step (d), the beads are removed from the liquid medium and washed with water, preferably deionized water.

[0155] In step (e), the beads are dried. The beads can be dried by any method known in the art. For example, the beads can be dried by air drying, spray drying, convection drying, vacuum drying, microwave drying, freeze drying 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.

[0156] In a preferred embodiment, the dried beads are further mixed with a flavoring agent to coat the beads with the agent. In a particularly preferred embodiment, the flavoring agent is garlic powder.

[0157] Once the aquatic animal feed additive is prepared according to 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 can be pelleted using any machine or device suitable for the process.The commercial fish feed used in the process, as well as the size and shape of the pellets, can be selected based on the requirements of the target aquatic animal population.

[0158] 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: preconditioner at >90°C for about 30 seconds, followed by extruder temperature of 90°C-120°C with cylinder pressure of >20 bar in the high pressure zone, followed by drying at 90°C-110°C for 44 minutes. The clearance between the screw and barrel in the extruder is less than 1 mm. In this embodiment, the aquatic animal feed additive composition is not substantially destroyed or degraded by these parameters. In this embodiment, the bead size is maintained at less than 500 μm (0.5 mm) in diameter, preferably less than 1 mm.

[0159] In another embodiment, the extruder is subjected to the following heat and pressure parameters: 70° C. and 20-30 bar pressure, with an extruder hold time (mixing through the extruder) of 25-30 seconds. In this embodiment, the aquatic animal feed additive composition is not substantially destroyed or degraded by these parameters. EXAMPLES

[0160] Further features of the present invention are more fully described in the following non-limiting examples, which are included solely for the purpose of illustrating the invention and should not be construed as a limitation on the broad description of the invention as set forth above.

[0161] Example 1 - Preparation of beads. To test the palatability and digestibility of fish feed additive compositions containing praziquantel (PZQ), five different bead formulations were prepared containing the following compositions: [Table 1-1] [Table 1-2]

[0162] Formulations A and E (chitosan beads) were prepared by dissolving chitosan powder in 0.2M acetic acid to prepare a 1% chitosan solution. Praziquantel and garlic derivatives were then mixed with the chitosan solution. The resulting suspension was then dropped into a bath of 2% w / v sodium triphosphate solution to form beads. Formulation A was allowed to complex for 3 hours, whereas Formulation E was allowed to complex overnight. The beads were then allowed to air dry in a fume hood for at least 7 days. Once dry, garlic powder equal to 1% of the total bead weight was mixed with the dried beads.

[0163] Formulation B was prepared by preparing a dry mix of praziquantel, chitosan, and garlic derivative. A 1% (w / v) alginate solution was then added to the dry mix and stirred to form a uniform suspension. The suspension was then added dropwise to a 1% (w / v) calcium chloride solution to form beads. The beads were allowed to sit in the calcium chloride solution for an additional 10-15 minutes and washed with deionized water. The beads were then allowed to air dry for at least 7 days in a fume hood. Once dry, garlic powder equal to 1% of the total bead weight was mixed with the dry beads.

[0164] Formulation B *was prepared by preparing a dry mix of praziquantel, chitosan, garlic derivative and Cremophor® RH40. A 1% (w / v) alginate solution was then added to the dry mix and stirred to form a uniform suspension. The suspension was then added dropwise to a 1% (w / v) calcium chloride solution to form beads. The beads were then left to sit in the calcium chloride solution for an additional 10-15 minutes and washed with deionized water. The beads were then allowed to air dry in a fume hood for at least 7 days. Once dry, garlic powder equal to 1% of the total bead weight was mixed with the dry beads.

[0165] Formulation C was prepared by preparing a dry mixture of praziquantel and garlic derivatives. A 2% (w / v) agar solution (when hot and flowable) was then added to the dry mixture and stirred to form a uniform suspension. The suspension was then added dropwise to cold garlic oil (which consisted of garlic oil: soybean oil: cod liver oil in a 5:3:1 ratio) to form beads. The beads were then washed with deionized water and then air-dried in a fume hood for at least 7 days. Once dry, garlic powder equal to 1% of the total bead weight was mixed with the dry beads.

[0166] Formulation D was prepared by preparing a dry mix of praziquantel, Cremophor® RH40 and a garlic derivative. A 1% (w / v) alginate solution was then added to the dry mix and stirred to form a uniform suspension. The suspension was then added dropwise to a 1% (w / v) calcium chloride solution to form beads. The beads were allowed to sit in the calcium chloride solution for an additional 10-15 minutes and washed with deionized water. The beads were then allowed to air dry for at least 7 days in a fume hood. Once dry, garlic powder equal to 1% of the total bead weight was mixed with the dry beads.

[0167] Example 2 - Disintegration test. Beads of formulations A, B, C, D and E were tested for their ability to disintegrate in 0.1M HCl.

[0168] The beads were placed in vials containing 10 mL of 0.1 M HCl or seawater and observed for 5 h and stirred at 100 rpm using a magnetic stirrer.

[0169] The results of the disintegration experiments are shown in Figure 1. The results showed that the beads of all formulations A, B, C, D and E did not disintegrate in seawater (Figure 1A), but at least swelled and softened in HCl solution (Figure 1B).

[0170] Example 3 - In vitro dissolution profiles of Formulations B and C. Approximately 10 mg of dried beads of formulation B and formulation C were weighed separately and transferred to a USP dissolution basket (708-DS model, Agilent Technologies, Mulgrave, Victoria, Australia), each then attached to a USP dissolution paddle shaft. This set-up ensured that the beads could be immersed in a low volume of dissolution media. No beads were removed during media sampling, the media could be stirred during dissolution, and beads could be transferred rapidly and en masse from one media to the next. Dissolution profiles were performed in triplicate at an agitation speed of 100 rpm and ambient temperature (22-25 °C). The beads were incubated in 500 mL seawater for 5 min, followed by 60 min in 100 mL simulated fish gastric fluid (SGF, seawater containing 0.1% (w / v) polysorbate 80 and 0.8 mg / mL pepsin, adjusted to pH 2.0 ± 0.05 with HCl) and finally 120 min in 100 mL simulated fish intestinal fluid (SIF consisting of PBS 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.8 ± 0.05, containing 0.1% (w / v) polysorbate 80 and 0.4 mg / mL trypsin). The respective dissolution media were sampled (1 mL) at 0 and 5 min (seawater), 35 and 65 min (SGF), and 125 and 185 min (SIF). The removed samples were filtered (0.45_m) and analyzed for praziquantel content using an HPLC assay. Control experiments were performed using pure PZQ powder (7-13 mg / basket). However, since the baskets could not be transferred from one dissolution medium to the next without losing powder, experiments with PZQ powder were performed separately in three dissolution media as follows: 5 min in 500 mL seawater, 60 min in 100 mL SGF, and 120 min in 100 mL SIF. Additionally, the dissolution profiles of Formulation B and Formulation C beads were determined over 3 h in seawater using the same equipment setup.

[0171] Dissolution of PZQ in seawater was undetectable after 5 min. However, 56.7 ± 4.1% of the drug powder was dissolved in 500 mL of seawater after 3 h (Figure 2a), which was comparable to the 53.7 ± 0.9% dissolution obtained after 1 h in 100 mL of SGF (Figure 2b). After 2 h of incubation in SIF, 78.2 ± 4.8% of the drug powder was dissolved (Figure 2b). Although there was incomplete dissolution of PZQ powder under simulated GIT conditions, the drug:vehicle ratio (7–13 mg in 100 mL) was lower than the solubility of PZQ in water (approximately 36 mg / 100 mL).

[0172] Beads of formulations B and C did not disintegrate even after 3 h of incubation in 500 mL seawater, with only 6.51 ± 0.59% and 1.36 ± 0.71% of the drug loading from the respective beads leaching into seawater at 3 h (Figure 2a). Simulation of bead passage from seawater to the fish GIT showed undetectable drug release from both formulations after 5 min in seawater. Formulation B beads remained intact after an additional 60 min of incubation in SGF, releasing only 2.3 ± 0.4% of the drug loading. However, bead disintegration was observed in SIF with release of 84.7 ± 2.9% of the drug loading at 185 min (Figure 2b). In contrast, beads of formulation C remained intact in seawater as well as in SGF and SIF, with low levels of drug release measured throughout the dissolution period. The cumulative percent drug release from formulation C beads was only 3.87 ± 0.24% at 185 min.

[0173] Example 4 - Differential Scanning Calorimetry (DSC) Analysis. Formulation B, Formulation C, blank agar, alginate and alginate-chitosan beads, and PZQ alone were analyzed with a differential scanning calorimeter (Discovery DSC25 System, TA Instruments, New Castle, DE, USA). Bead samples were analyzed within 14 days of manufacture. Samples (approximately 3 mg) were analyzed in standard aluminum pans (DSC Consumables Incorporated, Austin, MN, USA) over a heating rate of 10 °C / min over a range of 0 to 250 °C, with an empty aluminum pan as the reference. DSC thermograms were analyzed using TRIOS software (TA Instruments, New Castle, DE, USA).

[0174] DSC analysis was performed on formulations B and C and the thermograms were compared to the DSC thermograms of PZQ and the corresponding blank beads in Figure 3. PZQ (3.4 mg) showed a sharp melting endotherm with an onset at 138.9°C and a peak temperature of 141.99°C. The melting enthalpy was 99.870 J / g. The PZQ melting peak in formulation B (3.8 mg) had an onset at 138.35°C and a peak temperature of 141.50°C. Its enthalpy of 72.607 J / g was 72.70% of that of pure PZQ and corresponded to the drug loading of formulation B. The PZQ peak in formulation C (3.4 mg) had an onset at 138.03°C and a peak temperature of 141.93°C. Its enthalpy of 71.645 J / g was 71.74% of that of pure PZQ, which also closely corresponded to the drug loading of formulation C. Therefore, from the respective DSC thermograms, it can be concluded that PZQ retained its crystalline properties and did not interact with the matrix material in formulations B and C.

[0175] Example 5 - Fish palatability testing. Each of the dried beads of formulations A-E was mixed with ground commercial kingfish fish feed and pelleted. The feed contained the equivalent of 10 grams of pure praziquantel per kilogram of feed. Healthy kingfish were fed fixed doses based on the weight of the fish feed pellets containing the formulations and the water temperature. Fish were fed for a maximum of 3 minutes in one morning feeding over a 5-day period.

[0176] Fish feed containing formulations A and E was delivered to large healthy fish with an average weight of 2000 grams and 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.

[0177] Healthy kingfish were fed a fixed amount per tank per day, calculated based on their size and water temperature, with a single morning feeding over the course of a day.

[0178] The average time for the fish population to consume the entire supply of fish feed was measured. The average percentage of fish feed consumed was also recorded. Data was analyzed for each of the five day replicates. This was compared to a control group fed fish feed without the fish feed additive.

[0179] FIG. 5 shows the results for fish feed containing formulations A and E (chitosan beads). Kingfish fed fish feed formulation E never ate the complete feed supply during the 5-day study. As shown in FIG. 5A, a two-way ANOVA revealed that fish size (P=0.77) or diet (P=0.07) did not affect intake. Fish fed formulation E ate an average of 75% of the offered feed, whereas fish fed formulation A ate an average of only 68%. Fish in the unmedicated control treatment consumed 90% of the supply (pooled across small and large fish). These results were contrary to the hypothesis that larger fish were more susceptible to the taste and / or odor of praziquantel. Small control fish ate the entire supply of unmedicated feed, but only ate 60% and 70% of the treatment supplies for formulations A and E, respectively. As shown in Figure 5B, the time it took to consume the complete supply was significantly affected by treatment (P<0.0001) but not by fish size (P=0.27). When fish consumed the entire medicated supply, it took approximately 3 minutes. Fish given the unmedicated control treatment consumed their supply in approximately 1.5 minutes.

[0180] Figure 3 presents the results for fish diets containing formulations B, C, and D. There was no evidence that larger fish were more susceptible to the taste / odor of praziquantel in the formulations, so only small fish (260 grams) were used to evaluate the palatability of formulations B, C, and D. Fish consumed more than 99% of all treatment diets (Figure 5A) and there were no differences between treatments (P=0.28). Fish ate their complete ration at each feeding, but the time it took to consume a ration was significantly different between treatments (P=0.02). The amount of time required for fish fed diets containing formulation B (1.15 ± 0.14 min) and formulation C (1.16 ± 0.14 min) to ingest their feed was not different compared to the control (0.64 ± 0.07 min), however fish fed diets containing formulation D took significantly longer than the control to consume their feed (1.42 ± 0.04 min) (Figure 5B). This indicates acceptable palatability for each of formulations B, C, and D. Formulations B, C, and D were more palatable to the fish population based on both the greater percentage of the feed containing formulations B, C, and D being consumed, as well as the fact that these feeds were also consumed more quickly compared to the feeds containing formulations A and E.

[0181] Example 6 - Digestibility study. For formulations A and E, after the last feeding on day 5, fish were dissected to determine bead digestibility approximately 3 hours after feeding.

[0182] There was no evidence of beads in the digestive tract of either small or large fish fed Formulation A treatment, however, in larger fish undigested and partially digested beads were found throughout the entire length of the tract in the Formulation E treatment, as seen in Figure 6.

[0183] For Formulations B, C, and D, one fish per treatment was dissected prior to feeding on day 5 to determine whether any beads remained in the digestive tract after the previous day's feeding. Apart from a single Formulation D bead in the stomach, there was no evidence of digesta or beads in the digestive tract of fish from all treatments.

[0184] On day 5, all fish were fed their respective treatment diets and two fish per treatment were dissected at 3 and 6 hours post-feeding. The results are presented in Figure 7.

[0185] By 3 hours after feeding, the feed had traveled the entire length of the fish's intestines. For all treatments, there was an intact bead in the stomach, but no evidence of beads in any part of the tract. Similar findings were obtained 6 hours after feeding.

[0186] These results indicate that formulations A, B, C and D were digested by the fish as the beads did not pass through the intestine intact. Example 7 - Drug Loading Determination.

[0187] The drug loading of the beads of Formulations A-E was determined by dissolving the beads in 0.4M NaCl solution and then adding methanol to make a 50% methanol solution. The solution was then subjected to HPLC analysis to determine the drug loading. The results are presented in the table below: [Table 2]

[0188] Example 8 - Drug loading stability. The drug loading capacity (or more specifically the 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 presented in Table 3 below: [Table 3]

[0189] Example 9 - Palatability of fresh beads compared to stored beads. Fish feed containing formulations B and C was tested to compare the palatability of freshly prepared beads to approximately 4-month-old beads. Healthy kingfish were fed a fixed feeding of fish feed containing formulations B and C per tank per day in a single morning feeding for 5 days. The percentage of food consumed per feeding was measured, as well as the average time to consume the feeding for the last 2 days. The results are presented in Figure 8. The data show that there was no significant difference in the palatability of the 4-month-old beads and the fresh beads.

[0190] Example 10 - Second Preference Study. A second palatability study was conducted to compare formulations B and C with fish feed incorporating pure praziquantel (either not incorporated within the beads or with garlic extract). Formulations B or C beads were mixed with ground 3mm commercial kingfish feed and 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. A control diet was also made using the same process that did not contain praziquantel. Kingfish infected with Zeuxapta trematodes, weighing an average of 1600 grams, were fed a fixed supply of 155 grams per tank per day in a single morning feeding for six days.

[0191] If the fish consumed the entire feed supply within 3 min, the time was recorded. The weight of the dry unfed feed was also recorded. Unfed or regurgitated pellets in the tank were also collected, weighed, and converted to dry weight. Both weights were combined to determine the rate of feed consumption per supply and the actual praziquantel dose (mg / kg) received by the fish, calculated based on the amount of feed consumed. The results are presented in Figure 9.

[0192] The data show that food intake was significantly lower in fish fed pure praziquantel fish diet (17±4%) compared to plain fish diet (no additives). However, there was no significant difference between food intake in fish fed plain fish diet and fish diet incorporating formulations B or C (Figure 9A). Fish fed the non-medicated control diet consumed 79±6% of the feed and fish given formulations B and C treatments consumed approximately 62%. None of the diets were consumed 100%, which may be because these fish had not yet acclimatized to the tank conditions after being transferred from the sea cages. Figure 9B shows the daily feed intake. The graph demonstrates that feed intake increased in all treatments over the following days, except on day 2, when all tanks were poorly replenished and pure praziquantel remained low thereafter. The fact that intake of the fish feed containing formulations B and C increased over time similarly to the control feed is further evidence that the feed is very palatable, as intake with pure PZQ feed typically tends to decrease over time.

[0193] FIG. 9C shows the average daily dose of praziquantel in mg / kg ingested by fish receiving different treatments. The actual praziquantel dose ingested by fish fed pure praziquantel fish feed composition was 21 mg / kg. Meanwhile, fish populations fed fish feed containing formulations B or C ingested approximately 75 mg / kg praziquantel (P<0.001). Thus, the results demonstrate that formulations B and C clearly allow oral delivery of significantly increased amounts of praziquantel to fish. It is generally believed that fish must be dosed with approximately 50 mg / kg for three consecutive days to eliminate Zeuxapta. This dose was easily achieved with fish feed containing formulations B and C, but not with pure praziquantel treatment.

[0194] Example 11 - Treatment of fish populations affected by trematodes. After the palatability test, the fish populations from Example 10 were bathed with praziquantel to assess fluke infection. The fluke counts in each treatment group were determined.

[0195] The results are shown in Figure 10. Fish fed the unmedicated control diet had an average of 55±13 Zeuxapta trematodes per fish at the end of the study. There was a significant effect of diet on the percentage reduction in trematode counts in the three medicated treatments compared to the control (P<0.001). Fish fed Formulation B and Formulation C treatments had a 93±2% and 94±3% reduction, respectively, both significantly higher than the pure praziquantel control, where a 73±4% reduction was observed. These data demonstrate that both Formulations B and C are palatable, digestible, and effective against Zeuxapta infection.

[0196] Example 12 - Methods for preparing beads of different sizes. Using the method described in the following paragraphs, beads of different sizes (diameters) ranging from 0.4 to 2.5 mm were prepared. The bead sizes were adjusted by selecting appropriate chitosan particle size, extrusion needle and stirring speed. The chitosan size ranges were: (1) 355-500 μm; (2) 212-355 μm; (3) 150-355 μm; and (4) <150 μm.

[0197] First, a mixture of praziquantel, chitosan and garlic derivative (with and without Cremophor® RH40) was prepared. A 1% (w / v) alginate solution was then added to the mixture, stirred and sonicated to form a uniform suspension. Stirring of the suspension ranged from 30 minutes to overnight. The suspension was then added dropwise to a 1% (w / v) calcium chloride solution to form beads. Different sizes of syringe needles were used to add the suspension dropwise, for example, 18G or 21G needles were used to form smaller beads with smaller chitosan particles. The beads were allowed to sit in the calcium chloride solution for an additional 10-15 minutes and washed with deionized water. The beads were then allowed to air dry for at least 7 days in a fume hood. Once dry, garlic powder equal to 1% of the total bead weight was mixed with the dried beads.

Claims

Claim 1: A feed additive composition for aquatic animals capable of delivering a therapeutically effective amount of a therapeutically effective compound to aquatic animals, wherein at least the therapeutically effective compound and the carrier matrix are formed into a plurality of beads that are ingestible by aquatic animals, and the composition is (a) at least 45% w / w of the therapeutically effective compound, and (b) 1% - 10% w / w of a flavoring agent, and (c) 2% - 35% w / w of the carrier matrix and comprises a composition. Claim 2: (a) The beads are coated with a flavoring agent, (b) The beads contain a flavoring agent, (c) The beads contain a flavoring agent, the beads are coated with a flavoring agent, and the flavoring agent is the same drug, or (d) The beads contain a flavoring agent, the beads are coated with a flavoring agent, and the flavoring agent is not the same drug, the composition according to claim 1. Claim 3 The composition according to claim 2, wherein the carrier matrix is digestible by aquatic animals. Claim 4: The composition according to claim 2, wherein the carrier matrix is substantially stable at room temperature for at least 60 minutes in the aqueous environment in which the feed additive for aquatic animals is used. Claim 5 The composition according to claim 2, wherein the carrier matrix is selected from a combination of alginate and an acid-soluble polymer; alginate; or agar. Claim 6 The composition according to claim 2, wherein the carrier matrix comprises a marine hydrogel. Claim 7: The composition according to claim 2, wherein the flavoring agent, alone or in combination with the carrier matrix, masks the taste of the therapeutically effective compound. Claim 8 The composition according to claim 2, wherein the flavoring agent is a flavoring agent. Claim 9 The composition according to claim 2, wherein the flavoring agent is garlic or a derivative thereof. Claim 10: The composition according to claim 2, wherein the composition contains a wetting agent. Claim 11 The composition according to claim 10, wherein the wetting agent constitutes 5 - 10% w / w of each bead. Claim 12: The composition according to claim 10, wherein the wetting agent is a non-ionic wetting agent. Claim 13 The composition according to claim 2, wherein the therapeutically effective compound is an anthelmintic compound. Claim 14 The composition according to claim 13, wherein the anthelmintic compound is praziquantel. Claim 15 The composition according to claim 2, wherein the therapeutically effective compound is present in an amount of at least about 70% w / w of each bead.

16. The composition according to claim 2, wherein the carrier matrix constitutes about 10-25% w / w of each bead.

17. The composition according to claim 2, wherein the flavoring agent constitutes 2-8% w / w of each bead.

18. (a) The therapeutically effective compound includes praziquantel, (b) The flavoring agent includes a garlic derivative, (c) The carrier matrix includes a combination of an acid-soluble polymer and alginate, The composition according to claim 2.

19. (a) The therapeutically effective compound includes praziquantel, (b) The flavoring agent includes a garlic derivative, (c) The carrier matrix includes a combination of an acid-soluble polymer and alginate, (d) The composition includes a non-ionic wetting agent, The composition according to claim 2.

20. (a) The therapeutically effective compound includes praziquantel present in an amount of about 70-90% w / w of each bead, (b) The flavoring agent includes a garlic derivative present in an amount of about 5% w / w of each bead, (c) The carrier matrix includes alginate, and the alginate is present in an amount of 5-11% w / w of each bead, (d) The composition includes a non-ionic wetting agent in an amount of 5-10% w / w of each bead, The composition according to claim 2.

21. (a) The therapeutically effective compound includes praziquantel present in an amount of about 70-90% w / w of each bead, (b) The flavoring agent includes a garlic derivative present in an amount of about 2-5% w / w of each bead, (c) The carrier matrix includes a combination of agar present in an amount of 18-20% w / w of each bead, The composition according to claim 2.

22. (a) The therapeutically effective compound includes praziquantel present in an amount of about 70-90% w / w of each bead, (b) The flavoring agent includes a garlic derivative present in an amount of about 5% w / w of each bead, (c) The carrier matrix includes a combination of an acid-soluble polymer and alginate, the acid-soluble polymer is present in an amount of 8-10% w / w of each bead, and the alginate is present in an amount of 8-11% w / w of each bead, (d) The composition includes a non-ionic wetting agent in an amount of 5-10% w / w of each bead, The composition according to claim 2. **Claim 23**: The composition according to claim 2, wherein the therapeutically effective compound is an anthelmintic compound, the carrier matrix is digestible by aquatic animals, the flavoring agent is a fragrance agent, and the composition contains a wetting agent. **Claim 24** **Claim 25**: The composition according to claim 2, wherein the beads have a diameter selected from the group consisting of 5000 μm, <5000 μm, 1000 μm, <1000 μm; 500 μm; <500 μm; 355 - 500 μm; 212 - 355 μm; 150 - 355 μm; 150 μm; <150 μm; 100 μm and <100 μm. **Claim 26**: The composition according to claim 2, wherein the carrier matrix is digestible by aquatic animals, the carrier matrix contains a marine hydrogel, and the composition contains a wetting agent. **Claim 27**: The composition according to claim 2, wherein the carrier matrix is digestible by aquatic animals, the composition contains a wetting agent, the composition contains a flavoring agent, and the flavoring agent is a fragrance agent. **Claim 28**: The composition according to claim 2, wherein the carrier matrix is digestible by aquatic animals, the composition contains a flavoring agent, the flavoring agent is a fragrance agent, the composition contains a wetting agent, and the therapeutically effective compound is an anthelmintic compound. **Claim 29**: The composition according to claim 2, wherein the carrier matrix is digestible by aquatic animals, the composition contains a flavoring agent, the flavoring agent is a fragrance agent, the composition contains a wetting agent, and the therapeutically effective compound is an anthelmintic compound. **Claim 30**: A fish feed composition comprising the composition according to any one of claims 1 - 28. **Claim 31** **Claim 32**: A method for treating or preventing a fish disease, the method comprising administering to the fish the composition according to claim 31. **Claim 33** **Claim 34**: The method according to claim 33, wherein the disease is a parasitic infection. **Claim 35** **Claim 36**: The method according to claim 34, wherein the therapeutically effective compound is praziquantel. **Claim 37** **Claim 38**: The method according to claim 33, wherein the fish population is administered the therapeutically effective compound at a dose of about 50 mg per kg of body weight of the fish population to 150 mg per kg of body weight. **Claim 39** **Claim 40**: A method for preparing the feed additive composition for aquatic animals according to any one of claims 1 - 28, Step of preparing a dry mixture of (a) the therapeutically effective compound, the flavoring agent, and, if necessary, part of the carrier matrix and / or wetting agent; Step of adding a solution containing the carrier matrix and / or wetting agent to the dry mixture to form a homogeneous suspension; Step of dropping the homogeneous suspension into a liquid medium to form beads; Step of washing the beads with water; Step of drying the washed beads A method comprising.

35. The method according to claim 34, wherein the dried beads are mixed with a flavoring agent.

36. A method for preparing a fish feed composition according to claim 29, comprising: Step of mixing (a) the aquatic animal feed additive composition with the ground fish feed to form a mixture; Step of forming pellets from the mixture of (a); A method comprising.

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