USING WATER-SOLUBLE TETRAPYRROLE COMPOUNDS AND LIGHT

Field of the invention

The invention relates to a new method for removing or killing of ectoparasites from farmed fish. More particularly, the invention relates to removing or killing crustacean ectoparasites from farmed fish. The fish may be a salmonid fish, such as Atlantic salmon ( Salmo salar L.) or rainbow trout ( Oncorhynchus mykiss). The crustacean ec toparasite may be salmon lice ( Lepeophtheirus salmonis, Caligus rogercresseyi , Ca- ligus spp.). Even more particularly the invention relates to a method comprising treatment means where light from an artificial light source is used in combination with at least one chemical compound at toxicologically acceptable concentrations for fish, in particular salmonid fish. The method relates to the use of a combination of certain water-soluble tetrapyrrole compounds and light. In particular the invention relates to a composition in the form of an aqueous solution comprising the water-soluble tetrapyrrole compound or a pharmaceutical derivative thereof.

Background

Aquaculture, also referred to as aquafarming, is the farming of aqueous organism. Aquaculture involves cultivating fresh water and saltwater populations under con trolled conditions and is in contrast to commercial harvesting or fishing where the or- ganisms are naturally present. The farmed organisms can typically be fish, crusta ceans, mollusks, aquatic plants, and algae. The aquaculture farms can be in the form of tanks (closed or semi closed), fishponds, ocean cages or nets.

Diseases, especially infectious diseases, are a problem in aquaculture. Within fish aq uaculture, especially salmon farming, there has been a development of prophylactic therapy in the form of vaccines and development of treatment of disease in the form of various drugs. However, salmon lice infection remains a main problem regarding salmon farming. Relevant prior art publications and their relevant content

Currently several non-medicinal approaches are used to reduce the number of infected fish in the fish farm or to remove lice from infected fish. Several drugs are also cur rently used to combat salmon lice infections in fish farming. The regulatory approved drug substances include organophosphates, pyrethroids and hydrogen peroxide.

Azamethiphos is an organophosphatase inhibitor of acetylcholinesterase. Aza- methiphos is the active substance in the commercial product Azasure. Deltamethrin is a pyrethrin that interferes with the nerve membrane function in salmon lice resulting in paralysis and death. Deltamethrin is the active drug substance in the commercial product ALPHA MAX. Hydrogen peroxide has for many years been used to combat salmon lice infections in fish including salmon. The mechanism of action is probably related to an oxidative effect on lice components and the formation of oxygen emboli within the salmon lice.

Development of drug resistance is a general problem with farming of fish including salmon, and currently the development of resistance is known for all drug substances used in fish farming.

Although vaccines against salmon lice have been described in the patent literature, see for example WO2018035199 and others, no commercial vaccine are currently in use for treatment of fish, including salmon, infected by salmon lice.

There is a need for improved methods for treatment and prophylaxis of salmon lice infections in fish farming, and during the last 10 years there have been published sev eral new patent families within the field. See for example W02020048945, WO2019168406, WO2019146827, WO2019146826, WO2019146825, WO2019245891, WO2019245722, WO2019234396, WO2019045573, WO2019145730, W02019121900, WO2019121887, WO2019034694 and W02019031970.

The combination of light and drugs related to fish has been a topic in a few published scientific documents. Photodynamic therapy is generally a term for a therapy that in volves a so-called photosensitizer and light, and oxygen, where the light activates the photosensitizer to generate singlet oxygen and other reactive and toxic species. Pho todynamic therapy is well described in human medicine, see for example: Kwiatkowski S et al., 2018, Photodynamic therapy - mechanisms, photosensitizers and combina tions. Biomed Pharmacother. 106: 1098-1107. The clinical use of photodynamic thera py is, however, limited to some specific indications; especially some skin diseases with products comprising 5-aminolevulinic acid methyl ester as photosensitizer, and age- related macular degeneration with products comprising verteporfin as photosensitizer. Many of the publications on photodynamic therapy relates to cancer and some publica tions relate to infections, however, these diseases are not commonly treated by pho todynamic therapy.

D-P Hader et al., 2016, Fighting fish parasites with photodynamically active chloro- phyllin. Parasitol. Res. 115: 2277-2283, describe a study where water-soluble chloro- phyllin was used. The result was "In Ichthyobodo, 2 pg/mL proved sufficient with sub sequent simulated solar radiation to almost quantitatively kill the parasites, while in Dactylogyrus, a concentration of about 6 pg/mL was necessary. The LD50 value for this parasite was 1.02 pg/mL. Trichodina could be almost completely eliminated at 2 pg/mL. Only in the parasitic crustacean Argulus, no killing could be achieved by a pho todynamic reaction using chlorophyllin." The chlorophyllin was prepared from spinach and does not comprise copper.

Eliana Alves et al., 2015, Potential applications of porphyrins in photodynamic inacti vation beyond the medical scope. J. Photochem. and Photobiol. C: Photochemistry Reviews, 22:34-57 is a general review publication within the field on photodynamic therapy. Figure 4 on page 43 in this publication illustrates generally a treatment pro tocol for photodynamic treatment of infected fish. The text in page 43 refers to Elina Alves et al., 2011, Photodynamic antimicrobial chemotherapy in aquaculture; photoin activation studies if Vibrio fischeri in PLOS One 6, 6, e20970. The light is "solar irradia tion from 380 to 700 nm consisting in 13 OSRAM lamps." The photosensitizer used in these studies is a porphyrin derivative.

Peter KJ Robertson et al., 2009, A new generation of biocides for control of Crustacea in fish farms. J. Photochem and Photobiol. B, 95: 58-63. This publication relates to the field of photodynamic therapy where light and a photosensitizer are used to generate toxic species. The photosensitizer was methylene blue or nuclear fast red, the marine species as a model organism in place of salmon lice was the marine copepod Acartia clause, the lamp was a 500W tungsten halogen lamp. The result showed that the ma rine copepod mortality with methylene blue was high when the methylene blue con centration was 1 micromolar or above at 1 hour light activation. With nuclear fast red as photosensitizer, there was not observed any mortality during 1 hour light activa tion. All light sources used in the above discussed prior art relate to white light.

The invention has for its object to remedy or to reduce at least one of the drawbacks of the prior art, or at least provide a useful alternative to prior art. The object is achieved through features, which are specified in the description below and in the claims that follow.

Summary of the present invention

The invention relates to a new method for treatment of salmon lice (L. salmonis, C. rogercresseyi , Caligus spp.) in farmed fish such as Atlantic salmon (S. salar L.) using an artificial light source and at least one chemical substance at toxicologically ac ceptable concentrations for Atlantic salmon. More specifically, the present inventors have identified a novel therapy for the treatment of salmon louse in farmed Atlantic salmon populations that comprises a combination of artificial light together with water- soluble tetrapyrrole compounds with the proviso that the water-soluble tetrapyrrole compound is not chlorophyllin. Chlorophyll is not a water-soluble tetrapyrrole com pound.

The fish may be a salmonid fish, such as Atlantic salmon ( Salmo salar L.) or rainbow trout ( Oncorhynchus mykiss ). For convenience fish is referred to as Atlantic salmon or salmon in the preceding text and in the text to follow, however, without excluding rainbow trout, other salmonid species or other relevant fish species. The invention is defined by the independent patent claims. The dependent claims define advantageous embodiments of the invention.

The most preferred artificial light sources, according to the present invention, gener ate blue light. In the present description blue light comprises violet light (380nm to 450 nm, blue light (450nm to 485nm) and cyan light (485nm to 500nm). Blue light as used herein covers the wavelength from 380nm to 500nm.

The additional most preferred artificial light sources, according to the present inven tion, generate red light. Red light as used herein covers the wavelength from 625nm to 740nm.

More specifically, the invention relates to a composition in the form of an aqueous so lution comprising a chemical compound for use in a method of therapy using artificial light for an external crustacean parasite infection in salmonid fish, said external crus tacean parasite infection comprises an infection of salmon lice, Lepeophtheirus salm onis, Caligus rogercresseyi and Caligus spp. Said chemical compound is a water- soluble tetrapyrrole compound or a pharmaceutical acceptable derivative thereof, with the proviso that the water-soluble tetrapyrrole compound is not chlorophyllin or a de rivative of chlorophyllin. Said composition is administered in a bath treatment of the salmonid fish in need of such treatment, and the salmon lice is illuminated by artificial light.

In one embodiment said water-soluble tetrapyrrole compound may be a water-soluble tetrapyrrole derivative such as a toxicologically acceptable salt. In one embodiment said water-soluble tetrapyrrole compound may be a water-soluble tetrapyrrole com pound in the form of a toxicologically acceptable cyclodextrin complex.

In one embodiment said water-soluble tetrapyrrole compound may be a member of a group of porphyrins, or an acceptable pharmaceutical derivative thereof. In one em bodiment said water-soluble tetrapyrrole compound is a member of a group of chlo- rins, or an acceptable pharmaceutical derivative thereof. In one embodiment said wa ter-soluble tetrapyrrole compound is a member of a group of bacteriochlorins, or an acceptable pharmaceutical derivative thereof. In one embodiment said water-soluble tetrapyrrole compound is a member of a group of phthalocyanines, or an acceptable pharmaceutical derivative thereof.

In one embodiment said artificial light may be blue light. In an alternative embodi ment said artificial light may be red light. The most preferred light is blue light. In an alternative embodiment said artificial light is a combination of blue light and red light.

In one embodiment source of artificial light may comprise of LED lamps. In one em bodiment said artificial light source may comprise of a laser light system. The laser light system may be a laser light system targeting the salmon lice present on the fish, or the laser light system may be a laser light system that do not target salmon lice present on the fish.

The salmonid fish may be located in a receptacle. The receptacle may be one of a closed tank, a semi-closed tank, a chamber, a container, and a fishpond. The salmonid fish may be located in one of an ocean cage and a net pen.

The blue light source may comprise of LED lamps. The LED lamps may emit light of a wavelength of 380nm to 500nm, for example around 450 to 460 nm. Said water- soluble tetrapyrrole compound may be added in the water surrounding the salmonid fish. The water surrounding the salmonid fish may form the bath treatment.

In a second aspect the invention relates to a method for use of a composition as de scribed above, wherein the method of therapy comprises use of artificial light. Said method may be a photodynamic method. The method may comprise using a water- soluble tetrapyrrole compound or a pharmaceutical acceptable derivative thereof as a photosensitizer.

The artificial light may be blue light. The source of artificial light comprises of LED lamps. The artificial light may be red light. The method of therapy comprises salmonid fish located in a receptacle. The method of therapy comprises salmonid fish located in an ocean cage or a net pen.

The energy required for the artificial light source according to the present invention can vary from a few watts to several kilowatts depending upon the nature of the artifi cial light source and the volume of the enclosure or ocean cage or net. The intensity of the light diminishes with the distance between the light source and the target surface, e.g. the surface of the salmon lice. In some cases, and for some useful water-soluble tetrapyrrole compounds, such as positively charged tetrapyrrole compounds or a pharmaceutical acceptable salt thereof, or negatively charged tetrapyrrole compounds or a pharmaceutical acceptable salt thereof, or hydrophilic neutral tetrapyrrole com pounds or pharmaceutical acceptable derivates thereof, the light intensity may be too high at a too close distance, leading to a too rapid bleaching of the water-soluble tetrapyrrole compound, and thereby the water-soluble tetrapyrrole compound lose its effect. In some cases, and for some useful water-soluble tetrapyrrole compounds, the light intensity may be too low at a too far distance, leading to that water-soluble tetrapyrrole compound is not activated. It is therefore within the scope of the present invention to optimize the distance between the artificial light source and the salmon lice. The optimal distance is thus dependent on among other things the actual wave length, the actual water-soluble tetrapyrrole compound, and the actual specification of the lamp, e.g. output in lumen.

The light may comprise of a single light source or a plurality of light sources in a given three-dimensional geometric system to secure optimal efficacy for treatment of fish infected with salmon lice. The light may be placed in air above the bath with fish in fected with salmon lice, however, if the volume is large, as with an ocean cage, the light is preferably placed under water. A combination of light above and under water may in some cases be useful. The artificial light source may optionally be placed relat ed to a device where the fish can obtain a fixed dose of light. A typical device where the fish get a fixed dose of light could, for example, be a system comprising of pipes and baths where the water with infected fish is pumped through pipes and kept in baths during light irradiation. For some procedures it might be an advantage to use a combination of blue and red light as an alternative to blue light or red light alone. Surprisingly it has been found that not only do artificial light and water-soluble tetrapyrrole compounds according to the present invention together have an additional effect on salmon lice but that light from an artificial light source and water-soluble tetrapyrrole together have a synergistic effect. The exact mechanism of the combina tion of light together with the water-soluble tetrapyrrole compounds on salmon lice is not studied, but it can be speculated that the artificial light activates the water-soluble tetrapyrrole compounds through a photochemical process and that the formed reactive species are responsible for the observed increased toxicity on the salmon lice. This mode of action is seen in photodynamic therapy. The water-soluble tetrapyrrole com pound and derivates thereof may act as a photosensitizer. The inventors have per formed some simple experiments related to light penetration through lice and salmon skin. The overall conclusion is the unexpected result that light easily penetrate salmon lice while light of different wave lengths do not penetrate salmon skin. Based on this result and the speculated mechanism of action discussed above, the method described herein seems to be effective to kill salmon lice with an acceptable toxicity for the fish.

Said water-soluble tetrapyrrole compound may comprise a water-soluble tetrapyrrole derivative. Said water-soluble tetrapyrrole derivative may be a toxicologically accepta ble salt of a tetrapyrrole compound. By the term "toxicologically acceptable" in this text is meant that the aqueous concentration of the tetrapyrrole compound used to treat lice, is acceptable for the fish. The adverse events for the fish by applying the present treatment are equal to or less than adverse events seen in other state of the art methods for treatment of salmon lice infected fish. Said water-soluble tetrapyrrole derivative may be in the form of a cyclodextrin complex where the cyclodextrin com plex comprises a water-soluble tetrapyrrole compound.

The said water-soluble tetrapyrrole compound can be a derivative comprising one or more salt-forming functional groups or one or more neutral hydrophilic groups.

The artificial light source may comprise of LED lamps. The wavelength of the emitted light from the artificial light source may be between 200 and 800nm. The wavelength of the emitted light may be between 400 and 800nm. The wavelength of the emitted light may be between 200 and 400nm. The wavelength of the emitted light may be between 200 and 300nm.

Said artificial light source, for example a laser, may be a part of a device for automatic localization of salmon lice. Said artificial light source may be a light source used in cultivation or farming of salmonid fish. Said water-soluble tetrapyrrole compound may be added in a food pellet for feeding the salmonid fish. Said water-soluble tetrapyrrole compound may be added in the water surrounding the salmonid fish. An artificial light device may be used according to the methods described above.

The present invention may relate to a treatment of salmon louse in farmed fish popu lations that comprises a combination of water-soluble tetrapyrrole compounds accord ing to the present invention and artificial light.

The present invention may relate to a protocol for the present treatment. Suitably, the water-soluble tetrapyrrole compound is administered prior to, after or at approximate ly the same time as the artificial light.

The present invention may relate to a protocol for the present treatment. The present treatment can be performed when the Atlantic salmon is in receptacles or enclosures, e.g. closed tanks or semi-closed tanks, pipes, chambers, containers, fishponds, ocean cages or net pens or a combination of such enclosures.

The present invention may relate to the artificial light source. The light source accord ing to the present invention can be a light source positioned in the air or placed in wa ter. The light source can optionally be a plurality of light sources. The light source should generate light with wave lengths from 200 to 800nm. The light source might also generate electromagnetic radiation that is outside this range. The light source might be in the form of a laser. The light source or light systems may comprise halo gen lamps, mercury lamps, light emitting diodes (LEDs) or other suitable light sys tems. The light source can be a stationary light source or a movable light source.

The present invention may relate to a drug treatment protocol. Optionally, according to the present invention other drug substances or drug like compounds might, in addi tion to the water-soluble tetrapyrrole, be useful for the treatment according to the present invention. These drugs include other drugs that have been shown to be toxic to salmon lice including commercial products that are approved for salmon lice infec tions in Atlantic salmon.

The present invention may relate to the dose or concentration of water-soluble tetrapyrrole or pharmaceutically acceptable salts thereof in the clinical situation.

The present invention may relate to the exposure time of the treatment process. The time necessary to perform the present process varies from minutes to hours depend ing upon several factors like for example nature of the salmon lice infection, number of Atlantic salmon per cubic meter of water, concentration of the water-soluble tetrapyrrole or pharmaceutically acceptable salts thereof in the water and optionally other drugs and drug like compounds and nature of the artificial light source.

The present invention may relate to resistance. The method according to the present invention is new for treatment of Atlantic salmon infected by salmon lice and has ad vantages related to treatment of Atlantic salmon infected by resistant salmon lice and also related to generation of new forms of resistance. The possible mechanism of ac tion related to the present invention is biologically not a typical process where re sistance processes are generated.

The advantages by a combination of artificial light together with water-soluble tetrapyrrole compounds according to the present invention versus state-of-the-art treatment of salmon lice relate to one or more of the following topics: reduced amount of drugs, improved efficacy on salmon lice, reduced toxicity to the Atlantic salmon, resistance issues, cost and time.

Detailed description of the present invention

Surprisingly it has been found a new method for treatment of salmon lice (/.. salmonis and C. rogercresseyi , Caligus spp.) in farmed fish such as Atlantic salmon (S. salar L.) using an artificial light source and at least one chemical substance at toxicologically acceptable concentrations for Atlantic salmon.

The present invention relates to water-soluble tetrapyrrole compounds. More specifi cally, the invention relates in a first aspect to a composition in the form of an aqueous solution comprising a tetrapyrrole for use in a method of therapy using artificial light for an external crustacean parasite infection in salmonid fish, said external crustacean parasite infection comprises an infection of salmon lice, Lepeophtheirus salmonis, Ca ligus rogercresseyi and Caligus spp.

In one embodiment said water-soluble tetrapyrrole compound for use in a method of photodynamic therapy for an external crustacean parasite infection in salmonid fish is a water-soluble tetrapyrrole compound comprising one or more basic groups, such as aliphatic amino groups, or permanently charged nitrogen-comprising groups such as quaternary ammonium salts, one or more acidic groups such as sulfonic acid groups, phosphate groups or carboxylic groups, and/or one or more neutral hydrophilic groups like alcohols, phenols and/or polyethylene glycol.

The term "water-soluble tetrapyrrole compound" is herein any form of the tetrapyrrole compound on neutral or charged form. If the water-soluble tetrapyrrole compound is on charged form, the compound is in the form of a salt. Water-soluble tetrapyrrole salts are, according to the present invention any salt that is sufficient soluble in sea water, are toxic to salmon lice and are not toxic for fish including salmon with or with out light. For tetrapyrroles with basic groups typical salts are formed with acids like hydrochloric acid, sulfuric acid, phosphoric acid, succinic acid and other typical non- toxic acids used in pharmaceuticals for veterinary or human use. For tetrapyrroles with acidic groups typical salts are formed with bases like sodium hydroxide, calcium hydroxide, magnesium hydroxide, amine salts and other typical non-toxic bases used in pharmaceuticals for veterinary or human use. The term "water-soluble tetrapyrrole compound" can also be the tetrapyrrole derivative (as salt or not as salt) in the form of a complex with cyclodextrin.

The chemical group called cyclodextrin (CD) includes alpha-CD which is a 6-membered sugar ring molecule, beta-CD which is a 7-membered sugar ring molecule, gamma-CD which is a 7-membered sugar ring molecule and an almost unlimited number of chem ical derivatives with various degree of substitution on these three different sugar ring molecules.

Among the preferred water-soluble tetrapyrrole compounds according to the present invention are the corresponding cyclodextrin complexes. Regarding the cyclodextrin part of the water-soluble tetrapyrrole compound of cyclodextrin complexes are the following cyclodextrins preferred: unsubstituted cyclodextrin, methyl cyclodextrin and 2-hydroxypropyl cyclodextrin, and sulphobutyl-cyclodextrin. The most preferred cy clodextrins have a 7-membered sugar ring molecular structure.

The term "water-soluble tetrapyrrole compound" also includes compounds where the tetrapyrrole ring system incorporates a metal ion.

Very many photoactive compounds described in the scientific literature are so-called "tetrapyrroles". This term relates to cyclic compounds comprising four 5-membered rings comprising a nitrogen atom. A typical tetrapyrrole core is porphyrin shown be low.

Porphyrin The tetrapyrrole structures can be with various degrees of chemical double bonds as shown below forming different chemical ring structures. Chlorins and bacteriochlorins are reduced versions of porphyrin often referred to as hydroporphyrins. Chlorins have one reduced double bond while bacteriochlorins have two reduced double bonds. In phthalocyanines the four pyrrole ring structures are condensed with benzene rings.

The tetrapyrrole ring structure might optionally be condensed with other ring systems then benzene rings; for example a heterocyclic ring like pyridine.

Porphyrin Chlorin Bacteriochlorin

Phthalocyanine

The present invention relates to water-soluble tetrapyrrole compounds with or without additional ring systems. Methods for synthesis of water-soluble derivatives are well described in the scientific literature and patent literature, e.g. EP2464384. See for example Michael Luciano et al., 2017, Modifications of porphyrins and hydroporphyrins for their solubilization in aqueous media in Molecules: 22, 980, and references herein.

The abovementioned functional groups might be attached directly or indirectly to the tetrapyrrole ring or might be incorporated or attached to an additional ring system. Talporfin is a substance in a drug (Laserphyrin®) for treatment of lung cancer in hu mans in Japan. Talporfin is a chlorin derivative. This is an example where three of four water-solubilizing groups, i.e. four acidic groups are indirectly bond to the tetrapyrrole derived ring system and one acidic group is directly bond. Talporfin

Temoporfin is the substance in a drug (Foscan ®) for treatment of head and neck can cer in humans. This is an example where the solubilizing phenolic groups are attached to an additional ring system. Phthalocyanines can also be modified to become water- soluble compounds by covalent attachment of water-solubilizing functional groups di- rectly or indirectly to additional rings.

Temoporfin

Some preferred water-soluble tetrapyrrole compounds to be used according to the present invention include:

• Tetrapyrrole compounds with carboxylate functional groups (typically 1 to 6 carboxylic groups), except for chlorophyllin.

• Sulfonated tetrapyrrole compounds, typically with one to four sulphonic acid groups

• Phosphate/phosphonate tetrapyrrole compounds, typically with one to six phosphate and/or phosphonate groups

• 4-pyridinium-tetrapyrrole compounds, typically with one to four pyridinium groups

• Other pyridyl-substituted tetrapyrrole compounds

• Imidazolium-tetrapyrrole compounds

• Tetrapyrrole compounds comprising alcohol, phenol, ether; typically PEGylated tetrapyrrole; typically with more than one oxygen-comprising functional group The above-mentioned compounds are preferably in the form of a pharmaceutically acceptable salt if the tetrapyrrole compound comprises one or more acidic and/or basic group. For a given tetrapyrrole salt, the stoichiometric ratio between the ions might vary; for example, a tetrapyrrole phosphate ester might be in the form of monosodium salt or disodium salt.

The water-soluble tetrapyrrole compounds according to the present invention are easi ly available:

• Several compounds are commercially available from different vendors.

• Very many compounds are described in the scientific literature and patent liter ature with synthetic methods and chemical characterization methods and data.

• A person skilled in the art can utilize standard organic methods for preparation of compounds.

The present invention does not claim any new chemical compounds perse.

Salts of water-soluble tetrapyrrole compounds can easily be prepared by standard methods using an acidic compound and a basic compound. Such methods typically include mixing of relevant molecular ratios of acids and bases in an appropriate sol vent, optionally heating and then isolation by evaporation or precipitation. The water- soluble salt is dried using standard methods; air drying, head drying, vacuum drying and spray drying. The water-soluble tetrapyrrole cyclodextrin complexes according to the present invention can be prepared by standard methods for preparation of such complexes. These methods include co-evaporation, spray-drying, freeze drying and kneading. One simple method is to carefully mix the water-soluble tetrapyrrole with the cyclodextrin (typically molar ratio 1 to 5) with small amounts of water forming a very thick paste for 10 minutes using a mortar and pestle, dry the paste in an oven and prepare a powder of the dry material using the mortar and pestle.

The actual aqueous concentration of water-soluble tetrapyrrole compounds in the clini cal situation to remove salmon lice from fish, varies depending upon the various pa rameters like for example choice of water-soluble tetrapyrrole compound, tempera ture, disease stage, light source and the density of Atlantic salmon. Typical concentration range for the water-soluble tetrapyrrole compound according to the pre sent invention is in the range of 0.01 micromolar (10 ^s M) to 400 micromolar (400 times 10 ⁶ M). More preferred the concentration range for the water-soluble tetrapyrrole compounds according to the present invention is in the range 0.1 mi cromolar to 200 micromolar. The present invention relates to treatment of salmon louse in farmed fish populations that comprises a combination of water-soluble tetrapyrrole compounds and light from an artificial light source.

The present invention relates to a protocol for the present treatment. Suitably, water- soluble tetrapyrrole compound(s) are administered prior to, after or at approximately the same time as the light.

The light might be present after the water-soluble tetrapyrrole compound concentra tion around the Atlantic salmon is reduced. Any protocol using water-soluble tetrapyrrole and light is within the scope of the present invention. It is up to the skilled person on salmon lice treatment to select the best suited protocol for each treatment.

A first preferred protocol is to apply the water-soluble tetrapyrrole some time prior to the light treatment. In this case the light and the water-soluble tetrapyrrole are pre sent partly together and optionally continue to use light after the aqueous concentra tion of the water-soluble tetrapyrrole is reduced.

A second preferred protocol is to apply water-soluble tetrapyrrole and artificial light treatment together and optionally continue to use light after the aqueous concentra tion of the water-soluble tetrapyrrole is reduced.

A third preferred protocol is to apply light after the aqueous concentration of the wa ter-soluble tetrapyrrole is reduced but the water-soluble tetrapyrrole still is present within the salmon lice.

The most preferred protocol is to apply light, at least partly, when the concentration of the water-soluble tetrapyrrole compounds still is quite high in the water surrounding the Atlantic salmon and the salmon lice.

The present invention relates to a protocol for the present treatment. The present treatment can be performed when the Atlantic salmon is in receptacles or enclosures, e.g. closed tanks or semi-closed tanks, pipes, chambers, containers, fishponds, ocean cages or net pens or a combination of such enclosures.

The preferred methods of the present invention relate to treatment of Atlantic salmon with salmon lice within a tank/boat or ocean cages or net pens. The absolutely most preferred methods of the present invention relate to treatment of Atlantic salmon in fected with salmon lice within an ocean cage or a net pen. The present invention relates to the position of the artificial light source. One preferred position of the light source is in the air above the water. Another preferred position of the light source is on the walls or the bottom of a receptacle or enclosure, e.g. closed tank or semi-closed tank, pipe, chamber, container, or fishponds. Another preferred position of the light source is under water within the receptacle or the ocean cage or net by mechanical arrangements and/or floating devices. The most preferred position of the light source in a large ocean cage or net is under water.

The artificial light source should generate light that has wave lengths from 200nm to 800nm. One preferred artificial light source according to the present invention is that the artificial light source generates visible light within the wavelength band from 400nm to 800nm.

One preferred artificial light source according to the present invention is that the artifi cial light source generates light within one of the following colors: red, yellow, green, or blue. The artificial light source may generate white light. Another preferred light source according to the present invention is that the artificial light source generates UV light within the wavelength band from 200nm to 400nm.

Another preferred artificial light source according to the present invention is that the artificial light source generates UVC light within the wavelength band from 200nm to 300nm. The artificial light source might also generate electromagnetic radiation that is outside these wavelength bands. If so, the artificial light source must also generate some light within referred wave lengths or colors.

The artificial light source can be one single artificial light source or a plurality of artifi cial light sources. If the volume is huge, like in an ocean cage or net, it is according to the present invention preferred to use a plurality of artificial light sources.

One artificial preferred light source according to the present invention is that the artifi cial light source is based on laser.

Another preferred artificial light source according to the present invention is that the artificial light source is based on LEDs, halogen lamp or mercury lamp. The most pre- ferred type of lamps among these lamps are LED lamps.

The most preferred artificial light sources according to the present invention are LED lamps generating blue light; typically, in the wavelength range from 380nm to 500nm. Examples of such lamps are optionally dimmable, underwater lamps currently com mercially available and used in aquaculture for other purposes.

The light from the artificial light source may be stationary, i.e. same light in the same area over time, or the artificial light source may be movable.

The present invention relates to a drug treatment protocol. Optionally, according to the present invention, other drug substances or drug like compounds may, in addition to water-soluble tetrapyrrole, be useful for the treatment according to the present invention. One preferred treatment protocol according to the present invention is that the other drug substances or drug like compounds are selected among other drugs that are regulatory approved for use to treat Atlantic salmon infected by salmon lice. Another preferred treatment protocol according to the present invention is that the other drug substances or drug like compounds that are cholinesterase inhibitors, syn thetic pyrethroids, chitin synthase inhibitors or glutamate-based chlorine ion channel regulators.

Another preferred treatment protocol according to the present invention is that the other drug substances or drug like compounds are selected among compounds that are water-soluble tetrapyrrole compounds. Another preferred treatment protocol ac cording to the present invention is that the other drug substances or drug like com pounds are selected among compounds that are phototoxic.

The present invention relates to the dose or concentration of the water-soluble tetrapyrrole in the clinical situation.

The present invention relates to the timing of the treatment process. The time neces sary to perform the present process varies from minutes to hours depending upon several factors like for example nature of the salmon lice infection, number of Atlantic salmons per cubic meter, the nature and the concentration of the water-soluble tetrapyrrole and optionally other drugs and drug like compounds, and nature of the light source.

The present invention relates to resistance. The method according to the present in vention is new for treatment of Atlantic salmon infected by salmon lice and has ad vantages related to treatment of Atlantic salmon infected by resistant salmon lice and also relates to generation of new forms of resistance. The possible mechanism of ac tion related to the present invention is biologically not a typical process where re sistance processes are generated. The present invention relates to compositions. The composition might be in the form of liquid concentrated solution or in the form of a solid powder. The concentrated solu tion is diluted with sea water before use. The solid powder is typically dissolved to a concentrated solution and further diluted with sea water before use or optionally dis solved directly in sea water. The solutions comprise one or more solvents. The sol vents are selected among toxicologically acceptable solvents or solvent mixtures typi cally for example selected from water, sea water, ethanol, isopropanol, glycerol, dimethyl sulfoxide, dimethyl formamide, acetone and low molecular weight polyeth ylene glycols. The concentrate might typically comprise additional non-toxic additives like surfactants and solubilizers used in pharmaceutical compositions for human and animal medical products. These additives include polysorbates like for example Poly- sorbate 20 and Polysorbate 80, polyoxyethylene (PEG) castor oil derivatives, polyox- amers, glycerol esters and other ionic and non-ionic surfactants and solubilizers. An ionic surfactants are, according to the present invention, the most preferred group of ionic surfactants. However, the most preferred surfactants, according to the present invention, are non-ionic surfactants. Both ionic and non-ionic surfactants might work as solubilizers. The powder can be the water soluble tetrapyrrole active substance without any additives or a powder mixture comprising various additives. The additives to be optionally used in the present compositions include typical excipients used in pharmaceutical formulations like binders, carriers, coatings, diluents, disinteg rants, stabilizers, surfactants, solubilizers and other typical pharmacologically inactive excipi ents. Typical dry formulations are, according to the present invention, non- encapsulated dry powder (pure water-soluble tetrapyrrole derivatives or cyclodextrin derivatives thereof optionally mixed with additives), granulates comprising water- soluble tetrapyrrole derivatives or cyclodextrin derivatives thereof and dry powder or granulate comprising water-soluble tetrapyrrole derivatives or cyclodextrin derivatives thereof in water-soluble polymer bags, typically polyvinyl alcohol bags. Granulates can typically be prepared using standard granulation methods for example well known in the field of tableting. For granulation methods see for example Dilip M. Parikh (Ed.) Handbook of Pharmaceutical Granulation Technology, Second Edition (2005) Tay- lor&Francis Group, Florida, USA.

The advantages by a combination of light together with water-soluble tetrapyrrole de fined in the present invention relate to one or more of the following topics: reduced amount of drugs, reduction of environmental issues, no resistance development, im proved efficacy on salmon lice, and reduced toxicity to the Atlantic salmon. Example 1

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt - 2- hydroxypropyl-beta-cyclodextrin complex

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt (Aldrich 245356- 5G SDS) (mv 984.2, 492 mg, 0.5 mmol) and 2-hydroxypropyl-beta-cyclodextrin (Biosynth) (mw 1460, 3.65 g, 2.5 mmol) were mixed volumetrically in a mortar. Wa ter (2 ml) was added, and the blue paste was vigorously mixed in the mortar and then dried in an oven at 60 °C for 24 hours. The content was pulverized in the mortar. The title compound comprised 11.9 weight-% copper phthalocyanine-3,4',4",4"'- tetrasulfonic acid tetrasodium salt.

Example 2

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt - beta- cyclodextrin sulfobutyl ether sodium salt complex

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt (Aldrich 245356- 5G SDS) (mv 984.2, 98 mg, 0.1 mmol) and beta-cyclodextrin sulfobutyl ether sodium salt (Biosynth OC15979) (mw 2242, 2.24 g, 1 mmol) were mixed volumetrically in a mortar. Water (2 ml) was added, and the blue paste was vigorously mixed in the mor tar and then dried in an oven at 60 °C for 24 hours. The content was pulverized in the mortar. The title compound comprised 4.2 weight-% copper phthalocyanine- 3,4',4",4"'-tetrasulfonic acid tetrasodium salt.

Example 3

4,4',4",4"'-(porphine-5,10,15,20-tetrayl)tetrakis(benzene sulfonic acid) - be- ta-cyclodextrin sulfobutyl ether sodium salt complex

4,4',4",4"'-(porphine-5,10,15,20-tetrayl)tetrakis(benzene sulfonic acid) (Sigma- Aldrich)89456-100MG-F) (mw 935, 9 mg, 0.01 mmol) and beta-cyclodextrin sulfobutyl ether sodium salt (Biosynth OC15979) (mw 2242, 0.22 g, 0.1 mmol) were mixed vol umetrically in a mortar. Water (0.2 ml) was added, and the blue paste was vigorously mixed in the mortar and then dried in an oven at 60 °C for 24 hours. The content was pulverized in the mortar. The title compound comprised 3.9 weight-% 4,4',4",4"'- (porphine-5,10,15,20-tetrayl)tetrakis(benzenesulfonic acid). Example 4

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt - gamma-cyclodextrin complex

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt (Aldrich 245356- 5G SDS) (mv 984.2, 492 mg, 0.5 mmol) and gamma-cyclodextrin (Wacker Fine Chemicals, Cavamax W8) (mw 1297, 2.40 g, 1.85 mmol) were mixed volumetrically in a mortar. Water (2 ml) was added, and the blue paste was vigorously mixed in the mortar and then dried in an oven at 60 °C for 24 hours. The content was pulverized in the mortar. The title compound comprised 17 weight-% copper phthalocyanine- 3,4',4",4"'-tetrasulfonic acid tetrasodium salt.

Example 5

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt - beta- cyclodextrin complex

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt (Aldrich 245356- 5G SDS) (mv 984.2, 492 mg, 0.5 mmol) and beta-cyclodextrin (Wacker Fine Chemi cals. Cavamax W7 (mw 1135, 3,405g, 3 mmol) were mixed volumetrically in a mortar. Water (2 ml) was added, and the blue paste was vigorously mixed in the mortar and then dried in an oven at 60 °C for 24 hours. The content was pulverized in the mortar. The title compound comprised 11.9 weight-% copper phthalocyanine-3,4',4",4"'- tetrasulfonic acid tetrasodium salt.

Example 6

Phthalocyanine concentrate for dilution before use

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt 500 gram Macrogol glycerol ricinolaeate 15 gram Polysorbate 80 10 gram N-methylpyrrolidon ad. 5 liter

A stirred mixture of macrogol glycerol ricinolaeate, polysorbate 80 and N- methylpyrrolidone (3 liter) are heated to 60 °C. Copper phthalocyanine-3,4',4",4"'- tetrasulfonic acid tetrasodium salt is added. Additional N-methylpyrrolidon to a total volume of 5 liter is added and the mixture was stirred for 10 minutes, cooled and filled on one-liter bottles. One liter comprises 100 gram copper phthalocyanine-3,4',4",4"'- tetrasulfonic acid tetrasodium salt. Example 7

Bag comprising dry powder of porphine - cyclodextrin complex

4,4',4",4"'-(porphine-5,10,15,20-tetrayl)tetrakis(benzene sulfonic acid) - beta- cyclodextrin sulfobutyl ether sodium salt complex 100 gram

Kaolin 350 gram

Silica 50 gram

The components are volumetrically mixed and filled into polymer bags made from polvinylalcohol. Each bag comprises 100 gram dry powder (20 gram 4, 4', 4", 4"'- (porphine-5,10,15,20-tetrayl)tetrakis(benzenesulfonic acid) - beta-cyclodextrin sul fobutyl ether sodium salt complex.)

Example 8

Solubility and dispersibility studies in sea water

Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt. Visual solubility in sea water appr. 20 mg/liter at 20 °C. Dissolves over time. Blue solution.

The tetrapyrrole cyclodextrin complexes showed typically a visual solubility in sea wa ter more than appr. 100 mg/liter at 20 °C. The tetrapyrrole cyclodextrin complexes typically dissolve fast in sea water and are typically very dispersible in sea water.

Example 9

Evaluation of clinical efficacy of positively charged porphyrin to kill salmon lice (dark and blue light)

Drug: 5,10,15,20-tetrakis(l-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate)

20 microM Molecular weight is 1363.60 D.

(Synonym: meso-tetra(N-methyl-4-pyridyl)porphine tetratosylate salt; TMPyP) from Merck (Sigma Aldrich) 323497-250MG

Blue light: 100 blue LED lamp array (10 x 10) is mounted on a heat sink and cooled by an external fan. Specifications: 450-460nm, DC32-36V, 3500mA (100W), Lumi nous flux: 1000-1500lm.

Two dark controls (A and B): Sea water (A); and drug (20 microM) in sea water (B) Blue light control: Sea water no drug (C)

Blue light: Drug (20 microM) in sea water (D). All bioassays are conducted at a constant water temperature of 12°C. Numbers of live and dead salmon lice were evaluated during four hours after onset of experiments.

Expected result: No dead salmon lice are observed in controls (A, B and C). Dead salmon lice are observed in experiment with blue light and water-soluble tetrapyrrole compound (D).

Example 10

Evaluation of clinical efficacy of negatively charged porphyrin to kill salmon lice (dark and blue light)

Drug: 4,4',4",4"'-(porphine-5,10,15,20-tetrayl)tetrakis(benzenesul fonic acid)

(Synonym: meso-tetraphenylporphine-4,4',4",4"'-tetrasulfonic acid) from Merck (Sigma Aldrich) 89456-100MG

Two dark controls (A and B): Sea water (A) and drug (10 microM) in sea water (B) Blue light control: Sea water no drug (C)

Blue light: Drug (10 microM) in sea water (D).

All bioassays are conducted at a constant water temperature of 12°C. Numbers of live and dead salmon lice were evaluated during four hours after onset of experiments.

Expected result: No dead salmon lice are observed in controls (A, B and C). Dead salmon lice are observed in experiment with blue light and water-soluble tetrapyrrole compound (D).

Example 11

Evaluation of clinical efficacy of positively charged porphyrin to kill salmon lice (dark, white light and blue light)

Drug: Copper phthalocyanine-3,4',4",4"'-tetrasulfonic acid tetrasodium salt from Merck (Sigma Aldrich) 245356-1G

Two dark controls (A and B): Sea water (A) and drug (10 microM) in sea water (B) Blue light control: Sea water no drug (C)

White light control: Sea water no drug (D)

Blue light: Drug (10 microM) in sea water (E).

White light: Drug (10 microM) in sea water (F).

All bioassays are conducted at a constant water temperature of 12°C. Numbers of live and dead salmon lice were evaluated during four hours after onset of experiments. Expected result: No dead salmon lice are observed in controls (A, B, C and D). Dead salmon lice are observed in experiments with light and water-soluble tetrapyrrole compound. More and/or faster killing of salmon lice is observed with blue light plus water-soluble tetrapyrrole compound (E) than with white light plus water-soluble tetrapyrrole compound (F).