Dihydroisoxazole compounds for reducing infection of ectoparasites on fish

Abstract

Ectoplasmic parasite infections in fish farming can cause significant difficulties and problems for the aquaculture industry. This disclosure generally relates to the treatment or control of ectopic parasite infections (such as sea lice) in fish (such as salmonids) using specific dosage regimens of isoxazoline. Specifically, the dosage regimen comprises the active ingredient shown in structural formula (I): (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, including its salt, or N-oxide or solvate, at a dose of about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably about 0.025 mg / kg / day to about 0.125 mg / kg / day. Specifically, the dosage administration regimen includes oral administration for approximately 3 to approximately 10 consecutive days, preferably over a period of 7 consecutive days.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 538,786, filed September 15, 2023, and U.S. Provisional Patent Application Serial No. 63 / 610,853, filed December 15, 2023. The entire contents of each of the foregoing documents are incorporated herein by reference. Technical Field

[0002] This application generally relates to the field of treatment of ectoparasites in fish, and more specifically, to the use of isoxazoline compounds for the treatment of ectoparasite infections in fish, particularly farmed fish. Background Technology

[0003] Ectopic parasite infections in fish farming can cause significant difficulties and problems for the aquaculture industry. Ectopic parasites are external parasites that attach themselves to the skin, gills, or fins of fish, feeding on the fish's blood, mucus, or tissues. The most common ectoparasites in fish farming include copepods, such as the genus *Ceratophorus* (*Ceratophorus*). Lepeophtheirus ), Fish louse ( Caligus ) and the genus *Ariocarpus* ( Argulus Members of [unspecified group]. Some key issues associated with ectoparasite infections in fish farming can include reduced growth and feed conversion efficiency, skin and gill damage, secondary infections from open wounds, and mortality associated with severe infections. The economic impact of ectoparasite infections on fish farmers can be significant; for example, mortality, reduced growth rates, and treatment costs can lead to substantial economic losses.

[0004] Sea lice infestations are particularly challenging for aquaculture, especially salmonid aquaculture, as these crustacean ectoparasites feed on the mucus, tissues, and blood of their host marine fish. Sea lice belong to the class Copepoda (…). Copepoda ), *Dystomata* (order *Dystomata*) Siphonostomatoida Fish lice family ( Caligidae ) and several genera, including the genus *Cryptocoryne* ( Lepeophtheirus ) and fish lice ( Caligus ).

[0005] Since the large-scale farming of salmonids, the treatment or control of ectoparasites such as sea lice has primarily relied on the application of chemotherapeutic agents in immersion or medicated feed. Since its introduction in 2000, emamectin benzoate, or SLICE® (Merck Animal Health, USA), has been the most successful feed-based drug for controlling sea lice, and has subsequently been licensed in Canada, Norway, Scotland, Ireland, the Faroe Islands, and Chile. Emamectin benzoate is formulated in premixes prescribed by veterinarians and delivered to aquaculture feed mills for surface coating on fish feed. However, due to repeated use of SLICE®, and sometimes even alone, sea lice have gradually developed resistance to this chemotherapeutic agent. Similarly, resistance to hydrogen peroxide has emerged due to the heavy reliance on its use in immersion treatments. While the use of chemotherapeutic agents is decreasing due to resistance issues, the overall frequency of treatment events continues to rise under the ongoing pressure of ectoparasite infestations such as sea lice, leading to a significant increase in non-pharmacological treatments.

[0006] Mechanical lice removal systems are also widely used in major salmon farming areas. These systems include high-pressure water jets and warm water systems to separate sea lice. In Europe and more recently in eastern Canada, the stocking of cleaner fish in net cages has become a common practice. However, this comes with additional welfare considerations for the care of these cleaner fish, as well as the need to vaccinate them according to biosecurity measures to prevent outbreaks of infectious diseases in salmon.

[0007] In the foreseeable future, ectoparasite infections will continue to be a serious biological and economic problem in fish farming, particularly salmon farming. Ectoparasites, such as sea lice, have a strong ability to rapidly adapt to changes in the marine environment, allowing them to thrive under fish farming conditions despite human intervention.

[0008] Another challenge is that any treatment targeting ectoparasites, such as sea lice, may have unintended side effects on other commercially important crustaceans, such as lobsters and crabs. This disclosure attempts to address these prominent issues.

[0009] US Patent 9,920,037 discloses a member of the isoxazoline benzamide (IOB) class, namely: (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisooxazo-3-yl)thiophene-2-carboxamide, including its salts or solvates, with the following structural formula:

[0010] This specific active ingredient has not yet been granted marketing authorization for any species or indication. U.S. Patent 9,920,037 discloses the use of the above compound for reducing or eliminating ectoparasites on the body or surface of non-human animals, livestock, pets, and fish. U.S. Patent 9,920,037 discloses the application of the compound to Atlantic salmon at a dose of 1.0 mg / kg / day in medicated feed for 7 consecutive days, and the effect on sea lice (salmon lice) when lice counts were performed on the fish 10 days after the end of treatment. Lepeophtherius salmonis It has 100% efficacy on the attachment larvae (chalimus) and pseudo-adult / adult stages. Summary of the Invention

[0011] The present invention is provided to introduce a simplified form of the concept chosen, which will be further described below in the detailed description. The present invention is not intended to identify key or essential aspects of the claimed subject matter.

[0012] The following non-limiting embodiments of this disclosure are contemplated: 1. A composition comprising the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, its salt, N-oxide, or solvate, having the structural formula shown below:

[0013] The composition is intended for oral administration at a dose of the active ingredient, or a salt thereof, or an N-oxide or solvate thereof, at a dose of about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably about 0.025 mg / kg / day to about 0.125 mg / kg / day.

[0014] 2. The composition according to Embodiment 1, wherein the composition is prepared for feeding fish at a feeding rate in the range of 0.4%-2.6% body weight / day.

[0015] 3. The composition according to embodiment 1 or 2, wherein the oral administration is performed continuously for about 3 to about 10 days, preferably for 7 days.

[0016] 4. The composition according to any one of embodiments 1 to 3, comprising about 0.01 wt.% to about 99 wt.%, preferably about 0.01 wt.% to about 0.5 wt.%, more preferably about 0.1 wt.% to about 0.5 wt.%, of the active ingredient, or its salt, or N-oxide or solvate.

[0017] 5. The composition according to any one of embodiments 1 to 4, wherein the composition is medicated fish feed.

[0018] 6. The composition according to embodiment 5, wherein the medicated fish feed is feed pellets or feed granules.

[0019] 7. The composition according to any one of embodiments 1 to 6, wherein the dosage is about 0.025 mg / kg / d, about 0.050 mg / kg / d, or about 0.125 mg / kg / d.

[0020] 8. The composition according to any one of embodiments 1 to 7, wherein the composition is prepared for oral administration to fish.

[0021] 9. The composition according to embodiment 8, wherein the fish is a salmonid.

[0022] 10. The composition according to any one of embodiments 1 to 9, wherein the composition is prepared for treating or controlling ectoparasite infections in fish.

[0023] 11. The composition according to embodiment 10, wherein the ectoparasitic infection is a sea louse infection. For example, the sea louse infection includes infected copepods, chalimi, pseudo-adults, adults, or combinations thereof during the louse's life stage.

[0024] 12. A composition comprising the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt thereof, or an N-oxide or solvate thereof, having the following structural formula:

[0025] The composition is used to treat or control ectoparasitic infections in fish, wherein the composition is administered orally to fish at a dose of about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably about 0.025 mg / kg / day to about 0.125 mg / kg / day of the active ingredient, or a salt, or N-oxide or solvate thereof.

[0026] 13. The composition for the said use according to embodiment 12, wherein the fish is a salmonid.

[0027] 14. The composition for the stated purpose according to embodiment 12 or 13, wherein the ectoparasitic infection is a sea louse infection. For example, the sea louse infection includes copepods, appendage larvae, pseudo-adults, or adults, or combinations thereof, in the life stage of the infected louse.

[0028] 15. A composition for the purpose according to any one of embodiments 12 to 14, wherein the composition comprises about 0.01 wt.% to about 99 wt.%, preferably about 0.01 wt.% to about 0.5 wt.%, more preferably about 0.1 wt.% to about 0.5 wt.%, of the active ingredient, or a salt, or N-oxide or solvate thereof.

[0029] 16. The composition for the purpose according to any one of embodiments 12 to 15, wherein the composition is medicated fish feed.

[0030] 17. The composition for the purpose according to embodiment 16, wherein the medicated fish feed is in the form of feed pellets or feed granules.

[0031] 18. The composition for the purpose according to any one of embodiments 12 to 17, wherein the composition is prepared for feeding fish at a feeding rate in the range of 0.4% to 2.6% body weight per day.

[0032] 19. The composition for the purpose according to any one of embodiments 12 to 18, wherein the dosage is about 0.025 mg / kg / d, about 0.050 mg / kg / d, or about 0.125 mg / kg / d.

[0033] 20. The composition for the purpose according to any one of embodiments 12 to 19, wherein the composition is administered orally over a period of about 3 to about 10 days, preferably over a period of 7 days.

[0034] 21. A method for treating or controlling ectoparasitic infections in fish, comprising orally administering to the fish an active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt thereof, or an N-oxide or solvate thereof, having the following structural formula:

[0035] The dosage is from about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably from about 0.025 mg / kg / day to about 0.125 mg / kg / day.

[0036] 22. The method according to embodiment 21, wherein the ectoparasitic infection is a sea louse infection. For example, the sea louse infection includes copepods, larvae, pseudo-adults, or adults, or combinations thereof, at the infecting louse life stage.

[0037] 23. The method according to embodiment 21 or 22, wherein the oral administration is performed continuously for about 3 to about 10 days, preferably for 7 days.

[0038] 24. The method according to any one of embodiments 21 to 23, wherein the active ingredient, or its salt, or N-oxide or solvate, is included in the oral composition for fish.

[0039] 25. The method according to embodiment 24, wherein the oral fish composition comprises about 0.01 wt.% to about 99 wt.%, preferably about 0.01 wt.% to about 0.5 wt.%, more preferably about 0.1 wt.% to about 0.5 wt.%, of the active ingredient, or a salt, or N-oxide or solvate thereof.

[0040] 26. The method according to embodiment 24 or 25, wherein the oral composition for fish is fish feed.

[0041] 27. The method according to embodiment 26, wherein the fish feed is feed pellets or feed granules.

[0042] 28. The method according to any one of embodiments 21 to 27, wherein the dose is about 0.025 mg / kg / d, about 0.050 mg / kg / d or about 0.125 mg / kg / d of the active ingredient, or a salt, or N-oxide or solvate thereof.

[0043] 29. The method according to any one of embodiments 21 to 28, wherein the fish is a salmonid.

[0044] 30. The active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or its salt, N-oxide, or solvate, has the following structural formula:

[0045] Use in the production of a composition for treating or controlling ectoparasitic infections in fish, wherein the composition is used for oral administration to fish at a dose of about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably about 0.025 mg / kg / day to about 0.125 mg / kg / day of the active ingredient, or a salt thereof, or an N-oxide or solvate thereof.

[0046] 31. The use according to embodiment 30, wherein the ectoparasitic infection is a sea louse infection. For example, the sea louse infection includes copepods, appendage larvae, pseudo-adults, or adults, or combinations thereof, in the life stage of the infected louse.

[0047] 32. The use according to embodiment 30 or 31, wherein the oral administration is performed continuously for about 3 to about 10 days, preferably for 7 days.

[0048] 33. The use according to any one of embodiments 30 to 32, wherein the composition is an oral composition for fish.

[0049] 34. The use according to embodiment 33, wherein the oral composition for fish contains about 0.01 wt.% to about 99 wt.%, preferably about 0.01 wt.% to about 0.5 wt.%, more preferably about 0.1 wt.% to about 0.5 wt.%, of the active ingredient, or a salt, or N-oxide or solvate thereof.

[0050] 35. The use according to embodiment 33 or 34, wherein the oral composition for fish is fish feed.

[0051] 36. The use according to embodiment 35, wherein the fish feed is feed pellets or feed granules.

[0052] 37. The use according to any one of embodiments 30 to 36, wherein the dosage is about 0.025 mg / kg / d, about 0.050 mg / kg / d, or about 0.125 mg / kg / d.

[0053] 38. The use according to any one of embodiments 30 to 37, wherein the fish is a salmonid.

[0054] 39. The composition for the purpose according to any one of embodiments 12 to 20, wherein the composition is used to feed fish at a feeding rate in the range of 0.4% to 2.6% body weight per day.

[0055] All features of the exemplary embodiments described in this disclosure, which are not mutually exclusive, can be combined with each other. Elements of one embodiment can be used in other embodiments without further mention. Other aspects and features of the invention will become apparent to those skilled in the art by consulting the following detailed description in conjunction with the accompanying drawings. Attached Figure Description

[0056] The following detailed description of specific exemplary embodiments is provided herein with reference to the accompanying drawings, wherein: Figure 1 This is a non-restrictive plot showing the mean number of attached lice (attached larval stages 1 and 2) / number of fish on day 17 prior to continuous treatment with sea lice at dose rates of 0 (negative control), 0.010, 0.025, 0.050, and 0.125 mg / kg / d, respectively. Lice counts were performed on days 2, 12, and 16 post-treatment (DPT).

[0057] Figure 2 This is a non-restrictive plot showing the average number of active lice (pseudo-adults and adult lice) per fish on day 17 before continuous administration of sea lice at dose rates of 0 (negative control), 0.010, 0.025, 0.050, and 0.125 mg / kg / d, respectively. Lice counts were performed on days 2, 12, and 16 of the DPT.

[0058] Figure 3 This is a non-restrictive plot showing the mean total number of sea lice / fish number on day 17 before continuous administration of sea lice at dose rates of 0 (negative control), 0.010, 0.025, 0.050, or 0.125 mg / kg / d for 7 consecutive days. Lice counts were performed on days 2, 12, and 16 of the DPT.

[0059] Figure 4 This is a non-limiting graph showing the percentage reduction (%) in total sea lice / fish number relative to the untreated control when fish were infected with sea lice for 7 consecutive days prior to treatment at dose rates of 0.010, 0.025, 0.050, 0.125, and 1.0 mg / kg / d. For the 1.0 mg / kg / d dose, lice were counted on day 10 of the DPT, and for the 0.010, 0.025, 0.050, and 0.125 mg / kg / d doses, lice were counted on days 2, 12, and 16 of the DPT.

[0060] Figure 5 This is a non-restrictive plot showing the mean number of attachment lice (attachment larval stages 1 and 2) / fish number after continuous administration for 7 days at dose rates of 0 (negative control), 0.025, and 0.060 mg / kg / d, respectively, and infection on days 8 and 17 of the DPT. Lice counts were performed on day 21 post-infection (DPI).

[0061] Figure 6 This is a non-restrictive plot showing the average number of active lice (pseudo-adults and adult lice) / fish number after continuous administration for 7 days at dose rates of 0 (negative control), 0.025, and 0.060 mg / kg / d, respectively, and infection on days 8 and 17 of DPT. Lice counts were performed on days 21, 24, and 34 of DPI.

[0062] Figure 7 This is a non-restrictive plot showing the mean total number of sea lice / fish after continuous administration for 7 days at dose rates of 0 (negative control), 0.025, and 0.060 mg / kg / d, respectively, and infection on days 8 and 17 of DPT. Lice counts were performed at 21, 24, and 34 DPI.

[0063] Figure 8 This is a non-limiting graph showing the percentage reduction (%) in total sea lice / fish number relative to the untreated control, after continuous administration for 7 days at dose rates of 0 (negative control), 0.025, and 0.060 mg / kg / d, and infection on days 8 and 17 post-treatment. Lice counts were performed on days 21, 24, and 34 of the DPI.

[0064] Exemplary embodiments are shown in the accompanying drawings by way of example. It will be clearly understood that the description and drawings are for illustrative purposes only and to aid understanding. They are not intended to limit the scope of the invention. Detailed Implementation

[0065] The present technology is explained in more detail below. This description is not intended to be a detailed list of all different ways in which the technology can be implemented or all features that can be added to the technology. For example, a feature shown with respect to one embodiment may be incorporated into other embodiments, and a feature shown with respect to a particular embodiment may be removed from that embodiment. Furthermore, it will be apparent to those skilled in the art, in consideration of this disclosure, to various variations and additions to the many embodiments suggested herein, wherein such variations and additions do not depart from the present technology. Therefore, the following description is intended to illustrate some embodiments of the technology, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0066] The inventors have surprisingly and unexpectedly discovered that oral administration of the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide (also referred to herein as “S-roxapin”), including its salts, N-oxides or solvates, with the following structural formula: [The active ingredient is described in the original text.]

[0067] It is therapeutically active against ectoparasitic infections (such as sea lice) in fish (e.g., members of the salmon family). Throughout the product information, expressions such as "mg / kg / d" refer to the number of milligrams of the active ingredient, or its salt, or N-oxide, or solvate, administered per kilogram of fish body weight per day. Throughout the product information, expressions such as "at a certain dose," "at a certain dose rate," or "at a certain dose regimen" are used interchangeably.

[0068] In some embodiments, the active ingredient, or its salt, or N-oxide or solvate, is applied over a period of about 3 to about 10 days, preferably over a period of at least 7 days, more preferably over a period of 7 days.

[0069] Treatment for at least 7 consecutive days is preferred because there is a well-documented feeding level in the fish. Therefore, remedying this situation by treating for at least 7 consecutive days results in more uniform feed intake within the group.

[0070] This reduced dosage offers a technological advantage in commercial fish aquaculture environments due to the decreased likelihood of negative environmental impacts, such as side effects on non-target species. In other words, the reduced dosage of the active ingredient, or its salt, or N-oxide or solvate, described herein for controlling or treating ectoparasitic infections (such as sea lice) in fish (such as salmonids) reduces its release into the aquatic environment near the fish farm, which could otherwise negatively impact other aquatic organisms.

[0071] Unbound by any theoretical framework, this therapeutic activity is believed to be unexpected and unpredictable at the reduced doses described herein. The active ingredient, (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt thereof, or an N-oxide or solvate thereof, is a member of the isoxazoline benzamide (IOB) class. Its assumed pharmacological properties are inferred from its structure and the known effects of related compounds in the same class; however, the dosage must be derived empirically, resulting in unpredictable dose-effects. Unlike warm-blooded animals (mammals or birds), drug metabolism in fish, including salmon, is temperature-dependent, and little is known about the specific metabolic pathways by which drug compounds are cleared. This specific active ingredient, or its salt thereof, or an N-oxide or solvate thereof, has not been marketed for any species or indication. There is no publicly available data regarding its intended indications (treatment of ectoparasites, such as sea lice) in the target species (fish, such as salmonids) at these low dose rates.

[0072] Globally, four members of the IOB class have been marketed for the prevention of fleas and ticks in dogs and cats, namely NexGard. TM ), Simparica TM ), Loterana (Credelio) TM ) and Freranil (Bravecto TM Frerano (Bravecto) TM In Europe, it is approved for administration twice in drinking water at a dose rate of 0.5 mg / kg bw (animal body weight), 7 days apart, for a total dose of 1.0 mg / kg bw, for use in edible animal species to combat red spider mites on poultry. The aforementioned Class IOB members have higher dose rates in dogs and cats, with single oral doses ranging from 2 to 40 mg / kg bw (animal body weight) per treatment.

[0073] Although compounds in this class act with similar modes of action (in this case, gamma-aminobutyric acid (GABA) and glutamate-gated chloride channels), differences do exist among them affecting pharmacological activity. For example, compounds closely related to the disclosed compounds shown below showed no activity against copepod larvae of sea lice in similar doses of bath solution.

[0074]

[0075] The above compounds differ only slightly from the published compounds, which shows that even seemingly minor atomic differences can affect pharmacological activity.

[0076] More generally, among the four approved drugs, the labeled dosage ranges from 0.5 mg / kg twice in drinking water, 7 days apart, for a total dose of 1.0 mg / kg per treatment (poultry), to 2-40 mg / kg in a single oral dose for dogs and cats per treatment.

[0077] The inventors conducted a first set of experiments, administering 0.050, 0.125, and 0.250 mg / kg / day to salmon in medicated feed for seven consecutive days to determine the non-therapeutic dose for controlling or treating sea lice infestations. Since these lower doses represent a significant reduction in concentration compared to that described in U.S. Patent 9,920,037 (1.0 mg / kg / day), and also represent concentrations far below those approved for use in dogs, cats, and poultry by regulatory agencies such as the U.S. Food and Drug Administration, Health Canada, and the European Medicines Agency between 2014 and 2021, it was reasonable to expect to find a non-therapeutic dose at lower test doses. Unexpectedly, all dose rates evaluated in the first set of experiments were nearly 100% effective in reducing sea lice burden.

[0078] Then, the inventors conducted a second set of experiments, administering salmon to medicated feed for seven consecutive days at lower doses of 0.010, 0.025, 0.050, and 0.125 mg / kg / day to determine the non-therapeutic dose for controlling or treating sea lice infestations. The results showed that the doses of 0.025, 0.050, and 0.125 mg / kg / day were significantly more effective than the lowest dose (0.010 mg / kg / day). This difference indicates a first approximation of the non-therapeutic dose. It is tentatively inferred that the minimum effective dose observed is in the range of 20-40 times lower than the dose shown in U.S. Patent 9,920,037. It is not reasonable to predict that it will remain effective at concentrations 20-40 times lower than those shown in U.S. Patent 9,920,037. These results are as follows... Figure 4 As shown, the reduction in sea lice percentage at doses of 0.025, 0.050, and 0.125 mg / kg / d is similar to the results observed at the dose of 1.0 mg / kg / d described in U.S. Patent 9,920,037.

[0079] In some embodiments, this disclosure relates to compositions comprising the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt thereof, or an N-oxide or solvate thereof, having the following structural formula:

[0080] The composition is intended for oral administration at a dose of about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably about 0.025 mg / kg / day to about 0.125 mg / kg / day of the active ingredient, or a salt, or N-oxide or solvate thereof.

[0081] In some embodiments, this disclosure relates to compositions comprising the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt thereof, or an N-oxide or solvate thereof, having the following structural formula:

[0082] The composition is used to treat ectoparasitic infections (such as sea lice) in fish (such as salmonids), wherein the composition is administered orally to fish at a dose of about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably about 0.025 mg / kg / day to about 0.125 mg / kg / day of the active ingredient, or a salt, or N-oxide or solvate thereof.

[0083] In some embodiments, this disclosure relates to a method for treating ectoparasitic infections in fish, the method comprising orally administering the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt thereof, or an N-oxide or solvate thereof, having the structural formula shown below: .

[0084] In some embodiments, this disclosure relates to the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt, N-oxide, or solvate thereof, having the following structural formula:

[0085] Use in the production of compositions for treating or controlling ectoparasitic infections in fish, wherein the compositions are used for oral administration to fish at a dose of about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably about 0.025 mg / kg / day to about 0.125 mg / kg / day of the active ingredient, or a salt thereof, or an N-oxide or solvate thereof.

[0086] In some embodiments, those skilled in the art will recognize that the compositions described herein include, for example, liquid formulations or solid forms. For example, solid forms may include capsules, powders, including soluble powders, granules, particles, or encapsulation of the active ingredient, or its salts, or N-oxides, or solvates, in a polymer, such as cross-linked alginate particles. Liquid forms may include the active ingredient, or its salts, or N-oxides, or solvates, ready-to-use liquids or powders for reconstitution, which may be administered orally and parenterally (injection, immersion, and topical).

[0087] In some embodiments, for ease of administration, the composition may include medicated fish feed, for example, fish feed supplemented to include the active ingredient described herein, or its salt, or N-oxide, or solvate. Such fish feed is typically prepared by commercial feed mills according to veterinary instructions (e.g., prescriptions) and / or based on labels and delivered to fish farms. For example, in some jurisdictions, such commercial feed mills may be regulated and “licensed” by local regulatory bodies to allow them to produce medicated fish feed. Alternatively, where permitted, such medicated feed may be prepared on fish farms.

[0088] In some embodiments, the fish feed described herein may be in the form of feed pellets or granules. Fish feed is typically present in pellet or pellet form and contains components such as fish meal, fish oil, vegetable protein, poultry meal, feather meal, pork protein powder, sugars, and polysaccharides (such as mannan, dextran, and alginate). Additionally, the formulation may contain excipients such as pigments, vitamins, minerals, and binders. The active ingredients, their salts, N-oxides, or solvates may be integrated into the feed pre-granulation. Alternatively, the active ingredients, their salts, N-oxides, or solvates may be applied as a coating, alone or in a premix, to the feed pellets or granules. This premix, together with the active ingredients, their salts, N-oxides, or solvates, may contain veterinary-grade excipients such as starch, calcined silica, microcrystalline cellulose, lactose, and preservatives. The active ingredients, salts, N-oxides, or solvates may be incorporated into the feed mixture prior to the granulation process. Fish feed, such as salmonid feed, is known in the art and will not be described further in this document for the sake of brevity.

[0089] In some embodiments, the complete treatment duration for applying the active ingredient, salt, N-oxide, or solvate to the fish is from about 3 to about 14 days (about 2 weeks). In some embodiments, it ranges from about 3 to about 7 days, while in another embodiment, it is extended to from about 5 to about 14 days. In yet another embodiment, the treatment duration is from about 5 to about 10 days (about 1.5 weeks). Preferably, the treatment duration is at least 7 days. More preferably, the treatment duration is 7 days.

[0090] In some embodiments, the composition comprises fish feed containing the active ingredient described herein, or a salt thereof, or an N-oxide or solvate thereof. For example, the active ingredient, or a salt thereof, or an N-oxide or solvate thereof, can be incorporated into a premix, and the premix can be incorporated into or coated onto the fish feed. The premix can be applied to the fish feed by dry mixing followed by an oil coating, or by mixing the premix in oil and then spraying it onto the fish feed.

[0091] In some embodiments, when the fish feed comprises an active ingredient, or a salt thereof, or an N-oxide or solvate thereof, on at least its surface portion, the active ingredient, or its salt thereof, or an N-oxide or solvate thereof, can be coated with a suitable oil layer. Those skilled in the art will recognize that several commercially available oils suitable for this purpose are available, such as herring oil or vegetable oil.

[0092] As a general non-limiting rule, the compositions described herein contain from about 0.01 wt.% to about 99 wt.%, such as from about 0.01 wt.% to about 95 wt.%, of the active ingredient, or a salt thereof, or an N-oxide or solvate thereof. The reader will readily understand that the above ranges include any values ​​therein, such as about 0.01 wt.% (about 0.1 mg / g), about 0.02 wt.% (about 0.2 mg / g), about 0.025 wt.% (about 0.25 mg / g), about 0.03 wt.% (about 0.30 mg / g), about 0.04 wt.% (about 0.40 mg / g), about 0.05 wt.% (about 0.50 mg / g), about 0.06 wt.% (about 0.60 mg / g), about 0.07 wt.%, etc. t.% (approximately 0.70 mg / g), approximately 0.08 wt.% (approximately 0.80 mg / g), approximately 0.09 wt.% (approximately 0.90 mg / g), approximately 0.1 wt.% (approximately 1.0 mg / g), approximately 0.2 wt.% (approximately 2.0 mg / g), approximately 0.25 wt.% (approximately 2.5 mg / g), approximately 0.3 wt.% (approximately 3.0 mg / g), approximately 0.4 wt.% (approximately 4.0 mg / g), approximately 0.5 wt.% (approximately 5.0 mg / g), or greater. The reader will also readily understand that the above values ​​can form suitable ranges encompassing these values, such as approximately 0.01 wt.% (approximately 0.1 mg / g) to approximately 0.5 wt.% (approximately 5.0 mg / g) or approximately 0.1 wt.% (approximately 1.0 mg / g) to approximately 0.5 wt.% (approximately 5.0 mg / g). Readers will also readily understand that, based on current industrial practice, medicated feed (i.e., the compositions of this disclosure) can be prepared for feeding fish at a feeding rate (inclusive of any value or range) in the range of 0.4%-2.6% body weight per day (“bw / d”).

[0093] In some embodiments, the duration of efficacy against salmon sea lice obtained using the dosage described herein is at least 90% efficacy (e.g.) for at least 2 days after treatment, preferably at least 12 days after treatment, and can be expected to be up to 16 days after treatment.

[0094] In some embodiments, the active ingredient, or its salt, or N-oxide or solvate, is used to control or treat fish parasitic crustaceans, particularly to address sea lice infestations, which include infected copepods, appendage larvae, pseudo-adults, or adults, or combinations thereof, at the lice's life stage.

[0095] Unless otherwise stated, as used herein, the term "fish" refers to the taxonomic classes Chondrichthyes (cartilaginous fish, such as sharks and rays) and Osteichthyes (boneless fish), which live in water and have gills or mucus-covered skin, fins, and may have scales. This includes edible fish, spawning fish, and aquarium and pond fish, as well as farmed fish of all ages existing in freshwater, seawater (such as the ocean), and brackish water. This disclosure relates more specifically to marine fish, and more specifically to marine edible fish, particularly salmon. Within the scope of this invention, the term "salmon" should be understood to include all representatives of the Salmonidae family, particularly the following species: Atlantic salmon (… Salmo salar (Atlantic salmon); Brown trout ( Salmo trutta (Brown trout or sea trout); Rainbow trout ( Oncorhynchus mykiss (Rainbow trout); and Pacific salmon (chum salmon) Oncorhynchus ): hunchbacked salmon ( O. gorbuscha ); salmon ( O. keta ); Saffron salmon ( O. nerka ); Silver salmon ( O. kisutch ), large-scaled salmon ( O. tshawytscha ) and salmon ( O. masou It also includes artificially bred species, such as species of the genus *Salmon* (*Salmon*). Salvelinus spp.) and red salmon ( Salmo clarki The preferred hosts disclosed herein are Atlantic salmon, Pacific salmon, and sea trout.

[0096] A fish colony refers to a group of individual fish raised in an enclosed area such as a seawater tank, net cage, or fishing net. Net cages and fishing nets are anchored at the estuary, allowing the tides to flow over them daily, thus ensuring an adequate supply of oxygen and clean water. Net cages, on the other hand, either maintain a continuous flow of seawater or are periodically flushed with fresh seawater to ensure optimal water quality and oxygen levels for the fish's health. In this controlled environment, the fish are fed and, if necessary, medicated until they mature for sale as food or are selected for further breeding.

[0097] In one embodiment of this disclosure, the active ingredient, or its salt, N-oxide, or solvate, is applied to the fish population at the end of the freshwater phase or the beginning of the seawater phase of fish farming. According to another embodiment, the application is performed while the fish (e.g., salmon or sea trout) are kept in seawater.

[0098] According to this disclosure, the active ingredient, or its salt, or N-oxide or solvate, is particularly suitable for use in the control or treatment of ectoparasitic infections of fish, such as sea lice infections. The term "sea lice" refers to a group of fish-parasitic crustaceans that infect fish in marine waters, particularly ectoparasitic copepods. In this context, "sea lice" refers to the order Daphnia (sea lice). Siphonostomatoida Parasitic copepods within the family Cladosporidae, which feed on the mucus, skin, and tissues of their hosts. This group includes fish lice (Cladosporidae). Caligidae ) and the family Anthropoididae ( Lernanthropidae It is worth noting that the fish lobe family ( Caligidae Two members of the genus *Scalyflies*, species of which ( Lepeophtheirus spp.) and species of the genus *Ichthyophthirius* ( Caligus spp.)(C), leading to significant losses in salmon farming. Species of the genus *Schizothorax* (C) Lepeophtheirus An example of a subspecies within spp. (L) is the Pacific salmon louse ( lepeophtheirus salmonis oncorhynchi subsp. nov) and salmon scab louse ( L. salmonis salmonis Species of the genus *Ichthyophthirius* ( Caligus Examples within spp. include the red sea lice ( Caligus clemensi ), short fish lice ( Caligus curtus ), Dubrovettes fish lice ( Caligus dussumieri ), elongated fish lice ( Caligus elongatus ), long-tailed fish lice ( Caligus longicaudatus Chilean fish lice ( Caligus rogercresseyi ) and Strom sea lice ( Caligus stromii ) and tiny fish lice ( Caligus minimus Notably, salmon scab fish lice ( L. salmonis ) exists only in the Northern Hemisphere, while the Chilean fish lice ( C. rogercresseyi As the most influential sea lice species affecting the salmon industry, it is of particular importance in Chile.

[0099] The anthropod family (Anthropodidae) is of particular importance in Mediterranean fish farming. Lernanthropidae Representative species of the genus *Anthropoda* (human-shaped fish louse) Lernanthropus spp.). Species in the genus *Anthropoda* ( Lernanthropus spp.) contains humanoid fish lice ( Lernanthropus kroyeri ), Lernanthropus callinomymicola , Lernanthropus indefinitus , Lernanthropus cynoscicola and Lernanthropus gisleri .

[0100] Those belonging to the family Caterpillaridae ( ) are of particular importance in freshwater aquaculture. Argulidae Parasitic copepods, with the genus *Ariocarpus* (…). Argulus ) members, namely carp ( A. foliaceus), Japanese fish lice ( A. japonicus ) and whitefish ( A. coregoni Represented by ) and infected by the gnatidae family of salmon ( Lernaeopodidae ), representative Salmincola Members of the genus.

[0101] Infection is indicated when at least one stage of parasite development is observable on the surface of a fish. In specific cases, automated methods for sea lice counting can be used to identify and quantify infection by counting parasites. Generally, manual / visual methods for sea lice counting can be used to identify infection and assess the extent of infection by counting parasites.

[0102] As used herein, the term "control" refers to the reduction of ectoparasites, particularly fish-parasitic crustaceans, specifically sea lice colonies. This includes reducing, eliminating, or preventing initial and / or further infections, especially against fish-parasitic crustaceans, particularly sea lice, throughout their parasitic life cycle.

[0103] As used herein, the term “treatment” refers to active and responsive measures that encompass actions such as controlling, eliminating, protecting, and / or preventing infections or conditions caused by ectoparasites of fish, particularly ichthyophthirius multifiliis, specifically sea lice, in individual fish or fish populations. These terms include efforts to reduce the average number of parasites, such as sea lice, affecting each fish in the population, or to prevent an increase in the average number of current infections. This includes addressing existing ectoparasite infections and, optionally, preventing the initiation of new infections or associated symptoms caused by ectoparasites, such as sea lice. Additionally, it can include mitigating the severity of symptoms, conditions, or signs associated with ectoparasite infections. These terms may also cover preventing the recurrence of ectoparasite infections or associated symptoms in fish, as well as actions falling under the category of “control,” such as killing, expelling, decapitating, preventing, eliminating, mitigating, minimizing, and eradicating.

[0104] As used herein, the term "effective amount" refers to the quantity or dose of an active ingredient, its salt, N-oxide, or solvation that produces the desired effect when administered to fish or a fish population in a single or multiple doses. When determining the effective amount, a variety of factors may be considered, including (but not limited to) the species of fish, the degree of parasite infection, the response of the fish population, the method of administration, the bioavailability characteristics of the applied formulation, the selected dosage regimen, concurrent administration, and other relevant circumstances.

[0105] As used herein, the term "composition" means a product containing an active ingredient, its salts, N-oxides, or solvates, and veterinary-safe diluents, carriers, or excipients. In the context, unless otherwise specified, "veterinary-safe" means that the components must be chemically and / or toxicologically consistent with the composition or other components in the fish being treated. The term "pharmaceutical-safe" has the same meaning as "veterinary-safe." For example, the composition may be combined with a nutritionally suitable fish feed, including the active ingredient, its salts, N-oxides, or solvates.

[0106] In some embodiments, the active ingredient described herein, or its salt, or N-oxide, or solvate, may be used in combination with one or more other physiologically active agents. As used herein, the term "combined use" includes administration together with or in the same process as a therapy using the active ingredient described herein, or its salt, or N-oxide, or solvate.

[0107] These physiologically active agents can be another type of antiparasitic agent, specifically sea lice control agents, antibiotics, vaccine components including immune enhancers, or feed ingredients containing immunomodulators. Suitable parasite-killing agents include, for example, hydrogen peroxide; formaldehyde; organophosphates such as trichlorfon, malathion, dichlorvos, or methylpyridinium; macrolides such as ivermectin, emamectin benzoate, or moxifloxacin; pyrethroids such as cypermethrin or deltamethrin; neonicotinoids such as imidacloprid, acetamiprid, thiamethoxam, or thiamethoxam; spinosad such as spinosad; insect growth regulators (IGRs) such as ipofenam, tebufenozide, methoxyprotein, diflubenzuron, flufenuron, chlorfenapyr, fluzoflubenzuron, fluazinam, or clofenuron; carbamates such as phenoxycarb; isoxazolines such as afolanar (including essentially pure active enantiomers), saloranar, fleranar (including essentially pure active enantiomers), and loteranar; or cyclopropylamide compounds.These active agents are described in WO 2022162001A1, US Patent No. 7,964,204, US 2010 / 0254960, US 2011 / 0159107, US 2012 / 0309620, US 2012 / 0030841, US 2010 / 0069247, WO 2007 / 125984, WO2012 / 086462, US Patent Nos. 8,318,757, 8,466,115, 8,618,126, 8,822,466, 8,383,659, 8,853,186, 9,221,835, US 2011 / 0144349, and US Patent No. 8,053,452; US US Patent Nos. 2010 / 0137612, 8,410,153, 2011 / 152081, 2012 / 089623, 2012 / 089622, 8,119,671; 7,947,715; 2012 / 120135, 2012 / 107533, 2011 / 157748, 2011 / 0245274, 2011 / 0245239, 2012 / 0232026, 2012 / 0077765, 2012 / 0035122, 2011 / 0251247, 2011 / 15443 ... US Patent No. 2011 / 154434, US Patent No. 2012 / 0238517, US Patent No. 2011 / 0166193, WO 2011 / 104088, WO 2011 / 104087, WO 2011 / 104089, US Patent No. 2012 / 015946, US Patent No. 2009 / 0143410, WO 2007 / 123855A2, US Patent No. 2011 / 0118212, US Patent No. 7,951,828, US Patent No. 7,662,972, US Patent No. 2010 / 0137372, US Patent No. 2010 / 0179194 A2, US Patent No. 2011 / 0086886 A2, US Patent No. 2011 / 0059988, US US Patent Nos. 2010 / 0179195, 2015 / 0126523, WO 2010 / 003923, WO 2010 / 003877, WO 2010 / 072602, WO 2014 / 134236, WO 2017 / 147352, US Patent Nos. 7,897,630 and 7,951,828, the entire contents of which are incorporated herein by reference.

[0108] As used herein, the term "salt" refers to an acid or base salt, which is typically formed by, for example, mixing a compound with an acid or base, respectively, using various methods known in the art. The salt of the compound is pharmaceutically available to the extent intended for in vivo administration (i.e., administration to animals) for therapeutic benefit.

[0109] As used herein, the term "solvent" refers to a stable complex of an ingredient and a solvent, such that the complex remains intact after the unreinforced solvent is removed. These complexes are commonly referred to as "solvents." Solvents can be formed by the aggregation of the active ingredient described herein with solvent molecules, preferably in a weight-based ratio, such as water, alcohols like ethanol, aromatic solvents like toluene, ethers, and halogenated organic solvents like dichloromethane.

[0110] As used herein, the term “N-oxide” refers to a compound formed by oxidizing an amine or an N-heterocyclic compound, such as pyridine or pyrimidine, with an oxidizing agent, such as hydrogen peroxide, peracid, or an inorganic oxidizing agent, such as potassium persulfate (potassium persulfate preparation (oxone)).

[0111] Example The following examples describe some exemplary ways of preparing and practicing certain compositions described herein. These examples are for illustrative purposes only and are not intended to limit the scope of the compositions and methods described herein.

[0112] Example 1 Research Design A randomized, blinded, parallel-group, negative-control study was conducted in which laboratory-raised Atlantic salmon in seawater were given medicated feed. Salmo Salar The active ingredient was administered to post-smolt juvenile fish. Under laboratory conditions, the active ingredient was administered to sea lice (salmon scab louse) before feeding the medicated diet. Lepeophtheirus salmonis Experimental infection of juvenile salmon (smolt) was conducted to evaluate the efficacy of the active ingredient in reducing sea lice / fish populations.

[0113] Healthy Atlantic salmon juveniles (n=750) were recruited and sequentially distributed into ten (10) study tanks. Fish were individually weighed during the period from study day (SD)-95 when they were assigned to the tanks, with SD 0 used as the first day of treatment. Fish were weighed at SD-2, +8, +18, +22, and +30. The fish weight at SD-2 was used to estimate the average weight of all fish and to determine feeding amounts during the treatment period (SD 0 to +6). Lethal sampling was performed on a total of 10 fish / tank for lice counting, and weight and length were measured. At SD-2, the average weight of the fish in the tank was 829.9 ± 161.7 g.

[0114] Using an experimental challenge model, Atlantic salmon juveniles were infected once with sea lice. In SD-17, approximately 50 copepod larvae (salmon scab louse) were added to each tank per fish. Lepeophtheirus salmonis During the study, the water temperature varied between 8 and 12°C. Throughout the study, the water oxygen saturation remained above 80%.

[0115] Five treatment groups were randomly assigned to ten research tanks, resulting in two tanks for each treatment group. The researchers were unaware of the treatment methods used in each tank.

[0116] During the study, sea lice were counted four times. All sampled fish were euthanized and the lice were counted. Baseline counts occurred at SD-2 (10 fish per tank), corresponding to approximately 160 days post-infection (15 days). This time was sufficient for the lice to develop into larvae. Count 1 occurred at SD+8 (20 fish per tank), corresponding to approximately 260 days post-infection (25 days) and 2 days post-treatment. In the control tank, this time was sufficient for the lice to develop into appendage larvae and pseudo-adults. Count 2 occurred at SD+18 (20 fish per tank), corresponding to approximately 360 days post-infection (35 days) and 12 days post-treatment. In the control tank, this time was sufficient for the lice to develop into male and female adults. Count 3 occurred at SD+22 (20 fish per tank), corresponding to approximately 400 days post-infection (39 days) and 16 days post-treatment. This time was sufficient for the lice to develop into male and female adults, and the appendage larval stage was absent in the control tank.

[0117] Medicated feeds were prepared by dry coating commercially available fish feed pellets with a premix containing the said active ingredient to achieve concentrations of approximately 1.6, 4.0, 8.0, and 19.9 mg / kg. The fish pellets were then coated with feed-grade herring oil at a content of approximately 0.75% w / w (7.5 g / kg feed). The consumption rate of the medicated feed was set at 0.628% body weight.

[0118] For each treatment, the sea lice count was used to calculate the treatment efficacy as the ratio of total sea lice count to fish count and the ratio of total sea lice count to fish count during the active life stage (i.e., pseudo-adults and adults). The ratio of total attached sea lice count to fish count was calculated at the first count after treatment. The percentage efficacy of each treatment group compared to the control was calculated using the Abbott formula, as shown below: Efficacy percentage = 100 (MC-MT) / MC MC and MT are the arithmetic mean of sea lice counts in the untreated control group and the S-roxapin treatment group, respectively.

[0119] Treatment was considered effective when the percentage of efficacy in the treatment group was ≥90% compared to the untreated control group.

[0120] Results - Salmon scab lice on Atlantic salmon ( Lepeophtheirus salmonis ) activity Mean abundance of sea lice at different parasite life stages (attached, active, and total (attached and active together)) is reported and calculated as the number of lice on salmon divided by the number of fish sampled per tank. Attached lice include attached larval stages I and II. Active lice include male and female pseudo-adults and adults. This study considers the number of active lice, attached lice, and total lice count. At each sampling time, descriptive statistics such as mean, standard deviation, and efficacy percentage were calculated for the treatment groups.

[0121] Table 1 shows the mean (+ / - SD) number of active lice, attached lice, and total lice / fish count for each treatment group at the sampling time after each treatment, and the percentage of efficacy.

[0122] Table 1

[0123] SD – Standard Deviation.

[0124] Figures 1-4 The diagram illustrates these data.

[0125] The inventors of this pending application have discovered an unexpectedly low-dose active ingredient, (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, including its salts, or N-oxides or solvates, having the following structural formula:

[0126] It has therapeutic activity against ectoparasitic infections (such as sea lice) on fish (such as Atlantic salmon).

[0127] Example 2 Research Design A randomized, blinded, parallel-group, negative-control study was conducted in which Atlantic salmon were administered the active ingredient in their diet for seven consecutive days at three dose rates (0 mg / kg / day, 0.025 mg / kg / day, and 0.060 mg / kg / day). Following treatment, the fish were intentionally infected with sea lice on day 8 or 17 of the diurnal transplant period (DPT), all in a controlled laboratory setting using seawater. Efficacy was evaluated for each group at three specific time points post-infection by comparing lice counts between the treated and untreated control groups.

[0128] Healthy Atlantic salmon juveniles (n=1080) were recruited and distributed sequentially into eighteen (18) study tanks. Fish were individually weighed during tank assignment on study days (SD)-55 and -56. Fish were weighed again on SD-19 to estimate the mean weight of all fish and to determine feeding amounts during treatment (SD 0 to +6). The mean weight of the fish before treatment was 716.1 ± 152.5 g. Fish in the group infected on day 8 of DPT were additionally weighed on SD+8, +14, +27, +35, +41, and +48. Fish in the group infected on day 17 of DPT were additionally weighed on SD+8, +23, +36, +44, +50, and +57. For both groups, SD 0 was used as day 1 of treatment.

[0129] At each infection time (DPT day 8 or 17), use approximately 100 copepod larvae (salmon scab louse) per fish. Lepeophtheirus salmonis The fish were infected once with Atlantic salmon juveniles. During the study, the water temperature varied between 9 and 13°C, and the water oxygen saturation was above 80%.

[0130] By blocking paired cylinder exposure for DPT on days 8 and 17, the three treatment groups were randomly assigned to 18 study cylinders. Researchers were unaware of the treatment regimen for each cylinder.

[0131] During the study, sea lice were counted three times. Counts I, II, and II were performed on days 21, 27, and 34 post-infection, respectively. Counts were strategically scheduled to ensure that at least one of the three counts within the negative control treatment period represented the life stage, including the appended larvae, pseudo-adults, and adults. At all time points, 10 fish were euthanized and the sea lice were counted.

[0132] Medicated feed was prepared by dry coating commercially available fish feed pellets with a premix containing the active ingredient to achieve concentrations of approximately 4.2 and 10.0 mg / kg. The fish pellets were then coated with feed-grade herring oil at a content of approximately 0.75% w / w (7.5 g / kg feed). The consumption rate of the medicated feed was set at 0.6% body weight.

[0133] For the groups infected on day 8 of the DPT and the groups infected on day 17 of the DPT, treatment efficacy was calculated for each sea louse count (counts I, II & III) as well as for the total number of sea lice / fish and the total number of sea lice in the active life stage (i.e., pseudo-adults and adults) / fish. For the total number of attached sea lice / fish, treatment efficacy was calculated only for count I. The percentage efficacy of each treatment group compared to the control was calculated using the Abbott formula, as shown below: Efficacy percentage = 100 (MC-MT) / MC MC and MT are the arithmetic mean of sea lice counts in the untreated control group and the S-roxapin treatment group, respectively.

[0134] Treatment was considered effective when the percentage of efficacy in the treatment group was ≥90% compared to the untreated control group. Mean abundance of sea lice at different parasite life stages (attached, active, and total (attached and active together)) was reported and calculated as the number of lice on salmon divided by the number of fish sampled per tank. Attached lice included attached larval stages I and II. Active lice included male and female pseudo-adults and adults. This study considered the number of active lice, attached lice, and total lice count.

[0135] result: Duration of effectiveness against live lice For the group infected on day 8 of the DPT, within the same cohort, the percentage efficacy against active lice at each evaluation time point was between 86.9% and 91.8% in the 0.025 mg / kg / day treatment group and between 99.2% and 100% in the 0.060 mg / kg / day treatment group. However, for the group infected on day 17 of the DPT, only the 0.060 mg / kg / day dose showed some efficacy, with percentages ranging from 17.4% to 23%. The 0.025 mg / kg / day treatment dose did not show efficacy (Table 2).

[0136] Duration of efficacy against attachment lice Since the count of attachment lice was almost zero at the second and third evaluation time points (due to the transition to the pseudo-adult and adult life stages), only the efficacy percentage at the first evaluation time point was calculated. For the group infected on day 8 of the DPT, the efficacy percentage of the 0.025 mg / kg / day dose was 92.5%, while that of the 0.060 mg / kg / day dose was 99.8%. For the group infected on day 17 of the DPT, the efficacy percentages of the 0.025 mg / kg / day and 0.060 mg / kg / day doses were 21.2% and 46.2%, respectively (Table 2).

[0137] Duration of efficacy based on total lice count / fish count For the group infected on day 8 of the DPT, the efficacy percentages for the total number of sea lice were calculated to be greater than 86.8% and greater than 99.2% for the 0.025 mg / kg / day and 0.060 mg / kg / day treatment groups, respectively. For the group infected on day 17 of the DPT, only the 0.060 mg / kg / day treatment dose showed some efficacy, with percentages ranging from 17% to 23%. As shown in Table 2, no efficacy was observed for the 0.025 mg / kg / day treatment dose in DOE14.

[0138] Table 2 shows the mean (+ / - SD) number of active lice, number of attached lice, and total number of lice / fish at each count (21, 24, and 34 days post-infection) and efficacy percentage for the groups infected on days 8 and 17 of DPT, compared with the negative control group.

[0139] Table 2

[0140] SD – Standard Deviation.

[0141] Figures 5-8 The diagram illustrates these data.

[0142] Given the teachings of this specification, other embodiments of the implementation will become apparent to the reader and therefore will not be described further herein.

[0143] Note that headings or subheadings may be used throughout this disclosure for the convenience of the reader, but these should in no way limit the scope of the invention. Furthermore, certain theories may be presented and disclosed herein; however, whether they are correct or not, they should not in any way limit the scope of the invention, provided that the invention is practiced according to this disclosure, without regard to any specific theory or operational scheme.

[0144] For all purposes, all references cited throughout this specification are incorporated herein by reference in their entirety.

[0145] Throughout this specification, references to "some embodiments" and the like indicate that at least one embodiment described herein includes specific elements (e.g., features, structures, and / or characteristics) incorporating the invention, which may or may not be present in other embodiments. Furthermore, it should be understood that the inventive features can be combined in any suitable manner across multiple embodiments.

[0146] Those skilled in the art will understand that throughout this specification, the term "a" used before a term encompasses one or more embodiments referred to by that term. Those skilled in the art will also understand that throughout this specification, the term "comprising," synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unlisted elements or method steps.

[0147] Unless otherwise defined, all 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 pertains. In case of conflict, this document, including its definitions, shall prevail.

[0148] As used in this disclosure, the terms “about,” “approximately,” or “approximately” generally refer to a range of errors generally accepted in the art. Thus, the quantities of numerical values ​​provided herein generally include such error tolerances, so that the terms “about,” “approximately,” or “approximately” can be inferred even if not explicitly stated.

[0149] Although various embodiments of this disclosure have been described and illustrated, it will be apparent to those skilled in the art, in consideration of this specification, that many modifications and variations can be made. The scope of the invention is defined more specifically in the appended claims.

Claims

1. A composition comprising an effective amount of the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, including its salt, N-oxide, or solvate, having the following structural formula: The composition is intended for oral administration of the active ingredient, or a salt thereof, or an N-oxide or solvate thereof, at a dose of about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably about 0.025 mg / kg / day to about 0.125 mg / kg / day.

2. The composition according to claim 1, wherein, The composition is prepared for feeding fish at a feeding rate in the range of 0.4%-2.6% body weight / day.

3. The composition according to claim 1 or 2, wherein, The oral administration is carried out continuously for about 3 to about 10 days, preferably for 7 consecutive days.

4. The composition according to any one of claims 1 to 3, comprising about 0.01 wt.% to about 99 wt.%, preferably about 0.01 wt.% to about 0.5 wt.%, more preferably about 0.1 wt.% to about 0.5 wt.%, of the active ingredient, or a salt, or N-oxide or solvate thereof.

5. The composition according to any one of claims 1 to 4, wherein, The composition is medicated fish feed.

6. The composition according to claim 5, wherein, The medicated fish feed is in the form of small feed pellets or feed granules.

7. The composition according to any one of claims 1 to 6, wherein, The dosage is approximately 0.025 mg / kg / day, approximately 0.050 mg / kg / day, or approximately 0.125 mg / kg / day.

8. The composition according to any one of claims 1 to 7, wherein, The composition is prepared for oral administration to fish.

9. The composition according to claim 8, wherein, The fish in question belongs to the salmonid family.

10. The composition according to any one of claims 1 to 9, for oral administration to fish to treat or control ectoparasitic infections of fish.

11. The composition according to claim 10, wherein, The ectoparasitic infection mentioned is a sea lice infection.

12. A composition comprising the active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt thereof, or an N-oxide or solvate thereof, having the following structural formula: The composition is used to treat or control ectoparasite infections in fish, wherein, The composition is used to administer orally to fish at a dose of about 0.025 mg / kg / d to about 0.250 mg / kg / d, preferably about 0.025 mg / kg / d to about 0.125 mg / kg / d of the active ingredient, or its salt, or N-oxide or solvate.

13. The composition for said use according to claim 12, wherein, The fish in question belongs to the salmonid family.

14. The composition for said use according to claim 12 or 13, wherein, The ectoparasitic infection mentioned is a sea lice infection.

15. The composition for said use according to claim 14, wherein, The sea louse infection includes infected copepods, appendage larvae, pseudo-adults, adults, or combinations thereof during the louse's life stage.

16. The composition for said use according to any one of claims 12 to 15, wherein, The composition comprises about 0.01 wt.% to about 99 wt.%, preferably about 0.01 wt.% to about 0.5 wt.%, more preferably about 0.1 wt.% to about 0.5 wt.%, of the active ingredient, or its salt, or N-oxide or solvate.

17. The composition for said use according to any one of claims 12 to 16, wherein, The composition is medicated fish feed.

18. The composition for said use according to any one of claims 12 to 17, wherein, The dosage is approximately 0.025 mg / kg / day, approximately 0.050 mg / kg / day, or approximately 0.125 mg / kg / day.

19. The composition for said use according to any one of claims 12 to 18, wherein, The composition is prepared for feeding the fish at a feeding rate in the range of 0.4%-2.6% body weight / day.

20. The composition for said use according to any one of claims 12 to 19, wherein, The composition is to be used continuously for about 3 to about 10 days, preferably orally for 7 consecutive days.

21. A method for treating or controlling ectoparasitic infections in fish, comprising orally administering to the fish an active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or a salt thereof, or an N-oxide or solvate thereof, having the following structural formula: The dosage is from about 0.025 mg / kg / day to about 0.250 mg / kg / day, preferably from about 0.025 mg / kg / day to about 0.125 mg / kg / day.

22. The method according to claim 21, wherein, The ectoparasitic infection is a sea louse infection, preferably including copepods, appendage larvae, pseudo-adults, or adults or combinations thereof in the life stage of the infected lice.

23. The method according to claim 21 or 22, wherein, The oral administration is carried out continuously for about 3 to about 10 days, preferably for 7 consecutive days.

24. The method according to any one of claims 21 to 23, wherein, The active ingredient, or its salt, or N-oxide or solvate, is included in the oral composition for fish.

25. The method according to claim 24, wherein, The oral composition for fish contains about 0.01 wt.% to about 99 wt.%, preferably about 0.01 wt.% to about 0.5 wt.%, more preferably about 0.1 wt.% to about 0.5 wt.%, of the active ingredient, or its salt, or N-oxide or solvate.

26. The method according to claim 24 or 25, wherein, The oral composition for fish is medicated fish feed.

27. The method according to claim 26, wherein, The medicated fish feed is in the form of small feed pellets or feed granules.

28. The method according to any one of claims 21 to 27, wherein, The dosage is approximately 0.025 mg / kg / day, approximately 0.050 mg / kg / day, or approximately 0.125 mg / kg / day.

29. The method according to any one of claims 21 to 28, wherein, The fish in question belongs to the salmonid family.

30. The active ingredient (S)-3-methyl-N-(2-oxo-2-(prop-2-yn-1-ylamino)ethyl)-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)thiophene-2-carboxamide, or its salt, N-oxide, or solvate, has the following structural formula: Use in the production of compositions for the treatment or control of ectoparasitic infections in fish, wherein, The composition is used to administer orally to the fish at a dose of about 0.025 mg / kg / d to about 0.250 mg / kg / d, preferably about 0.025 mg / kg / d to about 0.125 mg / kg / d of the active ingredient, or its salt, or N-oxide or solvate.

31. The use according to claim 30, wherein, The ectoparasitic infection mentioned is a sea lice infection.

32. The use according to claim 31, wherein, The sea louse infection includes infected copepods, appendage larvae, pseudo-adults, adults, or combinations thereof during the louse's life stage.

33. The use according to any one of claims 30 to 32, wherein, The oral administration is carried out continuously for about 3 to about 10 days, preferably for 7 consecutive days.

34. The use according to any one of claims 30 to 33, wherein, The composition is medicated fish feed.

35. The use according to claim 34, wherein, The fish feed contains about 0.01 wt.% to about 99 wt.%, preferably about 0.01 wt.% to about 0.5 wt.%, more preferably about 0.1 wt.% to about 0.5 wt.%, of the active ingredient, or its salt, or N-oxide or solvate.

36. The use according to any one of claims 30 to 35, wherein, The dosage is approximately 0.025 mg / kg / day, approximately 0.050 mg / kg / day, or approximately 0.125 mg / kg / day.

37. The use according to any one of claims 30 to 36, wherein, The fish in question belongs to the salmonid family.

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