WO2009023013A1

WO2009023013A1 - Praziquantel and cmetidine compositions and methods

Info

Publication number: WO2009023013A1
Application number: PCT/US2007/023276
Authority: WO
Inventors: John Stephen Clark
Original assignee: Alpharma, Inc.
Priority date: 2007-08-13
Filing date: 2007-11-05
Publication date: 2009-02-19

Abstract

Disclosed is a multiparticulate in-feed additive composition comprising praziquantel or a salt thereof and cimetidine or a salt thereof, a medicated fish-feed comprising the multiparticulate in-feed additive composition, and a method of eliminating or reducing or preventing parasites in a fish population.

Description

PRAZIQUANTEL AND CMETIDINE COMPOSITIONS AND METHODS

BACKGROUND

[0001] The control of parasitic infestations in commercial fish farming operations, for example, is largely dependent on the use of chemical treatments. Typically, in fish farming operations, infestations are chemically treated by immersion bath treatments to reduce parasites. Immersion bath treatments, however, are very labor intensive, costly and cause considerable stress to fish. Furthermore, such treatments may not be feasible on exposed sites and during adverse weather conditions. An alternative to immersion bath treatments is in- feed treatment. In-feed treatment allows medication during adverse weather conditions and on exposed sites, and permits simultaneous medication of all cages on a site and all sites in a lake, reservoir or loch system or in a single bay, thus reducing any cross infestation that may occur during the several days necessary to apply bath treatments to all cages on a site.

[0002] Praziquantel (2-(cyclohexylcarbonyl)- 1,2,3,6,7,1 lb-hexahydro-4H- pyrazino[2,l-a]isoquinolin-4-one) is active against endoparasites. Praziquantel chemotherapy has been employed to control various internal helminth infections in mammals, and has recently been used to control monogenean diseases in fish by bath treatment. It has been reported that oral administration of praziquantel was effective in treating Microcotyle sebastis infestations in cultured rockfish Sebastes schlegeli. Since Microcotyle sebastis is a bloodsucking polyopisthocotylean, the parasite inevitably absorbs Praziquantel in the blood of treated fish in the process of blood feeding. However, all M. sebastis worms on the gills of a rockfish would not feed blood at the same time and the levels of Praziquantel in the blood would decline with the lapse of time. Considering this fast drop in Praziquantel levels in the blood, maintaining high levels of Praziquantel in blood during the treatment period and therefore increasing the amount of time to which the parasite is exposed to parasiticidal doses of the drug may be important for increasing treatment efficacy. [0003] Cimetidine (l-cyano-2-methyl-3-[2-[(5-methyl-lH-imidazol-4-yl) methylsulfanyl] ethyl] guanidine is a histamine H2 receptor antagonist. Cimetidine is widely used for the treatment of gastric hyperacidity syndrome in humans. Numerous studies have shown that cimetidine may interact with co-administered drugs by influencing absorption, metabolic clearance and/or renal clearance. Recent reports have described an increase in Praziquantel levels in plasma of mammals when cimetidine is simultaneously administered. Thus, it is advantageous to co-administer praziquantel and cimetidine.

[0004] There is, however, a need for improved praziquantel and cimetidine formulations, particularly those for aquaculture applications.

BRIEF SUMMARY

[0005] The above-described and other drawbacks are alleviated by the in- feed additive composition comprising praziquantel or a salt thereof and cimetidine or a salt thereof described herein.

[0006] In one embodiment, a multiparticulate in-feed additive composition comprises a plurality of granular cores, the cores comprising an inert hydrosoluble carrier, praziquantel or a salt thereof and cimetidine or a salt thereof, and a first coating disposed on the 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.

[0007] In another embodiment, a multiparticulate in-feed additive composition comprises a plurality of granular cores, the cores comprising an inert hydrosoluble carrier, praziquantel or a salt thereof and cimetidine or a salt thereof, and a first coating disposed on the 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.

[0008] In another embodiment, there is provided a medicated fish feed comprising the multiparticulate in-feed additive compositions described herein. [0009] In yet another embodiment, there is provided a method of eliminating or reducing parasites in a fish population comprising administering to a fish population the multiparticulate in feed additive compositions described herein.

[0010] The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 illustrates the Feed Conversion Ratio ("FCR") in test fish (Sea Bass, Lates calcarifer). Control refers to fish fed with a fish feed containing no multiparticulate in- feed additive composition, while Test refers to fish fed with a medicated fish feed containing a multiparticulate in-feed additive composition described herein. The medicated fish feed is administered on a seven day on, seven day off regimen.

[0012] Figure 2 illustrates the reduction of capsalid fluke infestations in test fish (Sea Bass, Lates calcarifer) administered a medicated fish feed containing a multiparticulate in- feed additive composition described herein.

DETAILED DESCRIPTION

[0013] Disclosed herein are improved praziquantel and cimetidine in-feed additive compositions suitable for aquaculture applications. The praziquantel and cimetidine in-feed additive composition comprises a multiparticulate in-feed additive composition. The term multiparticulate is intended to refer broadly to small particles regardless of their composition or the manner in which they are formed. The particles generally are of a mean diameter of about 50 to about 1000 μm, specifically about 100 to about 500 μm.

[0014] hi one embodiment, the multiparticulate in- feed additive composition comprises a plurality of granular cores comprising a carrier, praziquantel or a salt thereof and cimetidine or a salt thereof. Suitable carriers include, for example, "inert hydrosoluble carriers", meaning excipients, generally hydrophilic, pharmaceutically inert, crystalline or amorphous, in a particulate form, not leading to a chemical reaction under the operating conditions employed, and which are soluble in an aqueous medium, notably in a gastric acid medium. Examples of suitable carriers are derivatives of sugars, such as lactose, saccharose, hydrolyzed starch (malto-dextrine), and the like, and combinations comprising one or more of the foregoing carriers. In one embodiment, the inert hydrosoluble carrier is lactose. The individual particle size of the inert hydrosoluble carrier can be, for example, 50 to 500 microns.

[0001] The praziquantel and cimetidine can be in the form of pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" includes derivatives of an active agent, wherein the active agent is modified by making acid or base addition salts thereof, and further refers to pharmaceutically acceptable solvates, including hydrates, crystalline forms, noncrystalline forms, polymorphs, and stereoisomers of such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues; and the like, and combinations comprising one or more of the foregoing salts. The pharmaceutically acceptable salts include salts and the quaternary ammonium salts of the active agent. For example, acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, cesium salt, and the like; and alkaline earth metal salts, such as calcium salt, magnesium salt, and the like, and combinations comprising one or more of the foregoing salts. Pharmaceutically acceptable organic salts includes salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH₂)_n-COOH where n is 0-4, and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, and the like; and amino acid salts such as arginate, asparginate, glutamate, and the like; and combinations comprising one or more of the foregoing salts.

[0015] The carrier comprises 20 wt% to 70 wt%, specifically 25 wt% to 55 wt% of the total weight of the multiparticulate in-feed additive composition. The praziquantel comprises 2 wt% to 30 wt%, specifically 5 wt% to 25 wt% of the total weight of the multiparticulate in-feed additive composition. The cimetidine comprises 2 wt% to 30 wt%, specifically 5 wt% to 25 wt% of the total weight of the multiparticulate in-feed additive composition.

[0016] The granular cores are coated with one or more coatings to impart the desired properties to the multiparticulate in-feed additive composition. For example, the coatings provide the multiparticulate in-feed additive composition with desired release profile of the praziquantel and cimetidine in the digestive system of a target organism, taste-masking properties, and/or other desired properties.

[0017] In one embodiment, a multiparticulate in- feed additive composition comprises a plurality of granular cores, the cores comprising an inert hydrosoluble carrier, praziquantel or a salt thereof and cimetidine or a salt thereof, and a first coating disposed on the 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.

[0018] In one embodiment, the first coating disposed on the granular core comprises a hydrogel-forming polymer. Hydrogel-forming polymers are polymers that form an insoluble network that swells in aqueous solutions. Both natural and synthetic hydrogel-forming polymers that are capable of swelling and releasing the active agent in the gastrointestinal tract may be employed. Suitable hydrogel-forming polymers include, for example, alginate, carrageenan, agar, agarose, polysaccharides, natural gum, fucoidan, furcellaran, laminaran, hypnea, gum arabic, gum ghatti, gum karaya, gum tragacanth, locust bean gum, pectin, amylopectin, gelatin, casein, albumen, a hydrophilic colloid, polyvinyl alcohol, sodium polyacrylate, acrylate polymers and copolymers with hydrophilic groups, and combinations comprising one or more of the foregoing hydrogel-forming polymers. In one embodiment, the hydrogel-forming polymer comprises sodium alginate, potassium alginate, carrageenan, or a mixture thereof. In another embodiment, the first coating consists essentially of a hydrogel- forming polymer. In yet another embodiment, the first coating consists of a hydrogel-forming polymer. In a further embodiment, the first coating consists essentially of sodium alginate, potassium alginate, carrageenan, or a mixture thereof. In another embodiment, the first coating consists of sodium alginate, potassium alginate, carrageenan, or a mixture thereof.

[0019] The first coating optionally comprises one or more pharmaceutically acceptable excipients such as, for example, a binder. Exemplary binders include, for example, polyvinyl pyrrolidone, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose and hydroxyethyl cellulose, sugars, modified starch, gelatin, albumen, caseins, polyvinyl alcohol, and combinations comprising one or more of the foregoing binders.

[0020] The hydrogel-forming polymer in the first coating comprises 0.2 wt% to 20 wt%, specifically 0.25 wt% to 15 wt% of the total weight of the multiparticulate in- feed additive composition. The optional excipient comprises 0 wt% to 10 wt % of the total weight of the multiparticulate in-feed additive composition.

[0021] In one embodiment, a second coating comprising a barrier material is disposed on the first coating. A "barrier material" is a material that separates the first coating from subsequent coatings. In one embodiment, the barrier material imparts hydrophobicity to the coating without compromising digestibility. Suitable barrier materials include, for example, naturally occurring gums, e.g., gum acacia or gum tragacanth; naturally occurring phosphatides, e.g., lecithin or soybean lecithin; condensation products of ethylene oxide with e.g., a fatty acid, a long chain aliphatic alcohol, or a partial ester derived from fatty acids and a hexitol or a hexitol anhydride, for example polyoxyethylene stearate; polyoxyethylene sorbitol monooleate; polyoxyethylene sorbitan monooleate etc., and combinations comprising one or more of the foregoing. In one embodiment, the barrier material comprises lecithin. In one embodiment, the second coating consists essentially of a barrier material. In another embodiment, the second coating consists of a barrier material. In a further embodiment, the second coating consists essentially of lecithin. In another embodiment, the second coating consists of lecithin.

[0022] The barrier material of the second coating comprises 0.22 to 10 wt%, specifically 0.3 wt% to 8 wt% of the total weight of the multiparticulate in-feed additive composition. [0023] There are some specific advantages besides imparting hydrophobicity in the use of lecithin in the second coating. First, the use of lecithin improves lipid absorption rates and efficiencies through emulsification within the digestive system. Second, lecithins are excellent sources of phosphorus, choline, and inositol, which are advantageous nutrients in the fish feed compositions.

[0024] hi one embodiment, the third coating comprises a taste-masking polymer, hi one embodiment, the taste-masking polymer is soluble in gastric fluid at pHs less than or equal to 5. Suitable taste-masking polymers include a butyl methacrylate-(2- dimethylaminoethyl methacrylate)-methyl methacrylate copolymer (1:2:1) available under the tradename Eudragit^® E-100 (Rohm & Haas GmbH, Germany), and a 1:2:1 (butyl methacrylate-co-(2-dimethylaminoethyl)methacrylate-co-methyl methacrylate), having an average molecular weight of about 150,000, available under the tradename Eudragit^® E PO (Rohm & Haas GmbH, Germany), acrylic and/or methacrylic ester polymers, butyrates or propionates or copolymers of acrylates or methacrylates having a low quaternary ammonium content, and the like. The taste-masking polymer improves palatability of the multiparticulate in-feed additive composition, hi one embodiment, the taste masking polymer comprises Eudragit^® E and/or Eudragit^® EPO.

[0025] hi another embodiment, the taste-masking polymer is soluble at pHs greater than or equal to 7. Many polymers are employed for the taste masking of drugs. These polymers include the cellulose derivatives like cellulose esters either enteric or nonenteric and cellulose ethers. Examples of the nonenteric cellulose esters include cellulose acetate, cellulose triacetate, cellulose acetate butyrate and cellulose propionate. The enteric cellulose esters include cellulose acetate phthalate and hydroxy propyl methylcellulose phthalate. Available cellulose ethers include methylcellulose, ethyl cellulose, cellulose acetate, cellulose triacetate, hydroxy ethyl cellulose, hydroxy propyl cellulose and hydroxy propyl methylcellulose. Surelease by Dow Chemicals, and Aquacoat^® of FMC containing the ethylcellulose are most widely used. A variety of polymethacrylates and acrylic polymers are available under the trade name Eudragit^® from Rohm Pharma, eg. Eudragit^® S 100, FS30D, LlOO, NE 3OD, RS 30D and RL 30D. The acrylic polymers commonly employed are copolymers of methacrylic acid such as dimethylaminoethyl methacrylate and methacrylic acid ester.

[0026] In one embodiment, the third coating consists essentially of a taste-masking polymer. In another embodiment, the third coating consists of a taste-masking polymer. In a further embodiment, the third coating consists essentially of Eudragit^® E and/or Eudragit^® EPO. In another embodiment, the third coating consists of Eudragit^® E and/or Eudragit^® EPO.

[0027] The third coating provides gastric bypass and taste-masking. In one embodiment, the third coating forms a substantially complete coat on the particle. In those embodiments, each individual unit of the plurality of granular cores comprising the praziquantel and cimetidine is substantially completely coated. The term "substantially completely coated" means that the third coating substantially shields the praziquantel and cimetidine from contact with the outside environment. The substantially complete third coating helps ensure that the taste buds of an animal are not exposed to an objectionable- tasting material.

[0028] The taste-masking polymer in the third coating comprises about 5 wt% to about 25 wt%, specifically about 15 wt% to about 25 wt% of the total weight of the multiparticulate in-feed additive composition.

[0029] I think first is right in the next paragraph. As I see it from the examples, there are two different embodiments of the invention.

[0030] In an alternative embodiment, a multiparticulate in- feed additive composition comprises a plurality of granular cores, the cores comprising an inert hydrosoluble carrier, praziquantel or a salt thereof and cimetidine or a salt thereof, and a first coating disposed on the 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.

[0031] In this embodiment, the first coating disposed on the granular cores comprises a film-forming polymer. Suitable film-forming polymers include, for example, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate (lower, medium or higher molecular weight), cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate), poly (octadecyl acrylate), poly (ethylene), poly (ethylene) low density, poly (ethylene)high density, (poly propylene), poly (ethylene glycol poly (ethylene oxide), poly (ethylene terephthalate), poly(vinyl alcohol), polyvinyl isobutyl ether), poly(vinyl acetate), poly (vinyl chloride), polyvinyl pyrrolidone, and combinations comprising one or more of the foregoing polymers. In one embodiment, the film-forming polymer comprises ethylcellulose.

[0032] In another embodiment, the first coating consists essentially of a film-forming polymer. In yet another embodiment, the first coating consists of a film- forming polymer. In a further embodiment, the first coating consists essentially of ethylcellulose. In another embodiment, the first coating consists of ethylcellulose.

[0033] The film-forming polymer in the first coating comprises 0.2 wt% to 20 wt%, specifically 0.25 wt% to 15 wt% of the total weight of the multiparticulate in- feed additive composition. The optional excipient comprises 0 wt% to 10 wt % of the total weight of the multiparticulate in-feed additive composition.

[0034] In this alternative embodiment, a second coating disposed on the first costing comprises a taste-masking polymer. Suitable taste-masking polymers are those described above. In another embodiment, the second coating consists essentially of a taste-masking polymer. Li yet another embodiment, the second coating consists of a taste-masking polymer, hi a further embodiment, the second coating consists essentially of Eudragit^® E and/or Eudragit^® EPO. In another embodiment, the second coating consists of Eudragit^® E and/or Eudragit^® EPO. [0035] The taste-masking polymer in the second coating comprises about 5 wt% to about 25 wt%, specifically about 15 wt% to about 25 wt% of the total weight of the multiparticulate in-feed additive composition.

[0036] In this embodiment, a third coating comprises a hydrophobic component such as a lubricant. Suitable lubricants include, for example, stearic acid, a stearate salt, talc, mineral oil, saturated and unsaturated fatty acids containing about 8-22 carbon atoms, preferably 16-20 carbon atoms, saturated fats, or a combination comprising one or more of the foregoing lubricants. In another embodiment, the third coating consists essentially of a lubricant. In yet another embodiment, the third coating consists of a lubricant, hi a further embodiment, the third coating consists essentially of stearic acid, hi another embodiment, the third coating consists of stearic acid.

[0037] The lubricant in the third coating comprises 0.5 wt% to 6 wt%, specifically 1 wt% to 5 wt% of the total weight of the multiparticulate in- feed additive composition.

[0038] The coatings optionally comprise a plasticizer. Suitable plasticizers include, for example, polyethylene glycol, triacetin, vinylpyrrolidone, diethyl phthalate, dibutylsebacate, or a citric acid ester, and combinations thereof. Generally, the amount of plasticizer included in a coating is based on the total weight of the polymer contained in the coating, for example, typically about 0 wt% to about 50 wt% of the total weight of the polymer. Amounts of the plasticizer, however, can be determined by routine experimentation.

[0039] Each of the coatings individually optionally comprises additional excipients such as, for example, plasticizers (e.g., propylene glycol); anti-adhesives such as, for example, colloidal silicium dioxide (fumed silica), talc, and magnesium stearate; colorants (e.g., titanium dioxide); free flow agents (e.g., Sipernat^® 22S, available from The Cary Company); glazing agents e.g., Quick Shine from Alfred L. Wolff; materials that preserve the shelf life of the praziquantel and cimetidine, such as, for example, antioxidants (e.g., potassium metabisulfite, ascorbic acid, butylhydroxytoluene, butylhydroxyanisole, tocopherol), and combinations comprising one or more of the forgoing excipients. [0040] The multiparticulate in-feed additive composition described herein is simple to manufacture, cost-effective, and can be made with conventional tabletting machines and coating equipment.

[0041] The plurality of granular cores of the multiparticulate in-feed additive composition is prepared by techniques well known in the art such as, for example, rotary bed granulation, extrusion, or granulation and treatment in an intensive mixer. The application of the coatings is performed in a fluidized bed or by other suitable coating techniques such as pan coating, spray-drying, electrostatic coating, and the like.

[0042] The hydrosoluble carrier is combined with the praziquantel and cimetidine and granulated to form granules. The powder forms of each component are charged within a rotating chamber. A solution of a binding agent such as lactose is sprayed into the rotating mixture, until the mixture of components binds together to form suitably sized wet granules. Granule size is controlled by the speed of the rotating chamber. The granules are then dried in a warm flow of air and removed from the chamber.

[0043] Coated granules are formed by forming a solution/suspension containing the coating materials and then spraying the solution/suspension onto the granular cores to form a first coating. Suitable solvents include, for example, water, propylene glycol, ethanol, acetone, and combinations comprising one or more of the foregoing solvents. In one embodiment, the first coating is dried before applying a subsequent coating, for example, with an inlet temperature of 57°C. The mean particle size after applying the first coating should not be too small, e.g., less than 50 microns; typically, the mean particle size is about 150-300 microns.

[0044] The resulting material is overcoated with a first coating, such as a coating comprising a film-forming polymer. The film-forming polymer or other coating materials is dissolved/suspended in a solvent, and sprayed onto the granular cores. The cores coated with the first coating are optionally dried, for example at a temperature of 55-57°C, prior to deposition of the second coating. [0045] The resulting material is overcoated with a second coating, such as a coating comprising a barrier material to separate the praziquantel and cimetidine from additional coatings. The coating components are dissolved/suspended in a solvent, and sprayed onto the cores comprising the first coating. The cores coated with the optional second coating are optionally dried, for example at a temperature of 55-57°C, prior to deposition of the third coating.

[0046] Subsequently, a third coating comprising, for example, a taste-masking polymer is disposed on the first or second coating. The coating components are dissolved/suspended in a solvent, and sprayed onto the cores comprising the first coating and optionally the second coating. The third coating is dried at a temperature of 55-57⁰C.

[0047] IQ one embodiment, the multiparticulate in-feed additive is admixed with a basal fish feed. A basal fish feed refers to prepared or artificial diets that promote fish growth and health. Suitable diets include complete diets that contain the required nutrition for fish, e.g., protein, lipid, carbohydrate, ash, phosphorus, water, vitamins and minerals; and supplemental diets that are intended to help support the natural food (e.g, insects, algae, small fish) normally available to fish in ponds, tanks, cages , or outdoor raceways.

[0048] A medicated fish feed is a fish feed comprising a multiparticulate in-feed additive composition comprising praziquantel or a salt thereof and cimetidine or a salt thereof and a basal fish feed. The medicated fish feed can be prepared by incorporating a suitable amount of the multiparticulate in-feed additive composition comprising praziquantel or a salt thereof and cimetidine or a salt thereof into a basal fish feed to achieve the desired dosing levels. The amount of the in-feed additive composition comprising praziquantel and cimetidine incorporated into the fish feed will depend on the rate at which the fish are fed. For fish fed at the rate of 0.2% to 4% of biomass/day, the medicated fish feed specifically contains 50 to 300 mg of the praziquantel per kg of medicated fish feed, more specifically, 100 to 275 mg the praziquantel and per kg of medicated fish feed, and most specifically, 150 to 250 mg the praziquantel per kg of medicated fish feed. For fish fed at the rate of 0.2% to 4% of biomass/day, the medicated fish feed specifically contains 50 to 300 mg of the cimetidine per kg of medicated fish feed, more specifically, 100 to 275 mg the cimetidine per kg of medicated fish feed, and most specifically, 150 to 250 mg the cimetidine per kg of medicated fish feed.

[0049] Several embodiment of fish feed processing may be employed, hi one embodiment, a fish feed pelleting operation is employed. Conventionally, raw materials are passed through a hammer mill or pulverizer until >98% of the particles are reduced to sizes of 250microns and below. These raw materials are then passed to a ribbon mixer, where sensitive materials are added, especially vitamins, carotenoids and products such as praziquantel and cimetidine. Approximately 2% moisture is sprayed into this mixture to maximize pellet durability. The complete feed mix is then pelleted by compression at a die temperature of approximately 9O⁰C. The finished pellets are dried to remove excessive moisture, then cooled, sieved and bagged.

[0050] In another embodiment, a fish feed extrusion operation is employed. Conventionally, raw materials are passed through a hammer mill or pulverizer until >98% of the particles are reduced to sizes of 250 microns and below. These raw materials are then passed to a ribbon mixer, where sensitive materials are added, especially vitamins, carotenoids and products such praziquantel and cimetidine. Approximately 2-7% moisture is sprayed into this mixture to maximize pellet durability. The complete feed mix is then extruded by expansion at a die temperature of approximately 140°C. The finished pellets are dried to remove excessive moisture, then cooled, sieved and bagged.

[0051] In yet another embodiment, a feed mill top dressing operation is employed. Conventionally, raw materials are passed through a hammer mill or pulverizer until >98% of the particles are reduced to sizes of 250microns and below. These raw materials are then passed to a ribbon mixer, ready for extrusion. Approximately 2-7% moisture is sprayed into this mixture to maximize pellet durability. The incomplete feed mix is then extruded by expansion at a die temperature of approximately 140°C. The finished pellets are then passed to a fat coating device (spray nozzle facing into a rotating mixer) for addition of sensitive materials like praziquantel and cimetidine. The mix contains only fish oil, vitamins and the active substance. After oil absorption, pellets are dried to remove excessive moisture, then cooled, sieved and bagged. [0052] In another embodiment, an on-site, e.g., farmer, top dressing operation is employed. Many farmers do not trust feed mills to carry out such operations to their satisfaction, preferring instead to top-dress finished feed they purchase. They will buy their own mixers, and using fish oil or egg white, alginates or gelatin, they will mix the praziquantel and cimetidine with the binder and spray the resultant suspension onto the pellets. Pellets will then be sun dried and re-bagged. There is little high temperature exposure in this type of operation.

[0053] The multiparticulate Praziquantel in-feed additive composition is employed to eliminate or reduce many types of fish parasites, including ectoparasites, as well as endoparasites. The pathogenic endoparasites include cestodes, trematodes, nematodes, Acanthocephalae, in particular:

[0054] From the order of the Pseudophyllidea, for example Diphyllobothrium spp., Spirometra spp., Schistocephalus spp., Ligula spp., Bothridium spp., Diphlogonoporus spp.

[0055] From the order of the Cyclophyllidea, for example Mesocestoides spp., Anoplocephala spp., Paranoplocephala spp., Moniezia spp., Thysanosomsa spp., Thysaniezia spp., Avitellina spp., Stilesia spp., Cittotaenia spp., Andyra spp., Bertiella spp., Taenia spp., Echinococcus spp., Hydatigera spp., Davainea spp., Raillietina spp., Hymenolepis spp., Echinolepis spp., Echinocotyle spp., Diorchis spp., Dipylidium spp., Joyeuxiella spp., Diplopylidium spp.

[0056] From the subclass of the Monogenea, for example Gyrodactylus spp., Dactylogyrus spp.,Diplectanum spp., Polystoma spp., Benedenia spp and Neobenedenia spp. From the subclass of the Digenea, for example Diplostomum spp., Posthodiplostomum spp., Schistosoma spp., Trichobilharzia spp., Ornithobilharzia spp., Austrobilharzia spp., Gigantobilharzia spp., Leucochloridium spp., Brachylaima spp., Echinostoma spp., Echinoparyphium spp., Echinochasmus spp., Hypoderaeum spp., Fasciola spp., Fasciolides spp., Fasciolopsis spp., Cyclocoelum spp., Typhlocoelum spp., Paramphistomum spp., Calicophoron spp-, Cotylophoron spp., Gigantocotyle spp., Fischoederius spp., Gastrothylacus spp., Notocotylus spp., Catatropis spp., Plagiorchis spp., Prosthogonimus spp., Dicrocoelium spp., Eurytrema spp., Troglotrema spp., Paragonimus spp., Collyriclum spp., Nanophyetus spp., Opisthorchis spp., Clonorchis spp., Metorchis spp., Heterophyes spp., Metagonimus spp. From the order of the Enoplida, for example Trichuris spp., Capillaήa spp., Trichomosoides spp., Trichinella spp.

[0057] From the order of the Rhabditia, for example Micronema spp., Stronglyoides spp.

[0058] Ετom the order of the Strongylida, for example Stronylus spp., Triodontophorus spp., Oesophagodontus spp., Trichonema spp., Gyalocephalus spp., Cylindropharynx spp., Poteriostomun spp., Cyclococercus spp., Cylicostephanus spp., Oesophagostomum spp., Chabertia spp., Stephanurus spp., Ancyclostoma spp., Uncinaria spp., Bunostomum spp. ,Globocephalus spp., Syngamus spp., Cyathostoma spp., Metastrongylus spp., Dictyocaulus spp., Muellerius spp., protostrongylus spp., Neostrongylus spp., Cystocaulus spp., Pneumostrongylus spp., Spicocaulus spp., Elaphostrongylus spp., Parelaphostrongylus spp., Crenosoma spp., Paracrenosoma spp., Angiostrongylus spp., Aelurostrongylus spp., Filaroides spp., Parafilaroides spp., Trichostrongylus spp., Haemonchus spp., Ostertagia spp., Marshallagia spp., Cooperia spp., Nematodirus spp., Hyostrongylus spp., Obeliscoides spp., Amidostomum spp., Ollulanus spp.

[0059] From the order of the Oxyurida, for example Oxyuris spp., Enterobius spp., Passalurus spp., Syphacia spp., Aspiculuris spp., Heterakis spp.

[0060] From the order of the Ascaridia, for example Ascaris spp., Toxascaris spp., Toxocara spp., Par ascaris spp., Anisakis spp., Ascaridia spp.

[0061] From the order of the Spirurida, for example Gnathostoma spp., Physaloptera spp., Thelazia spp., Gongylonema spp., Habronema spp., Parabronema spp., Draschia spp., Dracunculus spp.

[0062] From the order of the Filariida, for example Stephanofilaria spp., Parafilaria spp., Setaria spp., Loa spp., Dirofilaria spp., Litomosoides spp., Brugia spp., Wuchereria spp., Onchocerca spp. [0063] From the order of the Gigantorhynchida, for example Filicollis spp., Moniliformis spp., Macracanthorhynchus spp., Prosthenorchis spp.

[0064] The multiparticulate praziquantel and cimetidine in-feed additive compositions disclosed herein are particularly effective as a treatment for Monogenea, such as, but not limited to, Dactylgyrids and Gyrodactylids, Benedeniiae (Allobenedenia, Allometabenedeniella, Ancyrocotyle, Benedenia, Benedeniella, Calicobenedenia, Dioncopseudobenedenia, Lagenivaginopseudobenedenia, Menziesia, Metabenedeniella, Neobenedenia, Oligoncobenedenia, Pseudoallobenedenia, Trimusculotrema), Capsalinae {Caballerocotylla.be, Capsala, Capsaloides, Nasicola, Tricotyla, Tristoma, Tristomella), Dioncinae (Dioncus), Encotyllabinae (Alloencotyllabe, Encotyllabe), Entobdellinae {Entobdella, Pseudoentobdella), Interniloculinae (Interniloculus), Nitzschiinae (Nitzschia), Pseudonitzschiinae {Pseudonitzschia), Trochopodinae {Allomegalocotyla, Macrophyllida, Mediavagina, Megalobenedenia, Megalocotyla, Megalocotyloides, Pseudobenedenia, Pseudobenedeniella, Pseudobenedenoides, Pseudomegalocotyla, Sessilorbis, Sprostonia, Sprostonella, Tetrasepta, Trilobiodiscus, Tocopella, Trochopus).

[0065] The administration can be carried out both prophylactically and therapeutically.

[0066] Fresh water and salt-water fish species are treated with Praziquantel to eliminate and/or reduce parasites. The fish include productive and breeding fish, fish for aquariums and ornamental fish of all ages which live in fresh water, salt water and brackish water. The productive and breeding fish include, for example, carp, eel, trout, white fish, salmonids, bream, roach, rudd, chub, sole, plaice, halibut, Japanese yellowtail (Seriola quinqueradiata), Japanese eel {Anquilla japonica), red seabream (Pagurus major), amberjacks, seabass (Dicentrarchus labrax), grey mullet (Mugilus cephalus), pompano, gilthread seabream (Sparus auratus), tilapia ssp., cichlid species, such as, for example, plagioscion, channel catfish.

[0067] The dose of praziquantel and cimetidine that is effective for reducing, eliminating, or preventing parasites is routinely determined, and varies depending on the species of fish treated, the particular parasites involved, and the degree of infestation. In specific embodiments, praziquantel or a salt thereof is administered at a dose of about 50 mg to about 300 mg per kg of fish biomass per day, more specifically, about 100 mg to about 275 mg per kg of fish biomass per day, most specifically, about 150 mg to about 250 mg per kg of fish biomass per day. In specific embodiments, cimetidine or a salt thereof is administered at a dose of about 50 mg to about 300 mg per kg of fish biomass per day, more specifically, about 100 mg to about 300 mg per kg of fish biomass per day, most specifically, about 150 mg to about 250 mg per kg of fish biomass per day.

[0068] The multiparticulate in-feed additive composition or a medicated fish feed is administered daily, for a period of 3 to 14 days, specifically for 7-14 days, most specifically for 1 week. The multiparticulate in-feed additive composition is effective in reducing parasite infection, for example, when sea bass is treated with the multiparticulate in-feed additive composition, 90.0-99.5 % of fluke infections are eliminated. The multiparticulate in- feed additive can be administered as a prophylactic measure to prevent the occurrence of parasites.

[0069] The invention is further illustrated by the following non-limiting examples.

EXAMPLES

[0070] Example 1. Manufacture process of a first multiparticulate in-feed additive composition containing praziquantel and cimetidine

[0071] Lactose (300 grams) is granulated with praziquantel and cimetidine (200 grams each) with a solution of lactose (80 grams) to produce a mixture of wet granules. These granules are spheronised, then dried at approximately 57⁰C. The granulate is coated with a first coating material containing sodium alginate (2.5 grams) dissolved in polyethylene glycol (15 grams) and water (100 grams) and the solution is sprayed onto the granulate to form a first coating under fluidised bed conditions using a warm air current at 55-57⁰C. The first coated beads are dried at 57⁰C and sieved so that the resulting beads have an average size of about 100 to 300 microns.

[0072] (2.5 grams) of powdered lecithin (Emulpur N, Lucas Meyer GmBH) is dissolved in (100 grams) water. The resulting solution is sprayed onto the granulate containing the first coating to form a second coating using fluidisation. The inlet temperature is 55-57⁰C. Beads were dried at 55-57⁰C.

[0073] Next, solutions containing Eudragit^® E 100 (or Eudragit^® E PO, Rohm & Haas GmbH, Germany) are prepared. (100 grams) of Eudragit^® E 100 and (100 grams) of ethyl cellulose are dissolved in ethanol, and the resulting solutions are sprayed onto the twice coated granulate to form a third coating. The beads are dried and sieved to give an average size of less than 400 microns.

[0074] Table 1 shows the composition of a multiparticulate in-feed additive composition manufactured according to the above-described process.

Table 1

* The amount represents wt% of an ingredient in the total weight of the multiparticulate in- feed additive composition.

Example 2: Manufacture process of a second multiparticulate in- feed additive composition containing Praziquantel and cimetidine

[0075] Lactose (475 grams) is granulated with praziquantel and cimetidine (100 grams each) with a solution of Lactose (80 grams) to produce a mixture of wet granules. These granules are spheronised, then dried at approximately 57⁰C. The granulate is coated with a first coating material containing hydroxypropylmethylcellulose (50 grams) dissolved in (1000 grams) water and 15 grams polyethylene glycol. The hydroxypropylmethylcellulose solution is sprayed onto the granulate to form a first coating under fluidised bed conditions using a warm air current at 55-57⁰C. The first coated beads were dried at 57⁰C and sieved so that the resulting beads had an average size of about 100 to 300 microns.

[0076] Next, solutions containing Eudragit^® E 100 (or Eudragit^® E PO, Rohm & Haas GmbH, Germany) are prepared. (75 grams) of Eudragit^® E 100 and (75 grams) of ethyl cellulose are dissolved in ethanol, and the resulting solutions are sprayed onto the coated granules to form a second coating and dried at 55-57⁰C.

[0077] Next, 30grams of stearic acid was dissolved in lOOgrams solvent. The resulting solution was sprayed onto the granulate containing the first and second coatings to form a third coating; the inlet temperature was 55-57⁰C. The beads were dried and sieved to give an average size of greater than 850 microns.

[0078] Table 2 shows the composition of a multiparticulate in-feed additive composition manufactured according to the above-described process.

Table 2

Example 3. Performance study: palatability and efficiency of the in- feed additive composition containing [0079] A palatability study was performed for 4 months. Two different types of fish feed were used in the study. Each fish feed had three replicate fish test groups.

[0080] Sea Bass {Lates calcarifer) fingerlings (1,000) each were placed in sea cages of 5x5x2m and fed for 16 weeks. Each cage was supplied with natural seawater at ambient temperature (25-32 ⁰C) and salinity (25-35 ppt), at flow rates according to tidal fluctuation. Fish were observed daily for behaviour and adverse drug reactions. Fish mortalities, weights, feed intake, FCR, bacterial counts, viral pathogens, fluke numbers, lice numbers and the occurrence of gross sea lice damage were also recorded.

[0081] The control fish were fed with a fish feed containing no praziquantel and no cimetidine (the Control) and the test fish were fed with a fish feed which contained 0.25% of the multiparticulate in-feed additive composition containing praziquantel and cimetidine (the Test) to give a dietary level of 200 micrograms praziquantel/Kg fish/Day and 20 micrograms cimetidine/Kg fish/Day. Test fish were fed on a 7-day on, 7 day off regimen.

[0082] The test fish gained weight during the experimental period, illustrating that there were no palatability problems during the 16-week test period, hi addition, the test fish had a lower Feed Conversion Ratio ("FCR") compared to the control fish (Figure 1). FCR is calculated from the number of kilograms of feed that are used to produce one kilo of whole fish. Specifically, FCR is the mass of the food eaten divided by the body mass gain, over a specified period of time (e.g., the 16-week test period). Thus, the test fish used the feed more efficiently.

[0083] Furthermore, efficacy of the Test formulation was high as evidenced in Figure 2, since sea lice numbers fall drastically (e.g., sea lice number of the test fish was down over 99% compared to the control fish) upon commencement of treatment and maintained at that low level during the whole 16-week period.

[0084] Particle sizes were measured by electron microscopy.

[0085] The multiparticulate in-feed additive composition disclosed herein provides an improved delivery system of praziquantel and cimetidine that is particularly suitable for aquaculture applications. For example, the in-feed additive composition is resistant to water and acid, and provides gastric bypass after ingestion by a fish. Additionally, the in-feed additive composition is resistant to the high temperatures up to 140°C under which a medicated fish-feed is manufactured.

[0086] Advantages of the formulations disclosed herein include low dust content, therefore inhalation exposure is considerably reduced compared to prior art formulations. Also, particle size is substantially more uniform than prior art formulations, and the coating ensures there is a pronounced barrier between the active substance and the environment. It is expected that the formulations disclosed herein will have a reduced environmental impact when compared to prior art formulations. Environmental concerns are paramount, especially in salmonid producing countries.

[0087] The use of the terms "a" and "an" and "the" and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality. The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0088] While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0089] All cited patents, patent applications, and other references are incorporated herein by reference in their entirety.

Claims

CLAIMS:

1. A multiparticulate in-feed additive composition comprising:

a plurality of granular cores, the cores comprising an inert hydrosoluble carrier, praziquantel or a salt thereof and cimetidine or a salt thereof, and

a first coating disposed on the 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.

2. The composition of claim 1, comprising

20 wt% to 70 wt% of the inert hydrosoluble carrier,

2 wt% to 30 wt% of the praziquantel,

2 wt% to 30 wt% of the cimetidine,

2 wt% to 20 wt% of the hydrogel-forming polymer,

2 to 10 wt% of the barrier material, and

5 wt% to about 25 wt% of the taste-masking polymer.

3. The composition of claim 1 , comprising

25 wt% to 55 wt% of the inert hydrosoluble carrier,

5 wt% to 25 wt% of the praziquantel,

5 wt% to 25 wt% of the cimetidine,

5 wt% to 15 wt% of the hydrogel-forming polymer,

3 to 8 wt% of the barrier material, and

15 wt% to about 25 wt% of the taste-masking polymer.

4. The composition of claim 1, wherein the hydrogel-forming polymer comprises sodium alginate, potassium alginate, carrageenan, or a mixture thereof.

5. The composition of claim 1, wherein the taste-masking polymer is soluble in gastric fluid at pHs less than or equal to 5.

6. The composition of claim 1, wherein the taste-masking polymer comprises butyl methacrylate-(2-dimethylaminoethyl methacrylate)-methyl methacrylate copolymer (1:2:1), comprises 1 :2:1 (butyl methacrylate-co-(2- dimethylaminoethyl)methacrylate-co-methyl methacrylate) having an average molecular weight of about 150,000, or a combination thereof.

of the total weight of the multiparticulate in- feed additive composition.

7. The composition of claim 1, wherein the barrier material comprises lecithin.

8. A medicated fish- feed comprising the multiparticulate in-feed additive composition of claim 1 and a basal fish- feed.

9. The medicated fish-feed of claim 8, wherein the multiparticulate in- feed additive composition comprises:

20 wt% to 70 wt% of the inert hydrosoluble carrier,

2 wt% to 30 wt% of the praziquantel,

2 wt% to 30 wt% of the cimetidine,

2 wt% to 20 wt% of the hydrogel-forming polymer,

2 to 10 wt% of the barrier material, and

5 wt% to about 25 wt% of the taste-masking polymer.

10. A method of reducing, eliminating, or preventing parasites in a fish population, comprising: feeding the multiparticulate in-feed additive composition of claim 1 to the fish population at a daily dose of praziquantel of about 50 mg to about 300 mg per kg of fish biomass per day and a daily dose of cimetidine of about 50 mg to about 300mg per kg offish biomass per day for a period of 3-14 days.

11. A multiparticulate in- feed additive composition comprising:

12. The composition of claim 1, comprising

20 wt% to 70 wt% of the inert hydrosoluble carrier,

2 wt% to 30 wt% of the praziquantel,

2 wt% to 30 wt% of the cimetidine,

2 wt% to 20 wt% of the film- forming polymer,

5 wt% to about 25 wt% of the taste-masking polymer, and

0.5 wt% to 6 wt% wt% of the lubricant.

13. The composition of claim 1 , comprising

20 wt% to 70 wt% of the inert hydrosoluble carrier,

2 wt% to 30 wt% of the praziquantel,

2 wt% to 30 wt% of the cimetidine,

5 wt% to 15 wt% of the film- forming polymer,

15 wt% to about 25 wt% of the taste-masking polymer, and

1 wt% to about 5 wt% of the lubricant.

14. The composition of claim 11, wherein the film-forming polymer comprises ethylcellulose.

15. The composition of claim 11, wherein the taste-masking polymer is soluble in gastric fluid at pHs less than or equal to 5.

16. The composition of claim 11, wherein the taste-masking polymer comprises butyl methacrylate-(2-dimethylaminoethyl methacrylate)-methyl methacrylate copolymer (1:2:1), comprises 1:2:1 (butyl methacrylate-co-(2- dimethylaminoethyl)methacrylate-co-methyl methacrylate) having an average molecular weight of about 150,000, or a combination thereof.

of the total weight of the multiparticulate in- feed additive composition.

17. The composition of claim 11, wherein the lubricant comprises stearic acid.

18. A medicated fish- feed comprising the multiparticulate in-feed additive composition of claim 11 and a basal fish- feed.

19. The medicated fish- feed of claim 18, wherein the multiparticulate in- feed additive composition comprises:

20 wt% to 70 wt% of the inert hydrosoluble carrier,

2 wt% to 30 wt% of the praziquantel,

2 wt% to 30 wt% of the cimetidine,

2 wt% to 20 wt% of the film- forming polymer,

5 wt% to about 25 wt% of the taste-masking polymer, and

0.5 wt% to 6 wt% wt% of the lubricant.

20. A method of reducing, eliminating, or preventing parasites in a fish population, comprising: feeding the multiparticulate in-feed additive composition of claim 11 to the fish population at a daily dose of praziquantel of about 50 mg to about 300 mg per kg of fish biomass per day and a daily dose of cimetidine of about 50mg to about 300mg per kg of fish biomass per day for a period of 3-14 days.