Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITION FOR REMOVING CRUSTACEAN ECTOPARASITES FROM FARMED
SALMONID FISH USING CURCUMIN OR A PHARMACEUTICAL ACCEPTABLE DERIVATE
THEREOF 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
io 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. More specifically the method relates to the use of a
combina-
tion of regulatory approved food additives or cyclodextrin complexes thereof
and light.
Background
Aquaculture, also referred to as aquafarming, is the farming of aqueous
organism.
Aquaculture involves cultivating freshwater and saltwater populations under
controlled
conditions and is in contrast to commercial harvesting or fishing where the
organisms
are naturally present. The farmed organisms can typically be fish,
crustaceans, mol-
lusks, aquatic plants, algae. The aquaculture farms can be in the form of
tanks (closed
or semi closed), fish ponds, 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.
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Relevant prior art publications and their relevant content
Currently there are several drug substances that are used to combat salmon
lice infec-
tion in salmon farms.
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 involves a so-
called pho-
tosensitizer and light, and oxygen, where the light activates the
photosensitizer to
generate singlet oxygen and other reactive and toxic species. Photodynamic
therapy is
well described in human medicine, see for example: Kwiatkowski S et al.
Photodyna-
mic therapy - mechanisms, photosensitizers and combinations. Biomed
Pharmacother.
2018 Oct; 106:1098-1107. The clinical use of photodynamic therapy is, however,
lim-
ited to some specific indications; especially some skin diseases with products
compris-
ing 5-aminolevulinic acid methyl ester as photosensitizer, and age-related
macular
degeneration with products comprising verteporfin as photosensitizer. Very
many of
the publications on photodynamic therapy relates to cancer and some
publications
relate to infections, however, these diseases are not commonly treated by
photody-
namic therapy.
D.-P-Hader et al. Fighting fish parasites with photodynamically active
chlorophyllin.
Parasitol. Res. 2016,115 page 2277-2283, describes a study where water-soluble
chlorophyll was used. The result was "In Ichthyobodo, 2 pg/mL proved
sufficient with
subsequent 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
inactiva-
tion beyond the medical scope. J. Photochem. and Photobiol. C: Photochemistry
Re-
views, 22:34-57 is a general review publication within the field on
photodynamic ther-
apy. Figure 4 on page 43 in this publication illustrates generally a treatment
protocol
for photodynamic treatment of infected fish. The text in page 43 refers to
Elina Alves
et al. Photodynamic antimicrobial chemotherapy in aquaculture;
photoinactivation
studies if Vibrio fischeri in PLOS One 2011, 6, 6, e20970. The light is "solar
irradiation
from 380 to 700 nm consisting in 13 OSRAM lamps." See below.
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Eliana Alves et al. 2011. Photodynamic Antimicrobial Chemotherapy in
Aquaculture:
Photoinactivation Studies of Vibrio fischeri. PLOS One,
https://doi.org/10.1371/journal.pone.0020970. The photosensitizer used in
these
studies is a porphyrin derivative.
Peter KJ Robinson et al. A new generation of biocides for control of crustacea
in fish
farms. J. Photochem and Photobiol. B:Biology,95(2009)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 sea lice was the marine
copepod
Acartia clause, the lamp was a 500W tungsten halogen lamp. The result showed
that
the marine copepod mortality with methylene blue was high when the methylene
blue
concentration 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 acti-
vation.
Methylene blue is a cationic (permanent positively charged) synthetic compound
and
nuclear fast red is a negatively charged sulphonic acid in sea water (pH 7.5
to 8.4).
Acartia clausi is a small animal (1 mm in length, 0.2 mm broad). The length of
salmon
lice is up to 18 mm.
All light sources used in the above discussed prior art relate to white light.
Hydrogen peroxide has for many years been used to combat sea lice infections
in fish
including salmon. The concentrates are diluted to a final concentration of
1500 mg
hydrogen peroxide per liter. Hydrogen peroxide is toxic for salmon lice. The
mecha-
nism of action is probably related to an oxidative effect on lice components
and the
formation of oxygen emboli within the salmon lice.
Drug resistance is a general problem with farming of salmon. See for example
Stian
Morch Aaen et al. Drug Resistance in sea lice: a threat to salmon aquaculture.
Recently it has also been reported resistance towards hydrogen peroxide in the
treat-
ment of salmon lice. See for example: K.O. Helgesen et al. 2017. Increased
catalase
activity-A possible mechanism in hydrogen peroxide resistant salmon lice
(Lepeo-
phtheirus salmonis). Aquaculture 468:135-140 and K.O. Helgesen et al. 2015.
First
report of reduced sensitivity towards hydrogen peroxide found in the salmon
louse
Lepeophtheirus salmonis in Norway. Aquaculture Reports, 1, 37-42.
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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
io 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 regu-
latory approved food additives.
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 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 de-
fine 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 in a first aspect to a composition in
the form of
an aqueous solution comprising a photosensitizer for use in a method of
photodynamic
therapy for an external crustacean parasite infection in salmonid fish, said
external
crustacean parasite infection comprises an infection of salmon lice,
Lepeophtheirus
salmonis, Caligus rogercresseyi and Caligus spp. Said photosensitizer is
curcumin or a
pharmaceutical acceptable derivative thereof, and said photosensitizer is
administered
in a bath treatment of the salmonid fish in need of such treatment, and the
salmon
lice is illuminated by light.
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In one embodiment said photosensitizer may be a curcumin cyclodextrin.
In one embodiment said light may be blue light. In an alternative embodiment
said
light may be red light.
The salmonid fish may be located in a receptacle. The receptacle may be one of
a
5 closed tank, a semi-closed tank, a chamber, a container and a fish pond.
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 450nm to 460nm.
Said photosensitizer may be added in the water surrounding the salmonid fish.
The
io water surrounding the salmonid fish may form the bath treatment.
In a second aspect the invention relates to use of an artificial light device
emitting
blue light in accordance to the first aspect of the invention.
In a third aspect the invention may relate to a composition in the form of an
aqueous
solution comprising a photosensitizer for use in a method of photodynamic
therapy 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. Said photosensitizer is riboflavin or a
pharmaceu-
tical acceptable derivative thereof, and said photosensitizer is administered
in a bath
treatment of the salmonid fish in need of such treatment, and the salmon lice
is illu-
minated by light.
In one embodiment said photosensitizer may be a riboflavin phosphate.
In one embodiment said light may be blue light. In an alternative embodiment
said
light may be red light.
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 fish pond. 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 450nm to 460nm.
Said photosensitizer may be added in the water surrounding the salmonid fish.
The
water surrounding the salmonid fish may form the bath treatment.
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In a forth aspect the invention may relate to a composition in the form of an
aqueous
solution comprising a photosensitizer for use in a method of photodynamic
therapy 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. Said photosensitizer is chlorophyllin
sodium cop-
per salt or a pharmaceutical acceptable salt or derivative thereof, and said
photosensi-
tizer is administered in a bath treatment of the salmonid fish in need of such
treatment, and the salmon lice is illuminated by light.
In one embodiment said light may be blue light. In an alternative embodiment
said
io light may be red light.
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 fish pond. 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 450nm to 460nm.
Said photosensitizer may be added in the water surrounding the salmonid fish.
The
water surrounding the salmonid fish may form the bath treatment.
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 sea lice. In some cases, and for some useful
photosensitizers,
such as curcumin or a pharmaceutical acceptable derivative thereof, or
riboflavin or a
pharmaceutical acceptable derivative thereof, or chlorophyllin sodium copper
salt or a
pharmaceutical acceptable salt or derivative thereof, the light intensity may
be too
high at a too close distance, leading to a too rapid bleaching of the
photosensitizer,
and thereby the photosensitizer lose its effect. In some cases, and for some
useful
photosensitizers, the light intensity may be too low at a too far distance,
leading to
that photosensitizer is not activated. It is therefore within the scope of the
present
invention to optimize the distance between the light source and the target
surface.
The optimal distance is thus dependent on among other things the actual
wavelength,
the actual photosensitizer, and the actual specification of the lamp, e.g.
output in lu-
men.
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Surprisingly it has been found that not only do light and regulatory approved
food ad-
ditives together have an additional effect on salmon lice but that light from
an artificial
light source and regulatory approved food additives together have a
synergistic effect.
The exact mechanism of the combination of light together with the regulatory
ap-
proved food additives on salmon lice is not studied, but it can be speculated
that the
light activates the regulatory approved food additives through a photochemical
pro-
cess and that the formed reactive species are responsible for the observed
increased
toxicity on the salmon lice.
Said photosensitizer may comprise a regulatory approved food additive. Said
photo-
io sensitizer may be a food additive cyclodextrin complex where the food
additive com-
prises the regulatory approved food additive.
The regulatory approved food additive may be at least one of E100 Curcumin,
E101(i)
Riboflavin, E101(ii) Riboflavin-5'-phosphate, E102 Tartrazine, E123 Amaranth,
E127
Erythrosine, E129 Allura Red AC, E131 Patent Blue V, E132 Indigotine; Indigo
Car-
mine, E133 Brilliant Blue FCF, E140 Chlorophylls and chlorophyllins, E141
Copper
complexes of chlorophyll and chlorophyllins, E142 Green S, E151 Brilliant
Black BN;
Black PN, E155 Brown HT, E160a Carotenes, E160b Annatto; Bixin; Norbixin,
E160c
Paprika extract; Capsanthian; Capsorubin, E160d Lycopene, E160e Beta-apo-8'-
carotenal (C30), E161b Lutein, E161g Canthaxanthin, E162 Beetroot Red;
Betanin,
E163 Anthocyanins and E180 Litholrubine BK.
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 800 nm. The
wavelength
of the emitted light may be between 400 and 800 nm. The wavelength of the
emitted
light may be between 200 and 400 nm. The wavelength of the emitted light may
be
between 200 and 300 nm.
Said artificial light source 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 photosensitizer may be added in a food pellet for feeding the salmonid
fish. Said
photosensitizer may be added in the water surrounding the salmonid fish.
An artificial light device may be used according to the methods described
above.
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In one aspect the present invention may relate to a treatment of salmon louse
in
farmed fish populations that comprises a combination of regulatory approved
food
additives as cyclodextrin complexes and artificial light.
In one aspect the present invention may relate to a protocol for the present
treat-
ment. Suitably, the food additive is administered prior to, after or at
approximately
the same time as the artificial light.
In one aspect the present invention may relate to a protocol for the present
treat-
ment. The present treatment can be performed when the Atlantic salmon is in
recep-
tacles or enclosures, e.g. closed tanks or semi-closed tanks, pipes, chambers,
con-
tainers, fish ponds, ocean cages or net pens or a combination of such
enclosures.
In one aspect the present invention may relate to the artificial light source.
The light
source according to the present invention can be a light source positioned in
the air or
placed in water. The light source can optionally be a plurality of light
sources. The light
source should generate light with wave lengths from 200 to 800 nm. 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
halogen lamps, mercury lamps, light emitting electrodes (LEDs) or other
suitable light
systems. The light source can be a stationary light source or a movable light
source.
In one aspect the present invention may relate to a drug treatment protocol.
Optional-
ly, according to the present invention other drug substances or drug like
compounds
might, in addition to regulatory approved food additives, 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 infections in Atlantic salmon.
In one aspect the present invention may relate to the dose or concentration of
the
regulatory approved food additive in the clinical situation.
In one aspect 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 depending upon several factors like for example nature of the salmon
lice infec-
tion, number of Atlantic salmon per cubic meter of water, concentration of
regulatory
approved food additive in the water and optionally other drugs and drug like
com-
pounds and nature of the artificial light source.
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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 light together with food additives
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 (L.
salmonis
and C. rogercresseyi) in farmed fish such as Atlantic salmon (S. salar L.)
using an arti-
ficial light source and at least one chemical substance at toxicologically
acceptable
concentrations for Atlantic salmon.
The present invention relates to the selection of food additives. Approved
food addi-
tives are listed with E-numbers. See for example:
https://www.food.gov.uk/business-
guidance/eu-approved-additives-and-e-numbers
Relevant food additives with E-numbers used together with light from an
artificial light
source for treatment of salmon lice (L. salmonis and C. rogercresseyi) in
farmed Atlan-
tic salmon according to the present invention are:
E100 Curcumin
E101(i) Riboflavin ,(ii) Riboflavin-5'-phosphate
E102 Tartrazine
E104 Quinoline yellow
E110 Sunset Yellow FCF; Orange Yellow S
E120 Cochineal; Carminic acid; Carmines
E122 Azorubine; Carmoisine
E123 Amaranth
E124 Ponceau 4R; Cochineal Red A
E127 Erythrosine
E129 Allure Red AC
E131 Patent Blue V
E132 Indigotine; Indigo Carmine
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E133 Brilliant Blue FCF
E140 Chlorophylls and chlorophyllins
E141 Copper complexes of chlorophyll and chlorophyllins
E142 Green S
5 E150a Plain caramel
E150b Caustic sulphite caramel
E150c Ammonia caramel
E150d Sulphite ammonia caramel
E151 Brilliant Black BN; Black PN
10 E153 Vegetable carbon
E155 Brown HT
E160a Carotenes
E160b Annatto; Bixin; Norbixin
E160c Paprika extract; Capsanthian; Capsorubin
E160d Lycopene
E160e Beta-apo-8'-carotenal (C30)
E161b Lutein
E161g Canthaxanthin
E162 Beetroot Red; Betanin
E163 Anthocyanins
E170 Calcium carbonate
E171 Titanium dioxide
E172Iron oxides and hydroxides
E173 Aluminium
E174 Silver
E180 Litholrubine BK
The preferred compounds according to the present invention are:
E100 Curcumin, E101(i) Riboflavin; (ii) Riboflavin-5'-phosphate, E102
Tartrazine, E123
Amaranth, E127 Erythrosine, E129 Allura Red AC, E131 Patent Blue V, E132
Indigo-
tine; Indigo Carmine, E133 Brilliant Blue FCF, E140 Chlorophylls and
chlorophyllins,
E141 Copper complexes of chlorophyll and chlorophyllins, E142 Green S, E151
Brilliant
Black BN; Black PN; E155 Brown HT, E160a Carotenes, E160b Annatto; Bixin;
Norbix-
in, E160c Paprika extract; Capsanthian; Capsorubin, E160d Lycopene, E160e Beta-
apo-8'-carotenal (C30), E161b Lutein, E161g Canthaxanthin, E162 Beetroot Red;
Betanin, E163 Anthocyanins and E180 Litholrubine BK.
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The most preferred compounds according to the present invention are:
E100 Curcumin, E101(i) Riboflavin; (ii) Riboflavin-5'-phosphate, E102
Tartrazine, E123
Amaranth ,E127 Erythrosine ,E129 Allura Red AC,E131 Patent Blue V, E132
Indigotine;
Indigo Carmine ,E133 Brilliant Blue FCF, E140 Chlorophylls and chlorophyllins,
E141
Copper complexes of chlorophyll and chlorophyllins, E142 Green S. E160a
Carotenes
E160c Paprika extract; Capsanthian; Capsorubin, E160d Lycopene, E161b Lutein,
E161g Canthaxanthin, E162 Beetroot Red; Betanin and E163 Anthocyanins.
The even most preferred compound is curcumin.
The concentration of the food additive varies depending upon the various
parameters
io like for example choice of food additive, temperature, disease stage,
light source and
the density of Atlantic salmon. Typical concentration range for the food
additives ac-
cording to the present invention is in the range 0.1 micromolar (1 time 10-7
molar
(M)) to 400 micromolar (400 times 10-6 molar(M)). More preferred the
concentration
range for the food additive cyclodextrin complex according to the present
invention is
in the range 0.5 micromolar to 300 micromolar.
The present invention relates to treatment of salmon louse in farmed fish
populations
that comprises a combination of regulatory approved food additives as
cyclodextrin
complexes and light from an artificial light source.
Cyclodextrins (CDs) are cyclic oligosaccharides what are known to form
complexes
with various chemical and drugs. Drug complexes with CDs are currently used in
hu-
man medicine. The potential advantages by using drug CD complexes relate to
chemi-
cal stability, aqueous solubility and oral bioavailability.
The chemical group called CDs include 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 chemical
deriva-
tives with various degree of substitution on these three different sugar ring
molecules.
The preferred and the most preferred food additives also relate to the food
additive
cyclodextrin complexes.
Regarding the cyclodextrin part of the food additive cyclodextrin complexes
are the
three unsubstituted cyclodextrins, methyl cyclodextrin and 2-hydroxypropyl
cyclodex-
trins the preferred cyclodextrins.
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Regarding the cyclodextrin part of the food additive cyclodextrin complexes is
beta-
cyclodextrin the most preferred cyclodextrin.
This means that typically curcumin beta-cyclodextrin, chlorophyll beta-
cyclodextrin
and chlorophyllin beta-cyclodextrin are among the most preferred food additive
cy-
clodextrin complexes to be used together with artificial light for treatment
of salmon
louse in farmed fish according to the present invention.
The food additive cyclodextrin complexes according to the present invention
can be
prepared by standard methods for preparation of such complexes. These methods
in-
clude co-evaporation, spray-drying, freeze drying and kneading. One simple
method is
io to carefully mix the food additive 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 concentration of the food additive cyclodextrin complex varies depending
upon the
various parameters like for example choice of food additive, temperature,
disease
stage, light source and the density of Atlantic salmon. Typical concentration
range for
the food additive cyclodextrin complex according to the present invention is
in the
range 0.1 micromolar (1 time 10-7 molar (M)) to 400 micromolar (400 times 10-
6mo-
lar(M)). More preferred the concentration range for the food additive
cyclodextrin
complex according to the present invention is in the range 0.5 micromolar to
300 mi-
cromolar.
The present invention relates to a protocol for the present treatment.
Suitably, food
additive is administered prior to, after or at approximately the same time as
the light.
The light might be present after the food additive concentration around the
Atlantic
salmon is reduced. Any protocol using food additive 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 regulatory approved food additive
some time
prior to the light treatment. In this case the light and the regulatory
approved food
additive are present partly together or the light is present only after the
aqueous con-
centration of hydrogen peroxide is reduced but still present within the salmon
louse.
A second preferred protocol is to apply the regulatory approved food additive
and to-
gether and optionally continue to use light after the aqueous concentration of
the reg-
ulatory approved food additive is reduced.
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A third preferred protocol is to apply light after the aqueous concentration
of the regu-
latory approved food additive is reduced but the regulatory approved food
additive still
is present within the salmon lice.
The most preferred protocol is to apply light, at least partly, when the
concentration of
the regulatory approved food additive 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, fish
ponds, ocean
io 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 infected 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 fish ponds. 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,
blue or white.
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
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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 the regulatory approved food additive, 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
reg-
ulatory 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.
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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 photosensitizers.
Another preferred treatment protocol according to the present invention is
that the
5 other drug substances or drug like compounds are selected among compounds
that
are phototoxic.
The present invention relates to the dose or concentration of the regulatory
approved
food additive in the clinical situation.
The present invention relates to the timing of the treatment process. The time
neces-
10 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 regulatory
approved
food additive and optionally other drugs and drug like compounds, and nature
of the
light source.
15 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 advantages by a combination of light together with the regulatory approved
food
additive according to the present invention relate to one or more of the
following top-
ics: reduced amount of drugs, improved efficacy on salmon lice, and reduced
toxicity
to the Atlantic salmon.
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Experimental
Preparation of compounds
Compound 1
Chlorophyllin sodium copper salt 2-Hydroxypropy1-8-cyclodextrin complex (2:3)
Chlorophyllin sodium copper salt (1.412 gram, 2mm01) and 2-Hydroxypropy1-8-
cyclodextrin (Average Mw ¨1,460) (4.38 gram, 3mm01) were mixed in a mortar
with a
pestle. Water (3 ml) was added and the formed paste was mixed in the mortar
for 5
minutes. The product was dried at 60 degrees centigrade in dark overnight. The
prod-
uct was in the form of a green powder. The product comprised 24.4%
chlorophyllin
io sodium copper salt.
Compound 2
Chlorophyllin sodium copper salt gamma-cyclodextrin complex (2:3)
Chlorophyllin sodium copper salt (1.412 gram, 2mm01) and gamma-cyclodextrin
(3.89 gram, 3mm01) were mixed in a mortar with a pestle. Water (3 ml) was
added
and the formed paste was mixed in the mortar for 5 minutes. The product was
dried at
60 degrees centigrade in dark overnight. The product was in the form of a
green pow-
der. The product comprised 26.6% chlorophyllin sodium copper salt.
Compound 3
Curcumin 2-Hydroxypropy1-8-cyclodextrin complex (2:3)
Curcumin (0.736 gram, 2mm01) and 2-Hydroxypropy1-8-cyclodextrin (Average
Mw ¨1,460) (4.38 gram, 3 mmol) were mixed in a mortar with a pestle. Water (3
ml)
was added and the formed paste was mixed in the mortar for 5 minutes. The
product
was dried at 65 degrees centigrade in dark overnight. The product was in the
form of
a yellow powder. The product comprised 14.4 % curcumin.
Compound 4
Curcumin gamma-cyclodextrin complex (2:3)
Curcumin (0.736 gram, 2mm01) and gamma-cyclodextrin (3.89 gram, 3mm01) were
mixed in a mortar with a pestle. Water (5 ml) was added and the formed paste
was
mixed in the mortar for 5 minutes. The product was dried at 60 degrees
centigrade in
dark overnight. The product was in the form of a yellow powder. The product
com-
prised 15.9 % curcumin.
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Compound 5
Methylene Blue 2-Hydroxypropy1-8-cyclodextrin complex (2:3)
Methylene Blue hydrate (640 mg, 2mm01) and 2-Hydroxypropy1-8-cyclodextrin
(Aver-
age Mw ¨1,460) (4.38 gram, 3mm01) were mixed in a mortar with a pestle. Water
(3
ml) was added and the formed paste was mixed in the mortar for 5 minutes. The
product was dried at 65 degrees centigrade in dark overnight. The product was
in the
form of a blue powder. The product comprised 12.8% methylene blue.
Compound 6
Methylene Blue gamma-cyclodextrin complex (2:3)
Methylene Blue hydrate (640 mg, 2mm01) and gamma-cyclodextrin (3.89 gram,
3mm01) were mixed in a mortar with a pestle. Water (3 ml) was added and the
formed
paste was mixed in the mortar for 5 minutes. The product was dried at 65
degrees
centigrade in dark overnight. The product was in the form of a green powder.
The
product comprised 14.1% methylene blue.
Compound 7
Curcumin-8-cyclodextrin complex (1:4)
Curcumin (0.368 gram, Immo!) and 8-cyclodextrin (4.54 gram, 4mm01) were mixed
in
a mortar with a pestle. Water (5 ml) was added and the formed paste was mixed
in
the mortar for 5 minutes. The product was dried at 65 degrees centigrade in
dark
overnight. The product was in the form of a yellow powder. The product
comprised
7.8% curcumin.
Compound 8
Curcumin-8-cyclodextrin complex (1:4) Polysorbate 80
Curcumin (0.368 gram, Immo!), Polysorbate 80 (250mg) and 8-cyclodextrin
(4.54 gram, 4mm01) were mixed in a mortar with a pestle. Water (5 ml) was
added
and the formed paste was mixed in the mortar for 5 minutes. The product was
dried at
65 degrees centigrade in dark overnight. The product was in the form of a
yellow
powder. The product comprised 7.4% curcumin.
Compound 9
Curcumin-8-cyclodextrin complex (1:4)
A mixture of curcumin (0.368 gram, Immo!) and 8-cyclodextrin (4.54 gram,
4mm01)
and absolute ethanol (30 ml) was stirred in a beaker for 20 hours at room
tempera-
ture. The mixture was headed to 70 degrees centigrade for 2 hours. The product
was
dried at 65 degrees centigrade in dark overnight. The product was in the form
of a
yellow fluffy powder. The product comprised 7.8% curcumin.
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Compound 10
Curcumin-p-cyclodextrin complex (1:4) Polysorbate 80
A mixture of curcumin (0.368 gram, Immo!), Polysorbate 80 (0.25g) and [3-
cyclodextrin (4.54 gram, 4mm01) and water (10 ml) was stirred in a beaker for
24
hours at room temperature. The product was dried at 65 degrees centigrade in
dark
overnight. The product was in the form of a yellow powder. The product
comprised
7.4% curcumin.
Compound 11
Curcumin-P-cyclodextrin complex (1:4)
A mixture of curcumin (0.368 gram, Immo!), Polysorbate 80 (0.25g) and [3-
cyclodextrin (4.54 gram, 4mm01) and water (10 ml) was stirred in a beaker for
24
hours at room temperature. The product was dried at 65 degrees centigrade in
dark
overnight. The product was in the form of a yellow powder. The product
comprised
7.8% curcumin.
Compound 12
Curcumin-P-cyclodextrin complex (1:8) Polysorbate 80
A mixture of curcumin (0.368 gram, Immo!), Polysorbate 80 (0.25g) and [3-
cyclodextrin (9.08 gram, 8mm01) and water (10 ml) was stirred in a beaker for
24
hours at room temperature. The product was dried at 65 degrees centigrade in
dark
overnight. The product was in the form of a yellow powder. The product
comprised
3.8% curcumin.
Compound 13
Curcumin-P-cyclodextrin complex (1:8)
A mixture of curcumin (0.368 gram, Immo!) and P-cyclodextrin (9.08 gram,
8mm01)
and water (10 ml) was stirred in a beaker for 24 hours at room temperature.
The
product was dried at 65 degrees centigrade in dark overnight. The product was
in the
form of a yellow powder. The product comprised 3.9% curcumin.
Compound 14
Riboflavin 2-Hydroxypropyl-13-cyclodextrin complex (2:3)
Riboflavin (0.752 gram, 2mm01) and 2-Hydroxypropyl-13-cyclodextrin (Average Mw
¨1,460) (4.38 gram, 3 mmol) were mixed in a mortar with a pestle. Water (3 ml)
was
added and the formed paste was mixed in the mortar for 5 minutes. The product
was
dried at 65 degrees centigrade in dark overnight. The product was in the form
of a
yellow powder. The product comprised 14.7% riboflavin.
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Example 1
In vivo testing on Salmon louse (white light and red light, low concentra-
tions)
Salmon louse (Lepeophtheirus salmonis) were collected from a salmon farming
facility
in Norway.
LABORATORY SETUP
The following three treatment compounds were tested in this study:
Control, seawater control.
Compound 3
Curcumin 2-Hydroxypropy1-8-cyclodextrin complex (2:3), concentration in
seawater:
7.4 mg curcumin per litre (curcumin solution; 20 micromolar).
Chlorophyllin sodium copper salt (Sigma C6003-25G), concentration in seawater:
4
mg per litre (chlorophyll solution; 5.5 micromolar).
5-Aminolevulinic acid hydrochloride (5-ALA) (Sigma-Aldrich). Concentration in
sea-
water: 168 mg per litre (5-ALA solution; 1 millimolar).
This pilot study involved exposing four treatment compounds to three different
light
sources for 24 h. In this study they were placed in the following 3 groups:
Group 1: White LED light (240V, 50W, 35001m)
Group 2: Red light. 100 red LED lamp array (10 x 10) mounted on a heat sink.
Speci-
fications: 620-625nm, DC32-36V, 3500mA, 1000-1500Im
Group 3: Dark
In the case of group 1 and 2, a water bath (10 C) provided with a light source
was set
up for each group. Group 3 was placed in a sealed temperature-controlled
cabinet. In
addition, each treatment received additional aeration throughout the exposure
period.
This provided aeration and ensures that the treatment solution remains in
suspension,
allowing for even distribution of the treatment compound.
BIOASSAY PROTOCOL
Nine glass flasks were filled with 300 ml seawater. Five lice were placed in
each flask.
The flasks were then randomly distributed among the three different treatment
groups, resulting in 4 containers in each group. Each flask was gently stirred
to ensure
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that all lice were attached, the contents were emptied over a sieve, after
which 300 ml
treatment compound was then introduced into the flask. The flasks were then
placed
in their respective water bath or dark cabinet.
Group 3 was placed in the darkened cabinet immediately following exposure to
the
5 treatment compound. Sixty minutes after the exposure period began a count
of the
number of affected/unaffected lice was conducted.
Groups 1 and 2 were exposed to treatment compound for 1 hour, after which the
lights were turned on and they were then exposed to a combination of treatment
compound and light source. One hour after the start of light exposure a count
of the
io number of affected/unaffected lice was conducted.
Additional counts of affected/unaffected lice in all 3 groups was conducted 4
hours into
the exposure period. A final count of affected/unaffected lice was conducted
at 24 h.
RESULTS
Results from group 1, white light treatment (Table 1), indicate that there was
no
15 treatment effect until conclusion of the 24 h exposure period. The
curcumin solution
(Compound 3) resulted in 60% parasites affected by the treatment. The
chlorophyll
solution (chlorophyllin sodium copper salt) treatment group showed no
treatment ef-
fect, in fact, a number of egg string had hatched in the chlorophyll solution,
all nauplii
present were alive and swimming. The 5-ALA solution showed no treatment
effect.
20 The concentration of 5-ALA was 1 millimolar which was 50 times higher
than the con-
centration of curcumin which was 20 micromolar.
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Table 1. Percentage (%) affected/unaffected parasites after 1 h, 4 h, and 24 h
expo-
sure to white LED light in combination with 3 treatment solutions.
1 h 4h 24h
Affected Unaffected Affected Unaffected Affected Unaffected
Seawater
0 100 0 100 0 100
control
Curcum in
0 100 0 100 60 40
solution
Chlorophyl!
0 100 0 100 0 100
solution
ALA
0 100 0 100 0 100
solutwn
.. Group 2, red light treatment (Table 2), resulted in no affected lice until
the 24 h
count. At the 24 h count, no affected parasites were observed in the seawater
control
solution. In comparison, 20% of the lice were affected in chlorophyll solution
and 40%
in the curcumin solution. The 5-ALA solution showed no treatment effect. The
concen-
tration of 5-ALA was 1 millimolar which was 50 times higher than the
concentration of
curcumin which was 20 micromolar.
Table 2. Percentage (%) affected/unaffected parasites after 1 h, 4 h, and 24 h
expo-
sure to red LED light in combination with 3 treatment solutions.
1 h 4h 24h
Affected Unaffected Affected Unaffected Affected Unaffected
Seawater
0 100 0 100 0 100
control
Curcumin
0 100 0 100 40 60
solution
Chlorophyll
0 100 0 100 20 SO
solution
ALA
0 100 0 100 0 100
solution
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Group 3, dark/no light treatment (Table 3), resulted in 20% affected lice at
the 1 h
count in the curcumin solution. However, at the conclusion of the bioassay all
lice ex-
posed to this solution were determined to be unaffected.
Table 3. Percentage (%) affected/unaffected parasites after 1 h, 4 h, and 24 h
expo-
sure to dark/no light in combination with 3 treatment compounds.
h 4h 24h
Affected Unaffected Affected Unaffected ,AS-f!.qo õUnaffected
Seawater
0 100 0 100 0 100
control
Curcumin
20 80 0 100 0 100
solution
Chlorophyll
õõõ 0 100 0 100 0 100
solution
ALA
0 100 0 100 20 80
solution
Example 2 In vivo testing on Salmon louse (white light, red light and blue
light, higher concentrations)
Salmon louse (Lepeophtheirus salmonis) were collected from a salmon farming
facility
in Norway.
LABORATORY SETUP
The following three treatment compounds were tested in this study:
Control (seawater)
Treatment I: Curcumin 2-hydroxypropyl-beta-cyclodextrin (2:3) (labelled MH-
103)
(Compound 3). Concentration: 80 micromolar (29.6 mg curcumin (204 mg complex)
per liter).
Treatment II: Curcumin beta-cyclodextrin labelled MH-107 (1:4) (Compound 7).
Con-
centration: 640 micromolar (22.7 mg curcumin (304 mg complex) per liter).
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Treatment III: Copper complex of chlorophyllin sodium salt 2-hydroxypropyl-
beta-
cyclodextrin complex (2:3) (Compound 1). Concentration: 22 micromolar (16 mg
copper complex of chlorophyllin sodium salt (64 mg complex) per liter).
In this study the four treatment compounds were exposed to the following light
sources for 24 h:
White LED light.(240V, 50W, 35001m)
Red light. 100 red LED lamp array (10 x 10) mounted on a heat sink.
Specifications:
620-625nm, DC32-36V, 3500mA, 1000-1500Im). (The power supply did not function
optimally in these experiments resulting in somewhat reduced intensity of the
red light
io (only 30 V, 0.51 A)).
Blue light: 100 blue LED lamp array (10 x 10) mounted on a heat sink.
Specifications:
380-500 nm, DC32-36V, 3500mA (100W). Luminous flux: 5000-6000Im.
Two water baths were set up with a constant water temperature of 12 C. Solid
barriers were placed in the water baths to separate the three light groups .
Each
treatment container received additional aeration throughout the exposure
period. This
not only provided aeration, but also ensured that the treatment solution
remained in
suspension, allowing for even distribution of the treatment compound.
BIOASSAY PROTOCOL
Eleven glass flasks were filled with 300 ml seawater. Ten lice were
transferred, using
forceps, into each flask. The flasks were randomly labelled and distributed
among the
3 light groups. Due to limited lice numbers only 2 control flasks were
available, so only
2 treatment groups had controls. Each flask was gently stirred to ensure that
all lice
were attached, the contents were emptied over a sieve, after which 300 ml
treatment
compound was then introduced into the flask. The flasks were then placed in
their
respective waterbath. Sixty minutes after the exposure period began a count of
the
number of affected/unaffected lice was conducted. All groups were exposed to
the
treatment compound for 30 min, after which the lights were turned on and they
were
then exposed to a combination of treatment compound and light source. One hour
after the start of light exposure a count of the number of affected/unaffected
lice was
conducted. A final count of affected/unaffected lice was conducted at 24 h.
RESULTS
When looking at the white LED group (Table 4), treatment I had 30% affected
parasites as compared to the other treatments with no affected parasites. The
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treatments were more effective at the 24 h count, with treatment I being most
effective with 100% affected lice, treatment II had 80% affected lice, and
treatment
III had 30% affected lice (Table 4). At the 24 h count it was observed that
treatment I
contained a lot of precipitate, whereas the other groups had none. Due to low
numbers of parasites, the red LED group did not include a control. Treatment
efficacy
at the 1 h control was low with treatment I resulting in 20% affected lice and
treatment III with 10% affected lice (Table 5). By the 24 h mark treatment III
resulted in 0% affected lice. However treatment I resulted in 80% affected
lice, and
treatment II had 50% affected lice (Table 5). Again, there were issues with
the
io presence of a high amount of precipitate in treatment I. The blue LED
group resulted
in no affected lice in either the control or treatment III for both the 1 h
and 24 h
counts (Table 6). Treatment I was the most effective with 70% affected lice at
the 1 h
count and 90% affected lice at the 24 h count. Treatment II resulted in 20%
affected
lice after 1 h and 30% affected lice after 24 h of exposure. In the blue light
group at
the 24 h count, treatment III had large amounts of precipitate out of solution
with lice
observed moving through it (Table 5).
Table 4. Percentage (%) affected/unaffected parasites after 1 h and 24 h
exposure to
white LED light in combination to four treatment solutions.
1 h 24h
Affected (%)
Unaffected (%) Affected (%) Unaffected ()%
Seawater control 0 100 0 100
Treatment I 30 70 100 0
Treatment II 0 100 80 20
Treatment III 0 100 30 70
Table 5. Percentage (%) affected/unaffected parasites after 1 h and 24 h
exposure to
red LED light in combination to 3 treatment solutions.
1 h 24h
Affected % Unaffected % Affected %
Unaffected %
Treatment I 20 80 80 20
Treatment II 0 100 50 50
Treatment III 10 90 0 100
Table 6. Percentage (%) affected/unaffected parasites after 1 h and 24 h
exposure to
blue LED light in combination to 4 treatment solutions.
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1 h 24h
Affected (%) Unaffected (%) Affected (%) Unaffected ()%
Seawater control 0 100 0 100
Treatment I 70 30 90 10
Treatment II 20 80 30 70
Treatment III 0 100 0 100
Example 3 In vivo testing on Salmon louse
Salmon louse (Lepeophtheirus salmonis) were collected from a salmon farming
facility
in Norway.
5 LABORATORY SETUP
The following three treatment compounds and in different concentrations were
tested
in this study:
Treatment I (Ti): Curcumin 2-hydroxypropyl-beta-cyclodextrin (MH-103).
Concentra-
tion: 160 micromolar curcumin (408 mg complex per liter).
io Treatment II (T2): Riboflavin 5'-phosphate sodium Ph. Eur.
Concentration: 100 mi-
cromolar (47.8 mg per liter).
Treatment III (T3): Riboflavin 5 Ph. Eur. Concentration: 100 micromolar (37.6
mg per
liter).
Treatment IV (T4): Riboflavin 5 Ph. Eur. Concentration: 300 micromolar (112.8
mg
15 per liter).
Treatment V (T5): Riboflavin 5'-phosphate sodium Ph. Eur. Concentration: 300
mi-
cromolar (143.4 mg per liter).
In this study the three treatment compounds were exposed to the following
light
sources:
20 White LED light. (100-240V, 50W, Luminous flux: 35001m)
Blue light. 100 blue LED lamp array (10 x 10) mounted on a heat sink.
Specifications:
450-460nm, DC32-36V, 3500mA (100W), Luminous flux: 1000-1500Im).
Dark (temperature-controlled cabinet)
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BIOASSAY PROTOCOL
Light regimens
Light regimen 1:
0-30 minutes: No light
30-120 minutes: Light
120-240 minutes: No light
Light regimen 2:
0-120 minutes: Light
120-240 minutes: No light
io The following protocol was followed for each of the two light regimen.
Twelve glass
flasks were filled with 300 ml seawater. Ten preadult 2 stage lice were
transferred,
using forceps, into each flask. The flasks were randomly labelled and
distributed
among the 3 light groups, resulting in 4 flasks per light treatment. Each
flask was
gently stirred to ensure that all lice were healthy and able to attach, the
contents were
emptied over a sieve, after which 300 ml treatment compound was then
introduced
into the flask. The flasks were then placed in their respective water
bath/darkened
cabinet and exposed to the respective lights as described below. Observations
of the
number of affected/unaffected lice were conducted at 10, 30, 40, 50, 60, 90,
120,
180, 240 min. At the completion of each round (240 min), the lice were rinsed
in
seawater, dried of excess fluid, placed in marked vials and frozen at -80 C
for further
chemical analysis.
RESULTS
Light regimen 1
Treatment I, curcumin cyclodextrin (160 micromolar) was found to be potent.
The
percentage of active salmon lice after 40 minutes was zero using blue light.
The
treatment I solution was also active using white light with 50% active lice
after 40
minutes and 20% active lice after 90 minutes.
The treatment II, riboflavin-5-phosphate solution and the treatment III,
riboflavin
solution were not active using the high intensity lights (Table 7). HPLC
analysis of sea
water solutions of both riboflavin and riboflavin phosphate showed extensive
degradation of both compounds after irradiation with the intensive 100W blue
LED
light after 10 minutes. Sea water solutions of both riboflavin and riboflavin
phosphate
showed no degradation in dark during 120 minutes. In a real aquaculture
situation,
the average light intensity will be much lower than in this example. The
inventors
expect that the lower blue light intensity will not degrade the riboflavin
molecules and
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thereby these compounds will work as photosensitizers for treatment of salmon
infected by salmon lice.
Table 7 Percentage active salmon lice recorded during nine observations during
a 4h
exposure to three different compounds and three different light sources /
conditions
Time (minutes)
30 40 50 60 90 120 180 240
Blue 100 70 0 0 0 0 0 0 0
Ti White 90 90 50 50 50 20 20 20 0
Dark 90 80 80 80 80 60 50 0 0
Blue 80 80 80 100 100 80 80 80 90
T2 White 100 100 100 100 100 100 100 100 100
Dark 100 90 90 90 60 60 80 80
100
Blue 100 80 80 80 80 80 80 80
100
T3 White 100 100 100 100 100 100 100 100 100
Dark 90 90 80 80 70 70 70 70 70
5
Light regimen 2
Exposure to the light regimen 2 confirmed that the riboplavin-5-phosphate
solution
(treatment IV) and riboflavin solution (treatment V) were not active in higher
concentrations (300 micromolar) (Table 8).
io
Table 8 Percentage active salmon lice recorded during nine observations during
a 4h
exposure to two different compounds and three different light sources /
conditions
Time (minutes)
10 30 40 50 60 90 120 180 240
Blue 80 90 90 80 90 100 100 100 90
T4 White 100 100 100 100 100 100 100 100 100
Dark 100 70 70 70 70 70 70 80
100
Blue 70 70 70 70 100 100 100 100 100
T5 White 70 70 70 70 90 90 90 90 90
Dark 100 70 70 80 80 80 80 80 90
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Example 4
SOURCING PARASITES
The following lice strains were used. The first was sourced from iLab, Bergen,
where
they cultured, collected, and shipped the lice to the laboratory based at NMBU
(Norwegian University of Life Sciences, Adamstuen, Oslo). The lice were
shipped in a
single 2.5L container filled with oxygenated seawater.. The second was sourced
from
the University of Bergen (UiB), where they cultured, collected, and shipped
the lice to
the laboratory based at NMBU. The lice were shipped in four 2.5L plastic
bottles, filled
with oxygenated seawater. All surviving lice were placed in a temperature-
controlled
io cabinet (12 C) with added air supply until commencement of the
bioassays.
LABORATORY SETUP
All bioassays were conducted at a constant water temperature of 12 C using two
water baths and a temperature-controlled cabinet and included the following
three
light treatments: white LED, blue LED and no light (Table 1). The bioassays
were
conducted in two rounds, one round for each parasite strain using the same
treatment
compounds being used as shown in Table 2.
In this study the salmon lice were exposed to the following light sources:
White LED light. (100-240V, 50W, Luminous flux: 35001m)
Blue light. 100 blue LED lamp array (10 x 10) mounted on a heat sink and
cooled by
an external fan. Specifications: 450-460nm, DC32-36V, 3500mA (100W), Luminous
flux: 1000-1500Im).
Dark (temperature-controlled cabinet)
The following three treatment compounds and in different concentrations were
tested
in this study:
Treatment I (Ti): Riboflavin 2-hydroxypropyl-beta-cyclodextrin. (Compound 14).
Con-
centration: 300 micromolar
Treatment II (T2): Riboflavin phosphate. Concentration: 300 micromolar
Treatment III (T3): Sea water control
Treatment IV (T4): 10 minutes: Curcumin 2-hydroxypropyl-beta-cyclodextrin.
Concen-
tration 160 micromolar (408 mg complex per liter)
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29
Treatment V (T5): 20 minutes: Curcumin 2-hydroxypropyl-beta-cyclodextrin.
Concen-
tration 160 micromolar (408 mg complex per liter)
Treatment VI (T6): 30 minutes: Curcumin 2-hydroxypropyl-beta-cyclodextrin.
Concen-
tration 160 micromolar (408 mg complex per liter)
BIOASSAY PROTOCOL
The following protocol was followed for each round of bioassays. Eighteen
glass flasks
were filled with 300 ml seawater. Ten preadult 2 stage lice were transferred,
using
forceps, into each flask. The flasks were randomly labelled and distributed
among the
3 light groups, resulting in 4 flasks per light treatment. Each flask was
gently stirred to
io ensure that all lice were healthy and able to attach, the contents were
emptied over a
sieve, after which 300 ml treatment compound was then introduced into the
flask.
Control group (T3) were then placed in their respective water bath/darkened
cabinet
and exposed to the respective lights for four hours. Treatment I (Ti) received
a
change of solution every every hour, treatment II (T2) received one solution
change
at the 2-hour mark. Treatment IV (T4) was exposed to the treatment solution
for 10
minutes, treatment V (T5) was exposed for 20 minutes, and treatment VI (T6)
for 30
minutes; after which they were rinsed with seawater, 300m1 clean seawater was
placed in the blue light treatment. Observations of the number of
affected/unaffected
lice were conducted at 30, 60, 120, 180, and 240 min, due to lack of
visibility counts
were made for group 4 and 5 when the treatment solutions were replaced.
RESULTS
The first round of experiments was conducted using lice supplied by iLab. A
malfunction occurred in the water bath containing the blue light treatments
with 2
containers being lost during the treatment. They have been marked with (x) in
Table
9. At the conclusion of the bioassay the control groups (T3) had 100% survival
in all
light treatments (Table 9). The riboflavin treatment (Ti) had no discernable
effect on
survival of the salmon lice. Curcumin cyclodextrin treatments (T4-T6) were the
most
effective with treatment IV dropping to 30% from the 30min observation
onwards.
Treatment V had 10% active lice until the final count where all lice were
affected.
Treatment VI was lost at the 120min observation, however the percentage active
lice
had dropped to 10% at the 60min observation.
HPLC analysis of sea water solutions of both riboflavin and riboflavin
phosphate
showed extensive degradation of both compounds after irradiation with the
intensive
100W blue LED light after 10 minutes. Sea water solutions of both riboflavin
and
riboflavin phosphate showed no degradation in dark during 120 minutes. In a
real
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aquaculture situation, the average light intensity will be much lower than in
this
example. The inventors expect that the lower blue light intensity will not
degrade the
riboflavin molecules and thereby these comounds will work as photosensitizers
for
treatment of salmon infected by salmon lice.
5 The second round of bioassays used lice from UiB. At the conclusion of
the bioassay
the control groups (T3) had 100% survival in all light treatments (Table 10).
The
riboflavin treatment (T2) had no discernable effect on survival of the salmon
lice. All
the curcumin cyclodextrin treatments (T4-T6) were effective at reducing
survival of
the lice, with the majority affected within 60min of exposure to blue light
(Table10).
io Table 9. Percentage active salmon lice recorded during five observations
during a 4h
exposure to eight different treatment compounds and three different light
sources /
conditions
Time (minutes)
30 60 120 180 240
Blue 100
Ti White 100 100 90 90
Dark 100 100 100 100
Blue 100 100 100 100 100
T3 White 100 100 100 100 100
Dark 100 100 100 100 100
T4 Blue 30 30 30 30 30
T5 Blue 0 10 10 10 0
T6 Blue 50 10
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Table 10. Percentage active salmon lice recorded during five observations
during a 4h
exposure to eight different treatment compounds and three different light
sources /
conditions
Time (minutes)
30 60 120 180 240
Blue - 100 100
T2 White - 90 100
Dark - 90 90
Blue 100 100 100 100 100
T3 White 100 100 100 100 100
Dark 100 100 100 100 100
T4 Blue 0 0 0 0 0
T5 Blue 20 10 10 10 10
T6 Blue 10 10 0 0 0
Example 5 Seawater solubility and light stability of curcumin cyclodextrins
Solubility
8 mg of curcumin, 55.5 mg of compound 3 ([2-hydroxypropyl] beta-cyclodextrin
curcumin) and 82.8 mg of compound 7 (beta-cyclodextrin curcumin) were each
dissolved in 12 mL sea water separately using vortex mixer at room
temperature. The
io solutions were saturated solutions. Samples were taken out from the
solutions,
centrifuged and analysed by HPLC. The peak area for compounds compound 3 and
compound 7 was almost 29 and 11 times more than the peak area for curcumin,
respectively (including all impurities in all three compounds).
Conclusion: 2-HP beta-cyclodextrin curcumin was 29 times more soluble than
curcumin in sea water. Beta-cyclodextrin curcumin was 11 times more soluble
than
curcumin in seawater.
Blue light stability
40 micromolar solutions of curcumin, compound 3 ([2-hydroxypropyl] beta-
cyclodextrin curcumin) (MH-103) and compound 7 (beta-cyclodextrin curcumin)
(MH-
107) in sea water/acetonitrile were prepared (100mL sea water and 1mL
acetonitrile).
From each solution 2 samples were taken, one kept in dark and one kept under
blue
light. Aliquots of each sample were analysed by HPLC after 10 min, 20 min, 40
min,
80 min and 120 min. Results are shown in Table 11.
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Table 11. HPLC analysis (peak area) of curcumin, compound MH-103 and compound
MH-107 over time when not exposed to light and exposed to blue light.
Dark Blue light
Time (min) Curcumin MH-103 MH-107 Curcumin MH-103 MH-
107
1040 662 702 663 398 679
903 410 662 379 381 607
40 736 281 670 92 116 366
80 691 637 462 0 82 369
120 667 182 368 0 86 248
Conclusion: Blue light is very effective in degradation of curcumin. The
curcumin cy-
5
clodextrin complexes are much more stable than curcumin in presence of blue
light.
Compound 7 (MH-107) comprising beta-cyclodextrin is much more stable than com-
pound 3 (MH-103) comprising 2-hydroxypropyl-beta-cyclodextrin in presence of
blue
light.
It should be noted that the above-mentioned embodiments illustrate rather than
limit
io the invention, and that those skilled in the art will be able to design
many alternative
embodiments without departing from the scope of the appended claims. In the
claims,
any reference signs placed between parentheses shall not be construed as
limiting the
claim. Use of the verb "comprise" and its conjugations does not exclude the
presence
of elements or steps other than those stated in a claim. The article "a" or
"an" preced-
15 ing an element does not exclude the presence of a plurality of such
elements.
The mere fact that certain measures are recited in mutually different
dependent claims
does not indicate that a combination of these measures cannot be used to
advantage.
