WO2024189101A1

WO2024189101A1 - Arylaminobenzamide or isoxazoline compounds for use in controlling a parasite infestation on a fish

Info

Publication number: WO2024189101A1
Application number: PCT/EP2024/056706
Authority: WO
Inventors: Bertrand Havrileck; Regina LIZUNDIA GORBEA; Stéphane Bonneau
Original assignee: Virbac SA
Current assignee: Virbac SA
Priority date: 2023-03-13
Filing date: 2024-03-13
Publication date: 2024-09-19
Anticipated expiration: 2025-09-13

Abstract

The present invention relates to Modoflaner, an arylaminobenzamide compound, for its use in controlling a parasite infestation on a fish and to an isoxazoline for its use in controlling a parasite infestation on a fish, said isoxazoline being administered parenterally.

Description

ARYLAMINOBENZAMIDE OR ISOXAZOLINE COMPOUNDS FOR USE IN CONTROLLING A PARASITE INFESTATION ON A FISH

Infestation of fish by parasites is a major problem for commercial fish farming. Fish farmers who are confronted with a parasite problem usually suffer substantial financial losses and carry additional expenses.

In particular, parasitic copepods are common on cultured and wild marine finfish, and are vastly described in the literature regarding their taxonomy and host ranges.

Many of these species have long been recognized to have the potential to affect the growth, fecundity and survival of wild hosts. And with the development of semi-intensive and intensive brackish water and marine aquaculture, the importance of parasitic copepods as disease causing agents has become more evident.

Sea lice are ectoparasites of the order Siphonostomatoida or of the family Argulidae. They are the most commonly reported species on marine and brackish water cultured fish throughout the world, accounting for approximately 61% of all reports. Members of this family have been responsible for most of the documented disease outbreaks. They affect fish, particularly farmed fishes of the Salmonidae family, negatively by feeding on the mucus, skin, tissue, and blood of the fish host. Sea lice can cause significant harm (i.e., serious fin damage, skin erosion, bleeding, and open wounds) to host fish, due to their attachment and feeding activities. Additionally, sea lice can cause chronic stress response in fish, which in turn can make them susceptible to other diseases. In addition, it appears that the sea lice have immunomodulatory effects on the host fish and can function as a vector in the transmission of other fish diseases.

Severe infestation kills the fish. Mortality rates of over 50%, based on sea lice infestation, have been reported from Norwegian fish farms. In a first phase, sea lice infestation is seen in the appearance of the parasites attached to the fish and later, even more clearly, from the damage caused to skin and tissue. The most severe damage is observed in smolts which are just in the phase in which they change from fresh water to sea water. The situation is made even worse by the specific conditions in the fish farms, where fish, for example salmon, of different age groups but of the same weight class are kept together; where fouled nets or cages are used; where high salt concentrations are to be found; and/or where flow through the nets and cages is minimal and the fish are kept in a very narrow space.

Damages due to parasitic infestations from sea lice thus result in considerable animal welfare issues, fish losses and increased expense.

Infestation with sea lice is considered one of the most important disease problems in the farming of fishes of the Salmonidae family, especially Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). In addition to the costs that are associated with treatment, lower classification ratings of slaughtered fish and reduced growth rate due to reduced feed intake contribute to the economic losses.

Infestation with sea lice can also occur in other fish species, for example, sea bass, tilapia, carp, and the like. In fact, sea lice are meanwhile widely prevalent and encountered in all fish farms.

There are a number of treatments already on the market for controlling sea lice including bath treatments, such as organophosphates (for example, dichlorvos and azamethiphos) and pyrethroids (for example, cypermethrin and deltamethrin), and in- feed-treatments, such as avermectins (for example, ivermectin and emamectin benzoate) and growth regulators (for example, lufenuron and teflubenzuron).

However, resistance to many of these treatments has been observed and most of these compounds do not have a long duration of activity.

In addition, most sea-lousicide treatments are administered topically in bathes or dips or orally in medicated feed which results in the dispersion of the active ingredient in the environment.

As opposed to topical or feed administration, parenteral administration, such as intraperitoneal or intramuscular administration, is environmentally safe.

Therefore, a need for new treatments remains, in particular treatments having a long duration of action and that are environmentally safe.

Accordingly, one aim of the present invention is the provision of new treatment options to control parasite infestation, in particular sea lice infestations, on fish, particularly in farmed fish populations.

Another aim of the present invention is to provide new treatments that can be used for prolonged control of parasites on fish, for example for at least one, two, three months or more.

Another aim of the present invention is to provide new treatments that are selective against the target parasite and/or capable of treating parasite populations, in particular sea lice populations, showing resistance or reduced sensitivity to the current products.

Another aim of the present invention is to provide new treatments that are safe to the fish recipients, their human caretakers and the environment.

Definitions:

As used herein, an "infestation" refers to the presence of parasites on the fish in numbers that pose a risk of nuisance or harm to said fish.

As used herein, “controlling or control of a parasite infestation” encompasses preventing or treating or both preventing and treating parasite infestations. The effect of the treatment can manifest itself directly by killing the parasites, either immediately or after some time has elapsed, or indirectly, for example by partially or completely inhibiting the development of parasites or reducing the number of eggs laid.

As used herein, “preventing or prevention of a parasite infestation” means avoiding the presence of parasites on a fish in numbers that pose a risk of nuisance or harm to said fish. The parasite infestation may be prevented by administering an effective dose of Modoflaner or the isoxazoline to a non-infested fish, e.g. before said fish enters in contact with a parasite or by administering to a fish that is or has been infested by a parasite an effective dose of Modoflaner or the isoxazoline to prevent a new parasite infestation or a re-infestation by the offspring of the infesting parasite.

As used herein, "treating or treatment of a parasite infestation" means reducing the number of parasites already present on the fish. The parasite infestation can be treated by administering to an infested fish an effective dose of Modoflaner or the isoxazoline.

As used herein, an “effective dose” refers to the amount of Modoflaner or the isoxazoline that needs to be administered to reduce the number (or count) of the existing or potential parasites on the fish by at least about 5%. Preferably, the dose is considered effective when the number of existing or potential parasites on the fish is reduced by at least about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 %, more preferably by at least about 90%. In a most preferred embodiment the dose is considered effective when the number of existing or potential parasites on the fish is reduced by at least about 95%.

As used herein, the term "about" refers in particular to a range of values ± 10% of a specific value. By way of example, the expression "about 20" includes the values 20 ± 10%, i.e. the values 18 to 22.

When the infestation is a sea lice infestation, an “effective dose” of Modoflaner or the isoxazoline refers to the amount of Modoflaner or the isoxazoline that needs to be administered to reduce or maintain the number (or count) of gravid (or ovigerous) female sea lice or adult sea lice below a regulatory threshold, i.e. a count of sea lice over which a treatment must be administered. Examples of thresholds are 0.1 gravid female / fish during outmigration period of wild salmon, 0.2 gravid female / fish during outmigration period of wild salmon and 0.5 gravid female / fish during the rest of the year, 0.3-0.5 gravid female / fish during outmigration period of wild salmon and 2.0 gravid female / fish during the rest of the year, 1.5 gravid female / fish, 3.0 gravid female / fish, 3.0 adult sea lice.

As used herein, a “sea louse” refers to a fish parasite of the order Siphonostomatoida or of the family Argulidae.

As used herein, the term fish, unless otherwise noted, refers to the taxonomic class Chondrichthyes (cartilaginous fishes, e.g., sharks and rays) and Osteichthyes (bony fishes) which live in water, have gills or mucus-covered skin for respiration, fins, and may have scales.

As used herein, “crystalline modification IV of Modoflaner” is synonymous with “crystalline form IV of Modoflaner”, or “crystalline polymorphic form IV of Modoflaner” and means in particular Modoflaner comprising from 20 to 100% (e.g. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) in weight of crystalline modification IV of Modoflaner based on the total weight of Modoflaner, the crystalline modification IV of Modoflaner being preferably in substantially pure form (e.g. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%).

As used herein, “fish body weight” refers to the bodyweight of an individual fish or weight of the fish biomass.

In a first aspect, the present invention concerns a parasiticide isoxazoline, preferably selected from the group consisting in fluralaner, afoxolaner, sarolaner and lotilaner, for use in controlling a parasite infestation on a fish, wherein the isoxazoline is administered parenterally to the fish.

The isoxazoline may be administered by intraperitoneal injection (e.g. an injection given into the mid ventral line, just below vent), subcutaneous injection, intramuscular injection (e.g. injection into the epiaxial musculature, generally approximately mid-way between the mid-dorsal line and the lateral line), or injection into the dorso-median sinus (e.g. an injection in the mid-dorsal line in the angle at the caudal margin of the cranial dorsal fin).

The isoxazoline is preferably administered intraperitoneally or intramuscularly.

The isoxazoline is preferably fluralaner.

The isoxazoline, especially fluralaner, is administered at a dose ranging from 1 to 100 mg/kg, preferably from 10 to 90 mg/kg, more preferably from 25 to 50 mg/kg, in particular about 40 mg/kg, of fish bodyweight.

In an embodiment, the isoxazoline, especially fluralaner, is administered at a dose ranging from about 40 mg/kg to about 120 mg/kg of fish bodyweight.

In particular, the isoxazoline is administered at a dose ranging from about 50, 60, 70, 80 or 90 mg/kg to about 120 mg/kg of fish bodyweight.

The parasite may be any fish-parasitic crustacean, notably a sea louse. It should be understood that, in the present invention, only parasites that are parasitic to fish are contemplated.

The sea louse may in particular belong to the family Argulidae or the order Siphonostomatoida.

Within the family Argulidae, the parasite belonging to the genus Argulus are of relevance, in particular Argulus foliaceus.

Within the order Siphonostomatoida, parasites of the family Caligidae, Ergasilidae, Lernaepodidae, or Lernanothropidae, particularly those belonging to the family Caligidae, are contemplated.

The family Caligidae includes the genus Lepeophtheirus and Caligus, in particular the species Caligus acanthopagri, Caligus antennatus, Caligus brevicaudatus, Caligus clemensi, Caligus curtus, Caligus dussumieri, Caligus elongatus, Caligus epidemicus, Caligus fugu, Caligus lalandei, Caligus latigenitalis, Caligus longicaudatus, Caligus longipedis, Caligus minimus, Caligus multispinosus, Caligus nanhaiensis, Caligus orientalis, Caligus oviceps, Caligus pageti, Caligus patulu, Caligus pelamydis, Caligus punctatus, Caligus rogercresseyi, Caligus rotundigenitalis, Caligus spinosus, Caligus stromii, Caligus teres, Lepeophtheirus atypicus, Lepeophtheirus cuneifer, Lepeophtheirus hippoglossi, Lepeophtheirus longiventris, Lepeophtheirus paralichthydis, Lepeophtheirus salmonis, Parapetalus occidentalis, Pseudocaligus apodus, Pseudocaligus fugu, Diergasilus kasaharai, Ergasilus australiensis, Ergasilus borneoensis, Ergasilus ceylonensis, Ergasilus labracis, Ergasilus lizae, Ergasilus lobus, Pseudoergasilus zacconis, Alella macrotrachelus, Alella sp., Bomolochus stocki, Clavella adunca, Lernanthropus atrox, Lernanthropus chrysophrys, Lernanthropus lappaceus, Colobomatus labrachis, Eobrachiella elegans seiolae, Hemobaphes disphaerocephalus, Holobomolochus confusus, or Neobrachiella fugu.

The parasite is a sea louse preferably selected from the group consisting in Lepeophtheirus salmonis, Caligus clemensi, Caligus curtus, Caligus dussumieri, Caligus elongates, Caligus longicaudatus, Caligus rogercresseyi or Caligus stromii, more preferably Lepeophtheirus salmonis, Caligus elongates, or Caligus rogercresseyi.

In another preferred embodiment, the parasite is a haematophagous parasite, notably a haematophagous sea louse, for example Lepeophtheirus salmonis.

The parasite infestation may be with several developmental stages like copepodites, chalimus, pre-adult, adult sea lice or a mixed infestation with various stages.

The fish include food fish, breeding fish, aquarium, pond, river, reservoir fish of all ages occurring in freshwater, sea water and brackish water. Such fish includes fishes of the Salmonidae family, in particular salmons, trouts, chars, freshwater whitefishes, graylings; or from sea breams, porgies, breams, scats, ray-finned fishes, cichlids, oreochromine cichlids, flounders, cods, groupers, basses, black basses, temperate basses, seabasses, for example Asian seabasses, tilapias, mullets, milkfishes, pufferfishes, pompanos, jacks, amberjacks, lates, grunters, perciformes, rabbitfishes, perches, cobias, carps, catfishes, smelts, sweetfishes, rockfishes, chubs, eels, gouramis, halibuts, plaices, roaches, rudds, soles and yellowtails.

Particular species of fish are black sea bream (Acanthopagrus schlegeli), common spade fish (Scataphagus argus), Malabar rock cod (Epinephelus malabaricus), Mozambique tilapia (Oreochromis mossambicus), Sobaity sea bream (Sparidentex hasta), olive flounder (Paralichthys olivaceus), Atlantic cod (Gadus morhua), Atlantic halibut (Hippoglossus hippoglossus), striped bass (Morone saxatilis), black porgy (Acanthopagrus schlegeli), blue tilapia (Oreochromis aurea), giant perch (Lates calcarifer), grey mullet (Mugil cephalus), large scale mullet (Liza macrolepis), milk fish (Chanos chanos), snubnose pompano (Trachinotus blochii), three-striped tiger fish, (Terapon jarbua), yellowfin bream (Acanthopagrus australis), tiger puffer (Takifugu rubripes), yellowtail amberjack (Seriola lalandi), striped jack (Pseudocaranx dentex), European sea bass (Dicentrarchus labrax), banded grouper (Epinephelus awoara), rabbit fish (Siganus fuscescens), thinlip mullet (Liza ramada), white sea bream (Diplodus sargus sargus), Japanese sea perch (Lateolabrax japonicus), Japanese sea bass (Lateolabrax japonicus), Japanese amberjack (Seriola quinqueradiata), dhufish (Glaucosoma hebraicum), black sea bass (Lates calcarifer) brown-marbled grouper (Epinephelus fuscoguttatus), humpback grouper (Cromileptes altivelis), leopard coral grouper (Plectropomus leopardus), Malibar grouper (Epinephelus malabaricus) orange-spotted grouper (Epinephelus coioides), haddock (Melanogrammus aeglefinus), greater amberjack (Seriola dumerili), spotted halibut (Verasper variegatus) olive flounder (Paralichthys olivaceus), cobia (Rachycentron canadum), tiger puffer (Takifugu rubripes), Borneo mullet (Liza macrolepis), tilapia (Oreochromis sp.), yellowfin bream (Acanthopagrus australis), greasy grouper (Epinephelus malabaricus), carp (Cyprinus sp.), Asian cichlid (Etroplus suratensis), southern flounder (Paralichthys lethostigma), ayu (Plecoglossus altivelis altivelis), Korean rockfish (Sebastes schlegeli), and Sobiaty sea bream (Acanthopagras cuvieri), as Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), humpback salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), chinook salmon (Oncorhynchus tshawytscha), masu salmon (Oncorhynchus masou), Taiwanese salmon (Oncorhynchus masou formosanum), Sockeye salmon or Red salmon (Oncorhynchus nerka); such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), coastal cutthroat trout (Oncorhynchus or Salmo clarkii clarkii), sea trout (Oncorhynchus mykiss); Salvelinus species such as Brook trout (Salmo fontinalis),; char, such as Arctic char (Salvelinus alpinus); freshwater whitefishes; graylings; taimens; lenoks

Particularly contemplated are the fish belonging to the Salmonidae family, including salmons (genus Salmo or Oncorhynchus) such as Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), humpback salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), chinook salmon (Oncorhynchus tshawytscha), masu salmon (Oncorhynchus masou), Taiwanese salmon (Oncorhynchus masou formosanum), Sockeye salmon or Red salmon (Oncorhynchus nerka); trouts (genus Oncorhynchus), such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), coastal cutthroat trout (Oncorhynchus or Salmo clarkii clarkii), sea trout (Oncorhynchus mykiss); Salvelinus species such as Brook trout (Salmo fontinalis),; char, such as Arctic char (Salvelinus alpinus); freshwater whitefishes; graylings; taimens; lenoks, Preferably, the Salmonidae is a salmon, in particular Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) or a trout, in particular rainbow trout (Oncorhynchus mykiss).

The fish is preferably selected from the group consisting in Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) or rainbow trout (Oncorhynchus mykiss), notably Atlantic salmon (Salmo salar).

In another preferred embodiment, the fish is preferably selected from the group consisting in Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) or rainbow trout (Oncorhynchus mykiss), notably Atlantic salmon (Salmo salar), and the parasite is a haematophagous parasite, notably a haematophagous sea louse, for example Lepeophtheirus salmonis.

The isoxazoline is preferably administered to juvenile fish. In a particular embodiment, the fish is a salmon and the isoxazoline is administered to a smolt, before said smolt is transferred from fresh-water tanks or cages to sea water cages. Typically, the smolt weighs between 50 and 350 g, preferably 50 and 150 g, more preferably between 80 and 120 g when the isoxazoline is administered.

The isoxazoline may be administered to the fish in a formulation that is a solution (the isoxazoline is dissolved in the formulation), a suspension (the isoxazoline is dissolved in the formulation) or is a long-injectable implant.

For intraperitoneal and intramuscular injection, the isoxazoline is formulated in a non-aqueous polar solvent. The isoxazoline may be present in the formulation in an amount of 10-1000 mg/ml of the formulation, preferably of 100 to 500 mg/ml of the formulation. The formulation may comprise a solvent having solubility for the isoxazoline, in particular selected from the group consisting in glycofurol (tetraglycol), acetone, acetonitrile, benzyl alcohol, butyl diglycol, dimethylacetamide, dimethylsulfoxide, dimethylformamide (DMF), N,N-diethyl-3-methylbenzamide, dipropylene glycol n-butyl ether, ethyl alcohol, isopropanol, methanol, phenylethyl alcohol, isopropanol, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, monomethylaceamide, dipropylene glycol monomethyl ether, liquid polyoxyethylene glycols, propylene glycol, N-methylpyrrolidone (NMP), 2-pyrrolidone, limonene, eucalyptol, diethylene glycol monoethyl ether, ethylene glycol, diethyl phthalate, polyethoxylated castor oil, methyl ethyl ketone, ethyl-L-lactate, and mixtures thereof, preferably a non-aqueous polar solvent selected from the group consisting in DMSO, NMP, tetraglycol, acetone, DMF and mixtures thereof. More preferably the non-aqueous polar solvent comprises DMSO. The non- aqueous polar solvent may be present in the formulation in an amount of 5-100% by weight, and preferably may be present in an amount of 10-50% by weight. The formulation may comprise a solubilizer, in particular selected from Cremophor EL, Tween, Brij C10, Kolliphor HS15, Cremophor RH40 and mixtures thereof. The solubilizer may be present in the formulation in an amount of 10-95% by weight, preferably may be present in an amount of 30-60% by weight. The formulation may also comprise a hydrophilic polymer, in particular having a molecular weight in the range of 50-2000, such as 100-1800. The hydrophilic polymer is preferably a PEG (polyethylene glycol), a PEG derivative, PPG (polypropylene glycol), or a PPG derivative and mixtures thereof. Preferably, the hydrophilic polymer is selected from the group consisting in monofunctional PEG, a homobifunctional PEG, a heterobifunctional PEG, a multi-arm PEG, a star-PEG, a polyether, a homobifunctional PPG, or a monofunctional PPG. More preferably the hydrophilic polymer comprises PEG, and the hydrophilic polymer may be PEG having a molecular weight of between 200 and 1000 g/mol. In specific embodiments, the hydrophilic polymer comprises PEG300, PEG400, or PEG600. The hydrophilic polymer may be present in the formulation in an amount of 5-100% by weight, preferably in an amount of about 70-90%, most preferably about 85% by weight. The formulation may comprise a combination of one or more hydrophilic polymer(s) and one or more non-aqueous polar solvent(s), preferably 75-90% by weight of one or more hydrophilic polymer(s) and 10-25% by weight of one or more non-aqueous polar solvent(s). In a particular embodiment, the hydrophilic polymer is present in the formulation in a total amount of about 85% by weight, and the non-aqueous polar solvent is present in the formulation in a total amount of about 15% by weight. For the sake of clarity, “xx mg/ml” of isoxazoline refers to the amount of isoxazoline (in mg) that is comprised in 1 ml of the formulation, i.e. in the one or more non-aqueous polar solvent(s), one or more hydrophilic polymer(s) and mixtures thereof.

Examples of formulations are those described in WO2016/042154 and WO2017/021498, incorporated herein in their entirety.

The isoxazoline may be administered in the form of an implant, notably in the form of microspheres, in particular a poly(lactic-co-glycolic acid) (PLGA)- and/or poly(lactic acid) (PLA)-based microspheres.

When administered at a dose ranging from 1 to 100 or 120 mg/kg, preferably from 10 to 90 mg/kg, more preferably from 25 to 50 mg/kg, in particular about 40 mg/kg or ranging from about 40 mg/kg to about 120 mg/kg, of fish bodyweight, the isoxazoline, especially fluralaner, provides effective control (e.g. it provides 90% reduction, preferably 95% reduction, of the parasite count or in particular reduces or maintain the number (or count) of gravid (or ovigerous) female sea lice or adult sea lice below 0.1 gravid female / fish during outmigration period of wild salmon, 0.2 gravid female / fish during outmigration period of wild salmon and 0.5 gravid female / fish during the rest of the year, 0.3-0.5 gravid female / fish during outmigration period of wild salmon and 2.0 gravid female / fish during the rest of the year, 1.5 gravid female / fish, 3.0 gravid female / fish, 3.0 adult sea lice.) for 1 month, 2 months, 3 months, 4 months, 5 months or 6 months.

The first aspect also encompasses the use of an isoxazoline for controlling a parasite infestation on a fish, as described above.

The first aspect also encompasses also a method of controlling a parasite infestation on a fish as described above, which comprises administering to the fish an effective dose of the isoxazoline, as described above.

The first aspect further encompasses the use of an isoxazoline for the manufacture of a medicament intended for controlling a parasite infestation on a fish, as described above.

In a second aspect, the present invention concerns Modoflaner, for use in controlling a parasite infestation on a fish.

The Inventors have found that a specific arylaminobenzamide compound, namely Modoflaner, exhibits excellent activity, in particular long-acting activity, against sea lice, and can be used for effective and prolonged control of sea lice infestation on fish.

The Inventors have also found that Modoflaner is nontoxic and well tolerated by fish, in contrast to many known antiparasitic agents that are toxic to fish.

As used herein, the term "Modoflaner" refers to (6-fluoro-N-(3-(2-iodo-4-(perfluoropropan-2-yl)-6-(trifluoromethyl)phenylcarbamoyl)-2-fluoro-phenyl)-nicotinamide, having the following formula:
Formula (I)

pharmaceutically acceptable salts, solvates, hydrates, tautomers and/or crystal polymorphs thereof.

Modoflaner can be prepared following the procedure described in WO2019/059412.

Acid and base salts can typically be formed by, for example, mixing Modoflaner with an acid or base, respectively, using various known methods in the art. To the extent a salt of Modoflaner is intended to be administered in vivo (i.e., to the fish) for a therapeutic benefit, the salt is pharmaceutically acceptable. In general, an acid addition salt can be prepared by reacting a free base compound with an approximately stoichiometric amount of an inorganic or organic acid. As used herein the term "salts" refers notably to acid addition salts of the pyridine residue of Modoflaner.

Modoflaner may be formulated into stable complexes with a solvent, such that the complex remains intact after the non-complexed solvent is removed. These complexes are often referred to as "solvates." It is particularly desirable to form stable hydrates with water as the solvent. A solvate of Modoflaner as mentioned above may be formed by aggregation of Modoflaner with solvent molecules such as water, alcohols, for example ethanol, aromatic solvents such as toluene, ethers, halogenated organic solvents such as dichloromethane, preferably in a definite proportion by weight.

Modoflaner may be made as various crystal polymorphs. Polymorphism is important in the development of veterinary products since different crystal polymorphs or structures of the same molecule can have vastly different physical properties and biological performances.

In an embodiment, Modoflaner is crystalline modification IV of Modoflaner as described in WO2022/101502.

The parasite may be any fish-parasitic crustacean, notably a sea louse. In general, Fish parasites such as copepods, in particular sea lice, have both free-swimming (planktonic) and parasitic life stages, all separated by moults. It should be understood that, in the present invention, only parasites that are parasitic to fish are contemplated.

Within the family Argulidae, the parasites belonging to the genus Argulus are of relevance, in particular Argulus foliaceus.

The family Lernanthropidae includes Lernanthropus spp. Species such as Lernanthropus kroyeri, Lernanthropus callinomymicola, Lernanthropus indefinitus, Lernanthropus cynoscicola and Lernanthropus gisleri. These parasites are of particular concern in Mediterranean fish farming.

The family Caligidae includes the genus Lepeophtheirus and Caligus, in particular the species Caligus acanthopagri, Caligus antennatus, Caligus brevicaudatus, Caligus clemensi, Caligus curtus, Caligus dussumieri, Caligus elongatus, Caligus epidemicus, Caligus fugu, Caligus lalandei, Caligus latigenitalis, Caligus longicaudatus, Caligus longipedis, Caligus minimus, Caligus multispinosus, Caligus nanhaiensis, Caligus orientalis, Caligus oviceps, Caligus pageti, Caligus patulu, Caligus pelamydis, Caligus punctatus, Caligus rogercresseyi, Caligus rotundigenitalis, Caligus spinosus, Caligus stromii, Caligus teres, Lepeophtheirus atypicus, Lepeophtheirus cuneifer, Lepeophtheirus hippoglossi, Lepeophtheirus longiventris, Lepeophtheirus paralichthydis, Lepeophtheirus salmonis, Parapetalus occidentalis, Pseudocaligus apodus, Pseudocaligus fugu, Diergasilus kasaharai, Ergasilus australiensis, Ergasilus borneoensis, Ergasilus ceylonensis, Ergasilus labracis, Ergasilus lizae, Ergasilus lobus, Pseudoergasilus zacconis, Alella macrotrachelus, Alella sp., Bomolochus stocki, Clavella adunca, Lernanthropus atrox, Lernanthropus chrysophrys, Lernanthropus lappaceus, Colobomatus labrachis, Eobrachiella elegans seiolae, Hemobaphes disphaerocephalus, Holobomolochus confusus, or Neobrachiella fugu. These parasites are of particular concern in Salmonidae farming.

In a preferred embodiment, the parasite is at least one of Lepeophtheirus salmonis, Caligus clemensi, Caligus curtus, Caligus dussumieri, Caligus elongates, Caligus longicaudatus, Caligus rogercresseyi or Caligus stromii, more preferably Lepeophtheirus salmonis, Caligus elongates, or Caligus rogercresseyi. These parasites are of particular concern in Salmonidae farming.

Particular species of fish are black sea bream (Acanthopagrus schlegeli), common spade fish (Scataphagus argus), Malabar rock cod (Epinephelus malabaricus), Mozambique tilapia (Oreochromis mossambicus), Sobaity sea bream (Sparidentex hasta), olive flounder (Paralichthys olivaceus), Atlantic cod (Gadus morhua), Atlantic halibut (Hippoglossus hippoglossus), striped bass (Morone saxatilis), black porgy (Acanthopagrus schlegeli), blue tilapia (Oreochromis aurea), giant perch (Lates calcarifer), grey mullet (Mugil cephalus), large scale mullet (Liza macrolepis), milk fish (Chanos chanos), snubnose pompano (Trachinotus blochii), three-striped tiger fish, (Terapon jarbua), yellowfin bream (Acanthopagrus australis), tiger puffer (Takifugu rubripes), yellowtail amberjack (Seriola lalandi), striped jack (Pseudocaranx dentex), European sea bass (Dicentrarchus labrax), banded grouper (Epinephelus awoara), rabbit fish (Siganus fuscescens), thinlip mullet (Liza ramada), white sea bream (Diplodus sargus sargus), Japanese sea perch (Lateolabrax japonicus), Japanese sea bass (Lateolabrax japonicus), Japanese amberjack (Seriola quinqueradiata), dhufish (Glaucosoma hebraicum), black sea bass (Lates calcarifer) brown-marbled grouper (Epinephelus fuscoguttatus), humpback grouper (Cromileptes altivelis), leopard coral grouper (Plectropomus leopardus), Malibar grouper (Epinephelus malabaricus) orange-spotted grouper (Epinephelus coioides), haddock (Melanogrammus aeglefinus), greater amberjack (Seriola dumerili), spotted halibut (Verasper variegatus) olive flounder (Paralichthys olivaceus), cobia (Rachycentron canadum), tiger puffer (Takifugu rubripes), Borneo mullet (Liza macrolepis), tilapia (Oreochromis sp.), yellowfin bream (Acanthopagrus australis), greasy grouper (Epinephelus malabaricus), carp (Cyprinus sp.), Asian cichlid (Etroplus suratensis), southern flounder (Paralichthys lethostigma), ayu (Plecoglossus altivelis altivelis), Korean rockfish (Sebastes schlegeli), and Sobiaty sea bream (Acanthopagras cuvieri), as Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), humpback salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), chinook salmon (Oncorhynchus tshawytscha), masu salmon (Oncorhynchus masou), Taiwanese salmon (Oncorhynchus masou formosanum), Sockeye salmon or Red salmon (Oncorhynchus nerka); such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), coastal cutthroat trout (Oncorhynchus or Salmo clarkii clarkii), sea trout (Oncorhynchus mykiss); Salvelinus species such as Brook trout (Salmo fontinalis),; char, such as Arctic char (Salvelinus alpinus); freshwater whitefishes; graylings; taimens; lenoks.

Particularly contemplated are the fish belonging to the Salmonidae family, including salmons (genus Salmo or Oncorhynchus) such as Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), humpback salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), chinook salmon (Oncorhynchus tshawytscha), masu salmon (Oncorhynchus masou), Taiwanese salmon (Oncorhynchus masou formosanum), Sockeye salmon or Red salmon (Oncorhynchus nerka); trouts (genus Oncorhynchus), such as rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), coastal cutthroat trout (Oncorhynchus or Salmo clarkii clarkii), sea trout (Oncorhynchus mykiss); Salvelinus species such as Brook trout (Salmo fontinalis); char, such as Arctic char (Salvelinus alpinus); freshwater whitefishes; graylings; taimens; lenoks.

Preferably, the fish is Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) or a trout, in particular rainbow trout (Oncorhynchus mykiss), notably Atlantic salmon (Salmo salar).

Modoflaner may be administered to the fish by any route which provides the sought control of parasite infestation, including enteral administration, notably oral administration, topical administration, or parenteral administration.

In determining the effective dose of Modoflaner, a number of factors are considered, including, but not limited to the species of fish; the degree of parasite infestation; the response of the fish population; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication and the duration of the sought effect.

For oral administration, Modoflaner may be administered in a medicated feed (freely taken by the fish or by gavage) or in a medicated water (the drug is dissolved or suspended in the water surrounding the fish).

Compositions suitable for oral administration notably include a medicated fish feed (e.g. a nutritionally complete feed comprising Modoflaner) typically in the form of granules or pellets and a medicated premix that is added to fish feed, to obtain a medicated fish feed.

When administered orally, the dose of Modoflaner is in the range of about 0.01 to 500 mg/kg, in particular 1 to 100 mg/kg, of the fish bodyweight. The medicated fish feed may be administered on one day or over the course of several days, until the amount of Modoflaner ingested by the fish is sufficient to provide the sought control of parasite infestation for the desired period. For example, the medicated fish feed is administered to the fish through multiple feedings (i.e. a divided daily dose). For example, a medicated fish feed comprising Modoflaner can be administered 1 time daily, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 days, notably 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 days.

For topical administration, Modoflaner may be administered by immersion or dipping (the fish is transferred from its pen or basin to a tank containing water in which Modoflaner is dissolved or suspended and allowed to stay in this tank during a period sufficient to reach an effective dose of Modoflaner), bath treatment (Modoflaner is dissolved and/or suspended in the water where the fish lives), flushing (the fish is immersed in running water or a raceway system wherein Modoflaner is dissolved or suspended), hyperosmotic infiltration (the fish is placed in an hypertonic solution then in water in which Modoflaner is suspended or dissolved or the fish is placed in a hypertonic solution or suspension of Modoflaner), transdermal application, inhalation or intranasal route.

Compositions for immersion, dipping, bath treatment, flushing, hyperosmotic infiltration, transdermal application, inhalation or intranasal route include powders, granulates, solutions, emulsions micro/nanoemulsions, emulsifiable concentrates, suspensions, nanosuspensions, or suspension concentrates, tablets, water-soluble films or films.

When administered by immersion, dipping, bath treatment, or flushing, the effective dose of Modoflaner is comprised between about 2ppb to about 500ppb, preferably between 20 and 250 ppb, more preferably between 50 and 150 ppb, based on the amount of water used for bathing, immersing, dipping or flushing the fish. The concentration of Modoflaner during administration to the fish by immersion, dipping, bath treatment, or flushing, depends on the manner and duration of treatment and also on the age and condition of the fish being treated. A typical immersion, dipping, bath treatment, or flushing time ranges from about 15 minutes to about 4 hours, preferably from about 15 minutes to 2 hours, and more preferably from about 30 minutes to about 1 hour.

For parenteral administration, Modoflaner may be administered by intraperitoneal injection (e.g. an injection given into the mid ventral line, just below vent), subcutaneous injection, intramuscular injection (e.g. injection into the epaxial musculature, generally approximately mid-way between the mid-dorsal line and the lateral line), or injection into the dorso-median sinus (e.g. an injection in the mid-dorsal line in the angle at the caudal margin of the cranial dorsal fin).

Compositions suitable for parenteral administration comprise solutions in a solvent having solubility for Modoflaner, such as glycofurol (tetraglycol), acetone, acetonitrile, benzyl alcohol, butyl diglycol, dimethylacetamide, dimethylsulfoxide, dimethylformamide (DMF), N,N-diethyl-3-methylbenzamide, dipropylene glycol n-butyl ether, ethyl alcohol, isopropanol, methanol, phenylethyl alcohol, isopropanol, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, monomethylaceamide, dipropylene glycol monomethyl ether, liquid polyoxyethylene glycols, propylene glycol, N-methylpyrrolidone (NMP), 2-pyrrolidone, limonene, eucalyptol, diethylene glycol monoethyl ether, ethylene glycol, diethyl phthalate, polyethoxylated castor oil, methyl ethyl ketone, ethyl-L-lactate, and mixtures thereof, preferably a non-aqueous polar solvent selected from the group consisting in DMSO, NMP, tetraglycol, acetone, more preferably DMSO or NMP.

When administered parenterally, the dose of Modoflaner ranges from about 0.1 mg/kg to 150 mg/kg, preferably from 10 mg/kg to about 150 mg/kg, more preferably from about 10 mg/kg to about 120 mg/kg, even more preferably from 60 to 120 mg/kg, of the fish bodyweight. The dose of Modoflaner to administer depends on several factors, including notably the parasite and the sought duration of activity.

In specific embodiments, the dose of Modoflaner is about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 110 mg/kg, about 120 mg/kg, about 130 mg/kg, about 140 mg/kg or about 150 mg/kg.

Preferably, Modoflaner is administered by parenteral administration, in particular by intraperitoneal injection or intramuscular injection. Preferably, the dose administered parenterally is comprised between 60 and 120 mg/kg. In one particular embodiment, the dose of Modoflaner is 90 mg/kg.

In an alternate embodiment, Modoflaner may be administered subcutaneously, intramuscularly or intraperitoneally to the salmon as an implant comprising Modoflaner and a biodegradable polymer, such as poly(lactic-co-glycolic acid) (PLGA)- and/or poly(lactic acid) (PLA)-based implant, in particular as poly(lactic-co-glycolic acid) (PLGA)- and/or poly(lactic acid) (PLA)-based microspheres.

Modoflaner may be administered at any time during the life of the fish, either prophylactically (e.g. to prevent the infestation by parasites) or therapeutically (e.g. to treat an already existing parasitic infestation).

When the fish is a salmon, Modoflaner is preferably administered prophylactically to smolts, before the smolts are transferred from fresh-water tanks or cages to sea water cages and may enter in contact with the parasite. Typically, the smolts weight between 50 and 350 g, preferably 50 and 150 g, more preferably between 80 and 120 g.

Modoflaner is well-tolerated by the smolts at doses as high as 120 mg/kg. In addition, this dose provides protection against parasites for at least four months despite the important weight gain of the salmon during this period of about tenfold.

Due to the high efficacy of Modoflaner against fish parasites, its long duration of activity and its low toxicity, it is possible to administer to smolts a single dose of Modoflaner that is sufficient to control a parasite infestation for several weeks (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 weeks) or months (e.g. 1, 2, 3, 4, 5, 6 months) without the need to administer again Modoflaner before such number of weeks or months have elapsed. This is particularly beneficial as the manipulation of the growing salmons by humans is a major cause of stress, which results in death or debilitation of the fish.

In a particular embodiment, a single dose of Modoflaner is sufficient to control a parasite infestation for at least 4 months, 5 months or 6 months.

In a particular embodiment, a single dose of 90 mg/kg to 120 mg/kg of Modoflaner is sufficient to control a parasite infestation for at least 4 months, 5 months or 6 months.

In a particular embodiment, Modoflaner provides a reduction or maintenance of the number (or count) of gravid (or ovigerous) female sea lice or adult sea lice below a regulatory threshold, i.e. a count of sea lice over which a treatment must be administered. Examples of thresholds are 0.1 gravid female / fish during outmigration period of wild salmon (ASC), 0.2 gravid female / fish during outmigration period of wild salmon and 0.5 gravid female / fish during the rest of the year (Norway), 0.3-0.5 gravid female / fish during outmigration period of wild salmon and 2.0 gravid female / fish during the rest of the year (Ireland), 1.5 gravid female / fish (Faroe Islands), 3.0 gravid female / fish (Chile and Scotland), 3.0 adult sea lice (British Columbia, Canada).

The second aspect also encompasses the use of Modoflaner for controlling a parasite infestation on a fish, as described above.

The second aspect also encompasses also a method of controlling a parasite infestation on a fish as described above, which comprises administering to the fish an effective dose of Modoflaner, as described above.

The second aspect further encompasses the use of Modoflaner for the manufacture of a medicament intended for controlling a parasite infestation on a fish, as described above.

In a third aspect, the present invention concerns a method of controlling a sea lice infestation in a farmed Salmonidae comprising administering intraperitoneally or intramuscularly to the fish a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, or a pharmaceutical composition comprising a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, and one or more pharmaceutical excipients.

Given the long duration of efficacy of Modoflaner or the isoxazolines against sea lice, e.g. of for several months, the frequency of administration to the fish is reduced. Furthermore, the parenteral route of administration of the pharmaceutical active ingredient has a limited environmental impact, notably on the surrounding ecosystems and wild fish when compared to the currently used routes of administration as bath or feed.

Preferably, the farmed Salmonidae is selected from the group consisting in Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss), notably Atlantic salmon (Salmo salar).

In a preferred embodiment, the pharmaceutical active ingredient is Modoflaner.

Preferably, the dose of Modoflaner ranges from about 0.1 mg/kg to 150 mg/kg, preferably from 1 mg/kg to about 100 mg/kg, more preferably from about 10 mg/kg to about 90 mg/kg, even more preferably from 30 to 50 mg/kg, of the fish bodyweight. In a specific embodiment, the dose of Modoflaner is 30 mg/kg or 40 mg/kg.

In specific embodiments, the dose of Modoflaner is about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 1100 mg/kg, about 120 mg/kg, about 130 mg/kg, about 140 mg/kg or about 150 mg/kg.

Preferably, the dose of Modoflaner administered parenterally is comprised between 60 and 120 mg/kg. In one particular embodiment, the dose of Modoflaner is 90 mg/kg.

In another preferred embodiment, the isoxazoline is fluralaner. Preferably, the dose of fluralaner ranges from 1 to 100 or 120 mg/kg, preferably from 10 to 90 mg/kg, more preferably from 25 to 50 mg/kg, or from about 40 mg/kg to about 120 mg/kg, of fish bodyweight, more particularly about 40 mg/kg, of fish bodyweight.

Preferably, the pharmaceutical active ingredient or the pharmaceutical composition is administered to juvenile fish, in particular to smolts, said juvenile fish or smolt having a weight ranging from 50 to 350 grams, preferably from 50 to 150 g, more preferably ranging from 80 to 120 g.

Preferably, the sea lice is selected from the group consisting in Lepeophtheirus salmonis, Caligus clemensi, Caligus curtus, Caligus dussumieri, Caligus elongates, Caligus longicaudatus, Caligus rogercresseyi or Caligus stromii, more particularly Lepeophtheirus salmonis or Caligus rogercresseyi, or combinations thereof.

Preferably, the sea lice is selected from haematophagous sea lice, for example Lepeophtheirus salmonis.

In a specific embodiment, the sea lice is selected from haematophagous sea lice, for example Lepeophtheirus salmonis, and the fish is selected from the group consisting in Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss), notably Atlantic salmon (Salmo salar).

In a specific embodiment, the method of controlling a sea lice infestation in a farmed Salmonidae comprises administering intraperitoneally or intramuscularly to the fish a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, or a pharmaceutical composition comprising a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, and one or more pharmaceutical excipients to a juvenile fish, in particulars to smolts for the initial treatment and thereafter every 3, 4, 5 or 6 months to the fishes collected in sea water cages.

The third aspect also encompasses the use of a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, or a pharmaceutical composition comprising a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, and one or more pharmaceutical excipients for controlling a sea lice infestation in a farmed Salmonidae, wherein the pharmaceutical active ingredient or pharmaceutical composition is administered intraperitoneally or intramuscularly to the farmed Salmonidae, as described above.

The third aspect also encompasses a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, or a pharmaceutical composition comprising a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, and one or more pharmaceutical excipients for use in controlling a sea lice infestation in a farmed Salmonidae, wherein the pharmaceutical active ingredient or pharmaceutical composition is administered intraperitoneally or intramuscularly to the farmed Salmonidae, as described above.

The third aspect further encompasses the use of a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, or a pharmaceutical composition comprising a pharmaceutical active ingredient selected from the group consisting in Modoflaner and an isoxazoline, preferably lotilaner, afoxolaner, sarolaner or fluralaner, and one or more pharmaceutical excipients for the manufacture of a medicament intended for intraperitoneal or intramuscular administration, for controlling a parasite infestation on a fish, as described above.

In one particular embodiment, the present invention concerns Modoflaner for use in controlling a Lepeophtheirus salmonis, Caligus elongates or Caligus rogercresseyi infestation in a Salmonidae, preferably Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) or a trout, in particular rainbow trout (Oncorhynchus mykiss), wherein a dose of Modoflaner ranging from about 60 to about 120 mg/kg, notably about 90 mg/kg or about 120 mg/kg; of fish bodyweight is administered parenterally, in particular intraperitoneally, to the Salmonidae, said dose providing control of the infestation for at least 4 months, or more than 4 months, 5 months or 6 months.

DESCRIPTION OF THE FIGURES

shows the X-ray powder diffractogram of Modoflaner form IV at 25 °C and Cu-Kα 1 radiation obtained using a single-crystal diffractometer Oxford Diffraction Gemini R Ultra with the following parameters: Anode: Cu; K-Alpha1 [Å]: 1.54060; Generator: 40 mA, 40 kV; Sample rotation: Yes; Scan axis: Gonio; Starting Position [°2Th.]: 2.0066; End Position [°2Th.]: 37.9906.

shows the average number of sea lice (Y-axis) of the experiment of example 3 at different time points (X-axis, days) for each development stage (non-motile = 1; motile = 2; mature adult female = 3 and mature adult female = 4) for the three groups (left histogram filled with dots = control; middle histogram filed with dashes = fluralaner 40 mg/kg and right histogram, filled in black = modoflaner 90 mg/kg).

EXAMPLES

Example 1: evaluation of the efficacy of Modoflaner in treating a sea lice infestation

Groups design:

60 Atlantic salmon (Salmo salar), of about 350g, were sea lice-infested (infection dose of 20 copepodids/fish, Lepeophtheirus salmonis), 4 weeks prior to the D0 injection described above. The fish were kept in sea water at ambient temperature (about 11 °C), 32 ‰ average salinity. Fish were fed with automatic feeders.

The number of lice at the time of injection (D0) was about 11.1 lice per fish.

The trial included 3 test groups:

Treated group T1: 10 mg/kg bodyweight; 20 fish in 2 separate tanks (10 fish in each tank);
Treated group T2: 30 mg/kg bodyweight; 20 fish in 2 separate tanks (10 fish in each tank);
Non-treated group C (control): non-treated control, 20 fish.

As described above, Modoflaner was injected at D0 in two doses (10 mg/kg and 30 mg/kg), as depicted in Table 1:

Group	n (number of fish)	Dose (mg/kg)	Formulation	Dose volume (mL/fish)	Dosing route
T1
	20	10	2% solution in DMSO	0.1	IP (intraperitoneal)
T2	20	30	6% solution in DMSO	0.1	IP (intraperitoneal)
C	20	-	DMSO	0.1	IP (intraperitoneal)

Table 1

The number of sea lice in each group was evaluated after 7 days and 14 days.

Results: Safety:

No fish died at time of injection or up to one month post injection of Modoflaner, for all groups. No intraperitoneal reactions associated with the injection has been observed, at time of injection or up to one month post injection. No significant difference between the control and the treatment groups was found in terms of ulcers.

Efficacy against an existing sea lice ( Lepeophtheirus salmonis ) infestation:

At day 7 and day 14, the average number of sea lice in the control group was 9.2 lice / fish and 7.2 lice / fish, respectively.

In the T1 group (10 mg/kg of Modoflaner), the average number of sea lice was 0.05 lice / fish at 7 days and 0 at 14 days.

In the T2 group (10 mg/kg of Modoflaner), no sea lice was observed on day 7 and day 14.

Modoflaner is effective in treating a sea lice infestation after 7 days at a dose as low as 10 mg/kg when administered intraperitoneally.

Example 2: evaluation of the efficacy of Modoflaner in preventing a Sea lice infestation.

In this study fish were first treated and challenged 3 weeks later with 20 copepodids/fish. 3 doses were investigated, each dose being administered either intraperitoneally or intramuscularly as depicted in Table 2:

Group	n (number of fish)	Dose (mg/kg)	Formulation	Dose volume (mL/fish)	Dosing route
T1
	20	10	2% solution in DMSO	0.1	IP (intraperitoneal, 20 fish) IM (intramuscular, 20 fish)
T2	20	30	6% solution in DMSO	0.1	IP (intraperitoneal, 20 fish) IM (intramuscular, 20 fish)
T3	20	90	18% solution in DMSO	0.1	IP (intraperitoneal, 20 fish) IM (intramuscular, 20 fish)
C	20	-	DMSO	0.1	IP (intraperitoneal, 20 fish) IM (intramuscular, 20 fish)

Table 2

Results: Safety:

No clinical signs associated with the injection of Modoflaner were observed, even at the highest dose of 90 mg/kg. Modoflaner is therefore well tolerated by salmons.

Efficacy against an induced sea lice ( Lepeophtheirus salmonis ) infestation:

28 days after injection of Modoflaner, the fish (200g at start of study) were challenged with copepodids, 20/fish, using standard methods with stagnant water, oxygenated keeping oxygen levels >80% saturation. Water flow was stopped during challenge. After 30 minutes, water flow was turned back on, oxygen tubes removed, and fish were kept without any further handling until counting at 28-32 days post challenge. Lice numbers were determined at 21 days post challenge, including stage of lice development. Relative reduction in lice numbers was contrasted with placebo group, kept in separate tanks. Statistical evaluation based on Poisson regression was used for assessment of statistical difference between treated and controls.

The efficacy corresponds to the reduction of the mean lice number obtained with Modoflaner in the T1-T3 groups, compared to the C group.

The results are presented in Table 3:

Group	Efficacy (%)
T1 - IM	75.8
T1 - IP	79.5
T2 - IM	98.4
T2 - IP	98.4
T3 - IM	97.3
T3 - IP	91.3
C	0

Table 3

One month after injection, Modoflaner is efficient for preventing attachment of sea lice on salmon. Notably, the dose of 30 mg/kg provides the most efficient control on sea lice attachment (98.4%), whatever the administration route (IP or IM).

Example 3: Efficacy and Safety of fluralaner and modoflaner against sea lice (Lepeophtheirus salmonis) infestations in farmed salmon in natural sea conditions in Norway.

For the trial treatments, two investigational products, Fluralaner (solution in NMP) and Modoflaner (solution in NMP/transcutol 60/40) were administered intraperitoneally at a dose of 0.1 ml. A control group received only N-methyl pyrrolidone (NMP/transcutol 60/40), dose size 0.1 ml injected intraperitoneally. The treatment took place over a period of four days. The fish did not receive feed for three days both before and after treatment. The doses were 40 mg/kg of the fish of fluralaner and 90 mg/kg of modoflaner.

The fish were Atlantic salmon (smolts, 500 to 1000 individuals per cage, 3 cages per group) having an average weight at start of 100 g. The fish were vaccinated with conventional vaccine: ALPHA JECT micro 7 from Pharmaq, dose size 0.05 ml injected intraperitoneally.

The fish were removed from the unit and put in a container with water added anesthetic (Finquel Vet) before the fish was individually weighed and treated (by injection of product). After treatment the fish was placed in a container with water before they were moved back into their respective trial unit.

The fish were then transferred from Land tanks to R&D Sea. First all fish were individually weighed and injected with the products at R&D Land at a bodyweight between 150 and 200 g and thereafter transferred to R&D Sea within two-three week after weighing. Arriving at R&D Sea the fish were placed in units based on treatment group.

Sampling and sea lice counting

At R&D Sea, sampling and sea lice counting ( Lepeophtheirus salmonis ) was performed every second week, 14±1 day). The first lice counting and scoring of fish is depending on fish health and status after transfer. On the day of lice counting the fish was starved, 30 fish per unit (randomized) were euthanized with an overdose of anesthetic (Finquel Vet) and sea lice counted and differentiated according to their development stage (4 stages):

1. Non motile (no differentiation between Chalimus I and II)

2. Motile (no differentiation between preadult male and preadult female)

3. Mature adult – female

4. Mature adult – male

The fish were transferred from the sea unit to land containers, one fish per container, and lice that has fallen of each fish were counted. In addition to sea lice counting the fish were weighed, opened and scored by Speilberg scale. No fish or lice was stored after sampling. Fish are maintained in the study units at R&D Sea and identified by unit number until the end of the trial. All remaining fish after trial termination will be euthanized.

Delousing: Any unit may be subject for delousing if the threshold of 1 adult female per fish in average is reached in the study unit or for animal welfare reason. If the threshold was reached for 2 consecutive samplings all units in the same group were subjected to delousing. All fish were sedated before the lice removed mechanically by point suction on each individual fish. The lice were not stored after counting.

Results:

The dose of 90 mg/kg of fish resulted in more than 6 months control, at an average of less than 0.5 sea lice per fish. Fluralaner, administered at the dose of 40 mg/kg of fish provided a lesser control and delousing was required at 4 months.

Hence, both fluralaner at 40 mg/kg of fish and modoflaner at 90 mg/kg of fish administered parenterally provide control of Lepeophtheirus salmonis infestation in sea. Both investigational products were well tolerated.

Example 4: Long-term product efficacy trial against sea lice (Caligus rogercresseyi) infestation upon Atlantic salmon (Salmo salar).

The objective of the study is to evaluate the efficacy of two experimental injectable products against C. rogercresseyi at chalimus and adult stages on Atlantic post-smolts salmons at 3 and 6-months post application.

The investigational products were evaluated against a negative control (0.9% NaCl solution), a positive control (NMP). The investigational products were either fluralaner (solution in NMP) administered at the 40 mg/kg dose or modoflaner (solution in NMP) administered at 90 mg/kg or 120 mg/kg of fish.

The fish (646 individuals) was Atlantic salmon (Salmo salar), post-smolts (strain Stofnfiskur). Fish size were ~ 60 g at stocking, ~ 130 g at injection of control product and ~ 180 g at injection of test products. All fish was vaccinated on stocking day with BlueGuard®5-1.

All study fish were stocked in a hatchery and vaccinated with BlueGuard® 5.1. For this, fish (n: 1200) were anaesthetised in groups of ~25 then intraperitoneally vaccinated with 0.1 ml of the vaccine. Fish were left to acclimate and immunize for 126 days until proper smoltification. Then, all fish were transferred to a 4000 L tanks to complete their acclimation and immunization periods before injection of the test / control products. On treatment day, test fish were marked, weighed, and injected with the test / positive control product. All fish were starved for 48 hours prior to the handling. Then, and in groups of ~20 fish at a time, they were anaesthetized and then injected with the corresponding test / control product (0.1 ml intraperitoneally).

The Study involved the performance of 3 sea lice challenges after 90 and 180 days (3 and 6 months) according to the following schedule:

Challenge n°	Study day	Number of tanks	Number of replicas	Number of fish per group per replica	Caligus stage counted
1	90	4	2	20-10	Adult
2	180	2	2	10-5	Adult

Before each sea lice challenge, the required number of fish per group was removed from the holding tank and stocked into challenge tanks for 14 days before challenge for acclimation purposes. For this, fish were removed from the holding tank in small groups at a time, anaesthetized. Weight was recorded for all selected fish to be challenged.

Fish were starved for 48 hours before each challenge. Fish were challenged in the study tanks at controlled temperature (~12.5°C) in sea water (~32‰) with C. rogercresseyi copepodites. Before challenge, copepodites were kept/reared under same water conditions as those mentioned above. On challenge day, the water flow was stopped, and water level lowered to reach a fish density ~80 kg/m3. The challenge room and tank lights were turned-off and challenge room and tank windows covered to avoid any light from entering the tanks. All study tanks were challenged at the same time. The fish were challenged by adding C. rogercresseyi copepodites on the tank’s surfaces at a ratio of 35-40 copepodites per fish. After 6 hours, the water level was slowly raised to normal level and water turnover was re-established and maintained at 0.8 tk/h for 18 further hours. 24 hours post challenge, water flow was fully re-established to between 1.0 – 1.3 tk/h and challenge room and tank lights turned on.

Sea lice counts were performed at the following points:

- for the two challenges: Lice attachment evaluation (4 days post-infestation -dpi-): lice load was assessed on 10 control fish.

- challenge n°1: efficacy evaluation on juvenile sea lice (9-10 dpi; Chalimus II-III stages) and efficacy evaluation on adult sea lice (21-25 dpi; Adult stage).

- challenge n°2: counting was performed at adult stages.

The results are presented in the next table:

Investigational product	% reduction of the number of adult caligus vs. negative control
	3 months	6 months
Fluralaner (40 mg/kg)	54.5	39.4
Modoflaner (90 mg/kg)	55	31.1
Modoflaner (120 mg/kg)	75	61
Negative control	-	-
Positive control (10 fish)	-	-

The results show that both fluralaner and modoflaner provide a significant reduction of the number of attached caligus with a long efficacy even after 6 months.

Claims

Modoflaner for use in controlling a parasite infestation on a fish.
Modoflaner for use according claim 1, wherein the parasite is a sea louse.
Modoflaner for use according to any of the preceding claims, wherein the parasite is at least one of Lepeophtheirus salmonis, Caligus clemensi, Caligus curtus, Caligus dussumieri, Caligus elongates, Caligus longicaudatus, Caligus rogercresseyi or Caligus stromii, more particularly Lepeophtheirus salmonis or Caligus rogercresseyi, preferably Lepeophtheirus salmonis,
Modoflaner for use according to any of the preceding claims, wherein the fish is selected from the group consisting in salmons, trouts, chars, freshwater whitefishes, graylings; or from sea breams, porgies, breams, scats, ray-finned fishes, cichlids, oreochromine cichlids, flounders, cods, groupers, basses, black basses, temperate basses, seabasses, for example Asian seabasses, tilapias, mullets, milkfishes, pufferfishes, pompanos, jacks, amberjacks, lates, grunters, perciformes, rabbitfishes, perches, cobias, carps, catfishes, smelts, sweetfishes, rockfishes, chubs, eels, gouramis, halibuts, plaices, roaches, rudds, soles and yellowtails.
Modoflaner for use according to any of the preceding claims, wherein the fish is selected from the group consisting in Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss).
Modoflaner for use according to any of the preceding claims, wherein Modoflaner is administered parenterally, in particular by intraperitoneal injection or intramuscular injection.
Modoflaner for use according to any of the preceding claims, wherein Modoflaner is administered at a dose ranging from about 0.1 mg/kg to 150 mg/kg, preferably from 10 mg/kg to about 150 mg/kg, more preferably from about 10 mg/kg to about 120 mg/kg,
Modoflaner for use according to any of the preceding claims, wherein Modoflaner is administered at a dose of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 110 mg/kg, about 120 mg/kg, about 130 mg/kg, about 140 mg/kg or about 150 mg/kg.
Modoflaner for use according to any of the preceding claims, wherein Modoflaner is administered to juvenile fish, in particular to smolts, said juvenile fish or smolt having a weight ranging from 50 to 350 grams, preferably 50 and 150 g, more preferably between 80 and 120 g.
Modoflaner for use according to any of the preceding claims, wherein Modoflaner provides control of the parasite infestation during 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 weeks or during 1, 2, 3, 4, 5, 6 months after administration.
Modoflaner for use according to any of the preceding claims, wherein Modoflaner reduces or maintains the number (or count) of gravid (or ovigerous) female sea lice or adult sea lice below 0.1 gravid female / fish during outmigration period of wild salmon, 0.2 gravid female / fish during outmigration period of wild salmon and 0.5 gravid female / fish during the rest of the year, 0.3-0.5 gravid female / fish during outmigration period of wild salmon and 2.0 gravid female / fish during the rest of the year, 1.5 gravid female / fish, 3.0 gravid female / fish or 3.0 adult sea lice.
Modoflaner for use in the control of an infestation of Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), Chinook salmon (Oncorhynchus tshawytscha) or rainbow trout (Oncorhynchus mykiss) by Lepeophtheirus salmonis or Caligus rogercresseyi, wherein from 1 mg to 100 mg, preferably from 10 mg to 90 mg, even more preferably from 30 mg to 50 mg of Modoflaner per kg body weight of the fish is administered to the fish, preferably parenterally.
Modoflaner and/or an isoxazoline, in particular selected from the group consisting in fluralaner, lotilaner, afoxolaner and sarolaner or a pharmaceutical composition comprising Modoflaner and/or an isoxazoline, in particular selected from the group consisting in fluralaner, lotilaner, afoxolaner and sarolaner and one or more pharmaceutical excipients, for use in controlling a sea lice infestation in a farmed Salmonidae, wherein Modoflaner and/or the isoxazoline or the pharmaceutical composition is administered to the fish intraperitoneally or intramuscularly.
Modoflaner and/or the isoxazoline, or the pharmaceutical composition comprising Modoflaner and/or an isoxazoline and one or more pharmaceutical excipients, for use according to claim 13, wherein Modoflaner and/or the isoxazoline, or the pharmaceutical composition comprising Modoflaner and/or an isoxazoline and one or more pharmaceutical excipients, is administered to juvenile fish and thereafter every 3, 4, 5 or 6 months to the fish collected in sea water cages.
Modoflaner and/or the isoxazoline, or the pharmaceutical composition comprising Modoflaner and/or an isoxazoline and one or more pharmaceutical excipients, for use according to claim 13 or 14, wherein Modoflaner is administered at a dose ranging from at a dose ranging from about 0.1 mg/kg to 150 mg/kg, preferably from 10 mg/kg to about 150 mg/kg, more preferably from about 10 mg/kg to about 120 mg/kg, and the isoxazoline is administered at a dose ranging from about 1 mg/kg to 100 mg/kg of bodyweight of the fish.