Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
  • A61K31/05—Phenols
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/14—Ectoparasiticides, e.g. scabicides

Definitions

• the present invention relates to a carvacrol and/or thymol or a composition
• the present invention further relates to the use Carvacrol and/or thymol for manufacture of a pharmaceutical composition and to a feed for fish.
• Sea lice (e.g. Caligus rogercresseyi and L. salmonis) infestation is for the time being one of the most important health challenges for the salmon production industry. This external parasite impairs production efficiency, causes stress and damages to the fish and is as a secondary infection a possible vector for other pathogens like the salmon alpha virus (for Pancrease Disease), Infectious Salmon Anaemia virus (ISAv) and the Piscirickettsia salmonis.
• infestation rates have increased significantly mainly affecting species like the Atlantic salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch).
• sea lice infections are considered as one of the most serious health problems in aquaculture of Salmon. This higher infestation rate is probably a consequence of a combination of factors including the development of extensive resistances of the sea lice to most of the applied chemical antiparasite agents used in treatment today.
• chemotherapeutic agents for the treatment of sea lice infections are e.g. belonging to the group of organophosphates, pyrethroids, hydrogen peroxides, chitin synthesis inhibitors and emamectin.
• resistances, often also including multiple and cross-resistances, of sea lice often result in limited success when applying many of the above-mentioned commonly applied anti-sea lice compounds.
• the objective technical problem of the present invention is to provide new effective agents and methods for killing copepod ecto-parasites, and/or to inhibit the infestation of ectoparasites in fish, such as sea lice and other copepod parasites.
• a first aspect of the present invention relates to carvacrol and/or thymol or a composition comprising one or both of the active compounds for use in the treatment and/or prophylaxis of ectoparasitic copepod infection and/or infestation in a fish.
• the ectoparasitic copepod is a sea lice.
• the sea lice is selected from Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi.
• the ratio of carvacrohthymol is in the range of 1 :3 to 3:1 , more preferable of 1 :2 to 2:1 , most preferred about 1 :1 based on weight.
• said fish is a Salmonidae and more preferred a fish selected from the group consisting of Atlantic salmon (Salmo salar), Rainbow trout (Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and Artie charr (Salvelinus alpinus).
• the active compounds are used for reduction of the total number of adult sea lice stages infested or re-infested on fish.
• carvacrol and/or thymol is added to a feed composition in a total concentration between 0.001 % to 0.8% of the feed, preferably 0.003 % to 0.6 % and more preferred between 0.003 and 0.012 wt % of the feed.
• the total concentration of carvacrol and/or thymol is 30 mg/kg or more, preferably 45 mg/kg feed or more, more preferred at least 100 mg/kg and most preferred at least 120 mg/kg feed.
• carvacrol and thymol is comprised in a ratio of 1 :1.
• the feed comprises other conventional feed ingredients in the fish feed.
• the feed further comprises one or several of the following ingredients selected from the group of nucleotides, prebiotics, vitamin C and immune stimulants, which can be specific or non-specific.
• the compound or composition is supplied to fish for at least 7 days, more preferred for at least 21 days and most preferred for at least 28 days.
• the treatment with the active compounds is combined with other chemical or medical agents against sea lice.
• the active compounds are specifically used to combat certain life stages of sea lice.
• the active compounds are used to reduce the infestation of gravid female sea lice on the fish.
• the fish are fed continuously the feed comprising the active compounds as part of their daily diet.
• the active compound or compounds are applied in form of a water bath treatment.
• a second aspect of the present invention relates to a method of killing, combating or controlling ectoparasitic copepods, wherein the method comprising applying carvacrol and/or thymol or a composition comprising one or both of the active compounds in order to kill the sea lice, make them lethargic or prevent infestation to fish.
• the ectoparasitic copepod is a sea lice.
• the sea lice is Lepeophtheirus salmonis, Caligus elongatus or Caligus rogercresseyi.
• said fish is a Salmonidae, preferably selected from the group consisting of Atlantic salmon (Salmo salar), Rainbow trout (Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and Artie charr (Salvelinus alpinus).
• said active compound, mixture of compounds or composition is applied to a locus to be protected from the parasites.
• the locus is an aquatic environment and wherein the active compound or composition comprising the active compound is applied directly to the aquatic locus.
• said locus contains fish and wherein the sea lice are located on said fish or said fish is susceptible for infestation or infection of said sea lice, wherein the active compound or mixture of compounds is applied directly to the fish, preferable by topical or oral application.
• the active compound or mixture of compounds are applied to the fish as a component of the feed fed to the fish.
• the ratio of carvacrohthymol is in the range of 1 :3 to 3:1 , more preferable 1 :2 to 2:1 , and most preferable about 1 :1 , based on weight.
• one or more of the active compounds or a composition comprising the active compounds are supplied to fish as part of feed for at least 7 days, more preferred for at least 21 days and most preferred for at least 28 days.
• a third aspect of the present invention relates to a use of carvacrol and/or thymol for manufacture of a pharmaceutical composition for the treatment and/or prophylaxis of ectoparasitic copepod infection and/or infestation in a fish.
• the ectoparasitic copepod is a sea lice.
• the sea lice is selected from Lepeophtheirus salmonis, Caligus elongatus and Caligus rogercresseyi.
• the ratio of carvacrol :thymol is in the range of 1 :3 to 3:1 , more preferable of 1 :2 to 2:1 , most preferred about 1 :1 based on weight.
• said fish is a Salmonidae.
• said fish is selected from the group consisting of Atlantic salmon (Salmo salar), Rainbow trout (Onchorhynkuss mykiss), Coho salmon (Oncorhynchus kisutch) and Artie charr (Salvelinus alpinus).
• the pharmaceutical composition is for a reduction of the total number of adult sea lice stages infested or re-infested on fish.
• the pharmaceutical composition comprising carvacrol and/or thymol is added to a feed composition in a total concentration of carvacrol and/or thymol from 0.001 % to 0.8% of the feed, preferably from 0.003 % to 0.6 % and more preferred from 0.003 and 0.012 wt % of the feed.
• the pharmaceutical composition comprising carvacrol and/or thymol is added to a feed composition, which has a total concentration of carvacrol and/or thymol of at least 30 mg/kg feed, preferably at least 45 mg/kg feed, more preferred at least 100 mg/kg and most preferred at least 120 mg/kg feed.
• carvacrol and thymol is comprised in a ratio of 1 :1.
• the feed comprises other conventional feed ingredients in the fish feed.
• the feed further comprises one or several of the following ingredients selected from the group of nucleotides, prebiotics, vitamin C and immune stimulants, which can be specific or non-specific.
• the compound or composition is supplied to fish for at least 7 days, more preferred for at least 21 days and most preferred for at least 28 days.
• the treatment with the active compounds is combined with other chemical or medical agents against sea lice.
• the active compounds are specifically used to combat certain life stages of sea lice.
• the active compounds are used to reduce the infestation of gravid female sea lice on the fish.
• the fish are fed continuously the feed comprising the active compounds as part of their daily feed.
• the pharmaceutical composition is applied in form of a water bath treatment.
• the present invention relates to a feed for fish comprising carvacrol and/or thymol in a total concentration of 0.001 % to 0.8% of the feed, preferably 0.003 % to 0.6 % and more preferred between 0.003 and 0.012 wt % of the feed.
• the total concentration of carvacrol and/or thymol is at least 30 mg/kg, preferably at least 45 mg/kg feed, more preferred at least 100 mg/kg and most preferred at least 120 mg/kg feed.
• the ratio of carvacrol :thymol is in the range of 1 :3 to 3:1 , more preferable of 1 :2 to 2:1 , most preferred about 1 :1 based on weight.
• Figure 1 shows the observed mortality of copepodids of C. rogercresseyi (as proportion of the number of lice at the start of the study), over a period of 48 h at different doses of Carvacrol. Each panel represents a time point (zero to 48 hours) from the start of the study.
• Figure 2 shows the percentage mortality of copepodids of C. rogercresseyi exposed to different doses of Carvacrol (pL/L) during 48 hours. The error bars represent the standard deviation obtained for the replicas in each dose of Carvacrol.
• Figure 3 shows the observed mortality of adults of C. rogercresseyi (as proportion of initial), over a period of 48 h at different doses of Carvacrol.
• Figure 4 shows the percentage mortality of adults of C. rogercresseyi exposed to different doses of Carvacrol (pL/L) during 48 hours. The error bars represent the standard deviation obtained for the replicas in each dose of Carvacrol.
• Figure 5 shows the proportion (%) of reduction in expected sea lice (C. rogercresseyi) infested over the fish when fed two doses of Carvacrol +Thymol in feed in comparison to the control feed.
• Figure 6 shows (for example 4) feed consumed by the fish in each treatment during the trial, a) Specific Feeding Rate (SFR) for fish fed with high dose of Carvacrol, b) SFR for fish fed with low dose of Carvacrol and c) SFR for fish fed with control feed.
• SFR Specific Feeding Rate
• Figure 7 shows (for example 4) sea lice (C. rogercresseyi) infestation expressed as total number of lice per tank which contained 35 fish , where dot is the average infestation and lines indicate credible intervals (95%) in each dose (low and high) and control.
• sea lice C. rogercresseyi
• Figure 8 shows expected average Caligus counts over whole monitoring period (posterior median) for each life stage and treatment with 95% credible intervals (error bar) (example 5, experiment 1 ).
• Figure 9 shows expected Caligus reinfestation counts (posterior median, solid line) for juveniles, adults and total lice by each treatment with 95% credible intervals (shadowed area, shown only for 0 and 120 ppm). Black line indicates control with 0 ppm (example 5, experiment 2).
• Figure 10 shows expected Caligus reinfestation counts for gravid females, females and adults for each treatment with 95% credible intervals (shadowed area). Black line means control (example 5, experiment 2).
• Figure 11 shows linear and quadratic coefficients of the dose-response model with 95% credible intervals for each sampling day (example 5, experiment 2).
• the quadratic effects are magnified (x100) to make them visible in the same plot as the linear terms. See text for explanation.
• the objectives of this study is to i) determine if Carvacrol induces mortality of copepodids of Caligus rogercresseyi, and ii) determine the LD50 (Lethal Dosis that causes 50% mortality) of Carvacrol against copepodids of Caligus rogercresseyi in vitro.
• LD50 was modelled with the help of a generalized linear model using binomial model with logit link (http://cran.r- project.org/doc/manuals/fullrefman.pdf).
• Carvacrol As a monoterpenoid, Carvacrol is lipophilic and therefore we believe that a possible mechanism of action is by passing through the cell wall and cytoplasmatic membrane, and disrupting structural layers of polysaccharides, fatty acids and phospholipids of the cells. No negative effect of Carvacrol has been reported for salmonids.
• Carvacrol induces high mortality over copepodids of Caligus rogercresseyi at the concentrations tested in this experiment.
• the objective of this experiment is to determine the LD50 of Carvacrol against adults of Caligus rogercresseyi in vitro.
• the results of the LD50 analysis for each exposure time are shown in Table 4.
• the estimates of LD50 doses vary widely and non-systematically between exposure times (Table 4). It can be observed that after 12 h of exposure, time with a minor variability, the LD50 has a lower and upper confidence limits of 0.45 ⁇ _/ ⁇ _ and 394.36 pL/L, respectively with an estimated LD50 of 8.26 pL/L.
• the estimated LD50 obtained after 12 h of exposure was 8.26 pL/L.
• Table 4 Estimated LD50 values of Carvacrol on adults of C. rogercresseyi for each exposure time from the categorical time model. Mean, median and 95% confidence interval.
• Example 3 Effect of a fish feed containing Carvacrol on sea lice
• Carvacrol has a surprisingly high potential to reduce sea lice (C. rogercresseyi) infestation.
• No previous studies have been done to test this phenolic compounds, e.g. Carvacrol and/or Thymol, directly against sea lice. Further, no previous studies have been done to assess the efficacy of Carvacrol and Thymol against sea lice, when fish are fed a feed containing such compounds.
• the objective of this study is to assess the efficacy of Carvacrol and Thymol against sea lice (Caligus rogercresseyi) by feeding fish with a fish feed containing different doses of phenol compounds, i.e. Carvacrol and Thymol.
• acclimatization period of 7 days was performed with a commercial feed.
• commercial feed was changed for the experimental feeds (see table 5 below): a) control, b) 0.17% Carvacrol+Thymol in feed and c) 0.61 % Carvacrol+Thymol in feed.
• Four replicas were applied per each treatment. After 12 days of feeding the fish with the experimental feeds, salmons were infested with copepodids of C. rogercresseyi (with an average infestation rate of 118 copepodids/fish) in 12 tanks of 0.35 m 3 with open sea water flow system and an average temperature of about 13°C.
• Table 5 Basal diet composition showing additionally the inclusion of Carvacrol and Thymol (%) for the two experimental diets.
• Carvacrol+Thymol reduces the infestation of Caligus rogercresseyi, when the fish were treated with Carvacrol and Thymol added as a component of the feed composition.
• Example 4 effect of Carvacrol in the reduction of Caliaus rogercresseyi infestation
• the object of the study was to measure the efficacy of Carvacrol in the reduction of Caligus rogercresseyi infestation, when Atlantic salmon (Salmo salar) fed a feed with the compound in two different doses.
• Atlantic salmons were initially allocated in 4 tanks of 3 m 3 , with 132 fish per tank and a maximum density of 16 Kg/m 3 , with flow trough system and filtrated sea water (60 pm). Fish were pit-tagged and weighted during the allocation in each tank. During 14 days fish were recovered (7 days) and acclimatized (7 days) in the same tanks, feeding a commercial feed. Then, Atlantic salmons were challenged with copepodids of C. rogercresseyi (72 copepodids/fish) and 20 fish were sampled (basal sampling) to obtain liver, mucus (5 ml_), muscle (each fillet) and skin. Samples were stored and frozen at -80°C for further chemical analysis.
• the data obtained were analyzed using the R language and its corresponding packages (http://www.r-project.org/ ).
• the lice counts made of 35 individual sampled fish were converted to a total count per tank by summing the individual counts (given the number of fish sampled is the same for all tanks).
• This method is for unbound counts, such as sea lice counts, and they are assumed to follow Poisson distribution.
• Lice counts can also be overdispersed, i.e. the variance does not follow the standard Poisson distribution. This does not affect the coefficient estimates as such but only their standard errors. Therefore the model was a generalized linear model estimated with the glm function of the R language.
• the family is Poisson as overdispersion was modeled.
• the first trial (Experiment 1 ) was conducted in a commercial sea site located in the XI Region of Chile.
• three (3) dietary treatments were compared: 1 ) a commercial control feed (EWOS Silva 1500 and 2500) with no supplementation of any kind; 2) the same base formulation as in 1 , but supplemented with two functional components, nucleotides and a prebiotic (EWOS pre-harvest); and 3) the same as in treatment 2, but supplemented with 30 g/ton of feed (0.003%) of pure carvacrol and thymol (50:50 wt) (pre-harvest + new).
• the base formulation of the feeds contained as the major ingredients fish meal, poultry by-products, soybean products, sunflower meal, corn gluten, wheat, wheat gluten and a mix of fish and vegetable oils (94.4% of the formulation).
• the control, pre-harvest (PH) and pre-harvest + carvacrol and thymol (PH + new) feeds were delivered to 7, 3 and 4 cages, respectively. All cages belonged to the same site. Each cage contained approximately 50.000 fish.
• the experiment was conducted from November 1 , 2009 to February 28, 2010. The fish weight increased from 1.7 kg to 2.5 kg during the experimental period. During this period, two pulses with the dietary treatments were given to the trouts. The first pulse was from
• the second trail was performed in January 20 1 in the X Region of Chile, at the experimental sea site of EWOS Innovation.
• the site has 16 cages of 15 m x 15 m with an automatic feeding system.
• Rainbow trout O. mykiss
• the average fish weight at the beginning of the trial was 2.0 kg.
• Each cage contained approximately 10,000 fish.
• four (4) dietary treatments were evaluated.
• One of the feeds was a commercial Control feed (EWOS Silva 2000) with no supplementation of any kind. Diets 2, 3 and 4 had the same base formulation as the control feed, but supplemented with a mix of nucleotides, prebiotic, vitamin C and an immune.stimulant (EWOS boost).
• the base formulation of the feeds contained as the major ingredients fish meal, poultry by-products, soybean products, sunflower meal, corn gluten, wheat, wheat gluten and a mix of fish and vegetable oils (94,6% of the formulation).
• the source of carvacrol and thymol used was the same as in Experiment 1 and contained 25% carvacrol and 25% thymol. Therefore, the concentrations of the source of carvacrol and thymol in the three experimental feeds were 60, 90 and 240 g/ton of feed, respectively. Thus, as a percentage of the feed, pure carvacrol and thymol were supplemented at 0.003, 0.006 and 0.012%, respectively.
• the EWOS Innovation sea site is located in a production area with natural infestation of Caligus.
• a bath treatment with deltamethrin (Alphamax®, 0.2 ml/m 3 for 30 min) was performed in all fish in a close tarpaulin system.
• an initial sampling of 30 fish per cage was performed to count the lice infestation rate. This initial sampling represented the basal infestation rate just after the anti-parasite treatment and before starting feeding the experimental feeds. All fish were anesthetized with Aqui-S and then weighted, tagged and counted for sea lice, as the standard procedure applied by EWOS Innovation.
• sea lice life stages recorded included the juvenile stages copepodids, Chalimus I, II, II and IV and the adult stages females, gravid females and males.
• the reinfestation counts can be negative if the lice count of a specific life stage decreased from the initial count.
• Figures 9 and 10 show that the fish fed with carvacrol and thymol had a lower reinfestation rate for juveniles, adults and total lice in a dose dependent trend ( Figure 9).
• An inclusion of as low as 0.003% of carvacrol and thymol in the feed was surprisingly effective on the reinfestation of sea lice in spite of the comparable low dose.
• the efficacy of carvacrol and thymol in the treatment of sea lice is increased by the presence of immune stimulants in the feed as shown in example 5.
• immune stimulants are not restricted to non-specific immune stimulants, but can also be selected from specific immune stimulants.
• the treatment with a feed comprising carvacrol and/or thymol is carried out on a continuous basis over an extended period of time, wherein the active compounds are preferably added to the feed consumed by the fish on a daily basis. It is preferred that fish are fed for at least 7 days with the diet, more preferred for 3 to 4 weeks. However, longer periods extending 28 days may also be applied.
• Carvacrol is the compound 5-isopropyl-2-methylphenol (C6H 3 CH3(OH)(C 3 H 7 )), and is a monoterpenoid phenol. It has a characteristic pungent, warm odor of oregano and a pizza-like taste. It is present in the essential oil of Origanum vulgare, oil of thyme, oil obtained from pepperwort, and wild bergamot.
• Thymol is the compound 5-isopropyl-2-methylphenol, and is a monoterpenoid phenol derivative of cymene, isomeric with Carvacrol, found in oil of thyme, and extracted as a white crystalline substance of a pleasant aromatic odor and strong antiseptic properties.
• Prebiotics are food ingredients that stimulate the growth and/or activity of bacteria in the digestive system being beneficial to health.
• Immune stimulants are substances (drugs and nutrients) that stimulate the immune system by inducing activation or increasing activity of any of its components. They may be non-specific or specific immune stimulants.
• Conventional feed ingredients are feed ingredients which are commonly used in feed compositions for a specific animal species such as lipids, proteins, vitamins, carbohydrates, minerals, etc.
• Nucleotides comprise any known phosphor ester of a nucleoside such as AMP, GMP, UMP, CMP, UMP.

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• 2010
  • 2010-03-26 NO NO20100451A patent/NO20100451A1/no not_active Application Discontinuation
• 2011
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  • 2011-03-28 CA CA2794481A patent/CA2794481C/en active Active
  • 2011-03-28 WO PCT/NO2011/000108 patent/WO2011119049A1/en active Application Filing
• 2012
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