ISOXAZOLINE SOLUTION CONTAINING VITAMIN E FOR USE WITH SANITIZED DRINKING WATER

Abstract

For the prevention of parasite infestation of animals, an isoxazoline can be administered by drinking water route. However the inventors have found that when the drinking water is sanitized, for instance by using hypochlorite, the isoxazoline becomes degraded. Surprisingly, the isoxazoline can be protected from degradation by the use of a vitamin E. A pharmaceutical composition can now be prepared containing a concentrated solution of the isoxazoline in a solvent and co-solvent, with vitamin E. The composition can be diluted in drinking water, even when sanitized, to prepare medicated drinking water for animals. This way an anti-parasitic treatment can be mass-administered, leading to a highly effective reduction of the parasite infestation of an animal, and its surroundings.

Claims

1. A Pharmaceutical composition comprising an isoxazoline in a pharmaceutically acceptable solvent and a co-solvent, and vitamin E, wherein the isoxazoline is the compound ##STR00015## wherein R.sup.1=halogen, CF.sub.3, OCF.sub.3, or CN; n=integer from 0 up to and including 3; m=1 or 2; R.sup.2=C.sub.1-C.sub.3haloalkyl; T=ring structure: 5-, or 6-membered, or bicyclic, which is optionally substituted by one or more radicals Y; Y=methyl, halomethyl, halogen, CN, NO.sub.2, NH.sub.2—C═S, or two adjacent radicals Y together form a chain; Q=X—NR.sup.3R.sup.4, NR.sup.5—NR.sup.6—X—R.sup.3, X—R.sup.3, or a 5-membered N-heteroaryl ring, which is optionally substituted by one or more radicals; X=CH.sub.2, CH(CH.sub.3), CH(CN), CO, CS; R.sup.3=hydrogen, methyl, haloethyl, halopropyl, halobutyl, methoxymethyl, methoxyethyl, halomethoxymethyl, ethoxymethyl, haloethoxymethyl, propoxymethyl, ethylaminocarbonylmethyl, ethyl aminocarbonyl ethyl, dimethoxyethyl, propynylaminocarbonylmethyl, N-phenyl-N-methyl-amino, haloethylaminocarbonylmethyl, haloethylaminocarbonylethyl, tetrahydrofuryl, methylaminocarbonylmethyl, (N,N-dimethylamino)-carbonylmethyl, propylaminocarbonylmethyl, cyclopropylaminocarbonylmethyl, propenylaminocarbonylmethyl, haloethylaminocarbonylcyclopropyl, alkyl sulfanyl alkyl, alkyl sulfinylalkyl, alkyl sulfonylalkyl, cycloalkyl, ##STR00016## ##STR00017## wherein Z.sup.A=hydrogen, halogen, cyano, or halomethyl; R.sup.4=hydrogen, ethyl, methoxymethyl, halomethoxymethyl, ethoxymethyl, haloethoxymethyl, propoxymethyl, methylcarbonyl, ethylcarbonyl, propylcarbonyl, cyclopropylcarbonyl, methoxycarbonyl, methoxymethylcarbonyl, aminocarbonyl, ethylaminocarbonylmethyl, ethyl aminocarbonyl ethyl, dimethoxyethyl, propynylaminocarbonylmethyl, haloethylaminocarbonylmethyl, cyanomethylaminocarbonylmethyl, or haloethylaminocarbonylethyl; R.sup.5=H, alkyl, or haloalkyl; R.sup.6=H, alkyl, or haloalkyl; or wherein R.sup.3 and R.sup.4 together form a substituent selected from the group consisting of: ##STR00018## and wherein the pharmaceutical composition comprises between about 1% and about 20% of vitamin E.

2. The Pharmaceutical composition according to claim 1, wherein Z.sup.A is CF.sub.3.

3. The Pharmaceutical composition according to claim 1, wherein the isoxazoline is one or more selected from the group consisting of: Fluralaner, Afoxolaner, Lotilaner, and Sarolaner.

4. A Method for the preparation of the pharmaceutical composition according to claim 1, the method comprising the step of dissolving the isoxazoline and the vitamin E in the pharmaceutically acceptable solvent and the co-solvent.

5. A Medicated drinking water comprising the pharmaceutical composition according to claim 1 and drinking water.

6. The Medicated drinking water according to claim 5, wherein the medicated drinking water further comprises a water sanitizer.

7. A Method for the preparation of the medicated drinking water according to claim 6, the method comprising the step of diluting into drinking water the pharmaceutical composition.

8. (canceled)

9. (canceled)

10. (canceled)

11. A Method for the treatment or prevention of parasite infestation of animals, comprising the administration to the animals of the pharmaceutical composition of claim 1 via medicated drinking water.

12. (canceled)

13. (canceled)

14. A Method for the treatment or prevention of parasite infestation of animals, comprising the administration to the animals of the medicated drinking water according to claim 6.

15. A Method for controlling and/or reducing parasitic arthropods in animal surroundings, the method comprising the administration to the animals of the medicated drinking water according to claim 6.

16. (canceled)

17. A Container comprising the pharmaceutical composition according to claim 1.

18. A Kit of parts, the kit comprising a container comprising the pharmaceutical composition according to claim 1, and instructions for use of the pharmaceutical composition according to claim 1 for preparing a medicated drinking water for animals.

Description

EXAMPLES

Example 1: Efficacy of Fluralaner in Medicated Drinking Water Against Poultry Red Mite Infestation of Chickens

[0248] As a comparative example, the results in this Example demonstrate the parasiticidal efficacy of Fluralaner, when administered orally via medicated drinking water, to control an artificially induced infestation of laying hens with poultry red mite (D. gallinae). The drinking water used was regular tap water not comprising a significant amount of oxidising sanitizer, and no vitamin E was used in the concentrated Fluralaner solution used.

[0249] 1.1. Materials and Methods

[0250] Experimental animals were chickens, particularly laying hens of about 30 weeks old. These were housed in a standard poultry house. Apparently healthy birds were assigned in a random way to treatment groups kept separately, and labelled individually. The birds were kept under natural light, and were given several days for acclimatization; drinking water consumption in each group was measured on three days prior to administration to calculate the average daily water consumption. Medicated water was prepared by diluting a concentrated Fluralaner solution (10 mg/ml) to the desired concentration of Fluralaner in regular tap water (hypochlorite <0.5 ppm).

[0251] The concentrated Fluralaner solution used was comparable to a pharmaceutical composition according to the invention, except that no vitamin E was present, and consisted of: 0.95% w/w Fluralaner, 24.76% w/w Transcutol V, and 74.29% w/w Tween 80.

[0252] Groups A-D (n=6) were treated with doses of 2, 1 or 0.5 mg Fluralaner/kg BW once, or with 1 mg Fluralaner/kg BW in a repeated dose of 0.5 mg/kg BW on 2 occasions, 7 days apart.

[0253] On day 0 (and group D additionally on day 7), the hens in groups A-D received Fluralaner via medicated drinking water. Group E received un-medicated drinking water ad libitum.

[0254] The dose to be administered was calculated based on average body weights of each treatment group, measured one day before treatment (day −1, day 6). A concentrated Fluralaner solution was diluted in the drinking water to prepare medicated drinking water ready for consumption, via the following dosing regimen: [0255] The volume of medicated water offered per group on day 0 (group D also on day 7) was approximately 50% of the calculated mean daily water intake measured previously in the respective group in order to ensure consumption of the full dose. [0256] Once all medicated water was consumed the other 50% volume of the mean daily water intake was supplied as unmedicated tap water in the same drinker.

[0257] On days 1, 5, 8, 12, 15, 19 and 22, four of the six hens per group were infested with approximately 200 vital, D. gallinae mites (unfed nymphs and adults that had starved before infestation for 7 days).

[0258] From each infested hen approximately 25 engorged mites were collected after a few hours of presence in the animal box, next they were incubated for approximately 24 hours, after which their status was observed. The dead, damaged and/or live mites were counted visually using a binocular.

[0259] Mites were classified as dead if no movement was determined or mites lay in a dorsal position. Mites were classified as damaged if their movement was uncoordinated.

[0260] The Mite Mortality and Mite Inhibition percentage was calculated for each treated group in comparison to a non-treated negative control group.

[0261] 1.2. Results

[0262] Fluralaner was well tolerated in the hens.

[0263] The % mortality and % inhibition of red mites assessed approximately 24 hours after the infestation of hens that received Fluralaner orally via drinking water are given in Tables 1 and 2. A fast onset of action was demonstrated for all administered doses.

TABLE-US-00003 TABLE 1 % mortality of D. gallinae assessed 24 hours after infestation % Mortality of mites 24 hours after Fluralaner infestation on day Group (mg/kg BW) 1 5 8 12 15 19 22 A 2 100 100 100 100 77 1 0 B 1 100 100 100 94 77 2 0 C 0.5 100 100 97 55 15 0 0 D 1 (2x 100 100 100 100 98 59 14 0.5)

TABLE-US-00004 TABLE 2 % inhibition of D. gallinae assessed 24 hours after infestation % Inhibition of mites 24 hours after Fluralaner infestation on day: Group (mg/kg BW) 1 5 8 12 15 19 22 A 2 100 100 100 100 81 14 0 B 1 100 100 100 95 81 3 0 C 0.5 100 100 100 75 19 0 0 D 1 (2x 100 100 100 100 99 66 27 0.5)

[0264] On each assessment time point, mites observed from the untreated control group were vital and showed their normal behaviour.

Example 2: Efficacy of Fluralaner in Medicated Drinking Water Against Northern Fowl Mite Infestation of Chickens

[0265] As a further comparative example, studies comparable to those described in Example 1 were performed to demonstrate the high effectivity of isoxazoline in unsanitized medicated drinking water also against a natural infestation of layer chickens with Northern fowl mite (O. sylviarum). No vitamin E was present in the concentrated Fluralaner solution used to prepare the medicated drinking water.

[0266] The effectivity of an oral dosing regimen of Fluralaner was tested by administering medicated drinking water (via gavage) to laying hens, in 2 repeated doses of 0.25, 0.5 or 1.0 mg/kg body weight, administered 7 days apart, and challenging them with infestation by Northern fowl mites, via source birds and natural infestation. The birds were kept in separate pens, at 11 birds per pen.

[0267] The statistical analysis of results was based on mite count from each bird in the study, using pen as a random factor in the model, and mite counts were performed and recorded for individual birds.

[0268] Mite vent count reduction of the treated groups compared to the control group was the primary efficacy criterion. Effectiveness was demonstrated as there was a significant difference in O. sylviarum (p<0.05) mite vent counts between the Fluralaner treated- and the control groups.

[0269] Again Fluralaner was well tolerated in the hens, and provided significant efficacy against O. sylviarum when administered orally as 2 single doses of 0.25, 0.5, or 1 mg/kg BW, 7 days apart.

[0270] Early onset of action was observed in all doses, with statistically significant reductions in mite vent counts observed in all treated groups already beginning at day 1. The concentrations of Fluralaner in the birds' plasma increased proportionally to the dose administered.

[0271] An efficacy against Northern fowl mites (reduction in mite vent count) of 90% was demonstrated in all treated groups for at least 19 days, even despite the presence of untreated source birds in the same pen. Also, the source birds (5 per pen) maintained a natural mite infestation pressure throughout the study.

[0272] Duration of efficacy was dependent on the Fluralaner dose administered, and lasted for up to 19, 22 or 22 days after first treatment administration, for the 0.25, 0.5 or 1.0 mg/kg BW dose groups, respectively.

Example 3: Effect of Drinking Water Quality on Degradation of Isoxazoline

[0273] To assess what caused the degradation of Fluralaner in dilution in drinking water, the impact of different types and qualities of drinking water was tested. This was done by incubation of samples containing 1 μg/g of Fluralaner, in water that contained different ions at increased concentrations from average normal conditions. Samples were mixed and incubated for 7 days, either at 2-8° C., or at 40° C./75% relative humidity (RH). Fluralaner contents were measured before and after incubations.

[0274] 3.1. Fluralaner Measurements

[0275] Isoxazolines such as Fluralaner can be detected and quantified by fast liquid chromatography methods, using standard equipment and procedures, such as UHPLC. Standard liquids for washing and as carrier are e.g.: LC grade Acetonitrile, Methanol, Formic acid, and MiliQ™ water can be prepared in house. Fluralaner is characterised based on its UV spectrum and retention profile, and quantified in reference to dilutions of standards.

[0276] 3.2. Testing of Different Water Qualities

[0277] LoQ=Limit of Quantitation, the lowest concentration at which the analyte can reliably be detected. For Fluralaner by UHPLC the LoQ is 100 ng/ml.

TABLE-US-00005 TABLE 3 Different water qualities tested for effect on Fluralaner. Fluralaner content (μg/g) after 7 days at Drinking water quality 2-8° C. 40° C./75% RH Purified water 0.90 0.92 Water at pH = 4 1.23 1.26 Water at pH = 10 0.78 0.79 Soft water/low pH (1) 0.88 0.89 Hard water/high pH (2) 0.92 0.96 Fe.sup.3+ water at 1 mg/l 0.91 0.89 Na.sup.+ water at 300 mg/l 0.85 0.84 K.sup.+ water at 300 mg/l 0.90 0.80 Cu.sup.2+ water at 10 mg/l 0.90 0.78 Al.sup.3+ water at 1 mg/l 0.99 1.01 NO.sub.3.sup.− water at 100 mg/l 0.92 0.93 PO.sub.4.sup.− water at 100 mg/l 1.14 1.15 Cl.sup.− water at 300 mg/l 0.97 0.98 ClO.sup.− water at 250 ppm Not detected Not detected ClO.sup.− water at 150 ppm <LOQ <LOQ ClO.sup.− water at 50 ppm <LOQ <LOQ ClO.sup.− water at 5 ppm <LOQ <LOQ ClO.sup.− water at 0.5 ppm 0.99 0.82 (1) Soft water contained 30 mg CaCl.sub.2•2H.sub.2O/l; pH was 6.2 (2) Hard water contained 500 mg CaCl.sub.2•2H.sub.2O/l; pH was 8.1

[0278] Although these conditions of incubation applied were very demanding, it is evident from these results that especially hypochlorite is incompatible with an isoxazoline such as Fluralaner. Unfortunately hypochlorite concentrations over 0.5 ppm, up to 100 ppm and above may actually be used for water sanitizing purposes in animal farming operations. Because degradated isoxazoline cannot be active anymore as parasiticide, therefore this degradation needed to be prevented in some way, in order to allow mass application via the local drinking water used.

[0279] Of the effects detected, either positive of negative, only those of hypochlorite were found to be reproducible.

[0280] 3.3. Analysis of Breakdown Products of Fluralaner

[0281] The breakdown products of Fluralaner by hypochlorite were analysed by LC-MS and UHPLC-MS. The aim was to determine molecular ion peaks of decomposition products in order to deduce their chemical identity.

[0282] Samples of medicated drinking waters from the incubations in different types of drinking water were analysed directly, but were found to be too dilute for mass detection.

[0283] To overcome this, direct incubations of samples of Fluralaner with hypochlorite were made: 1.5 mg/ml Fluralaner was dissolved in tetrahydrofurane containing water to dissolve the isoxazoline, and these were incubated with 5 ppm hypochlorite for 24 hours. These samples were tested by UHPLC-MS. For comparison, specific standards were also analysed of truncated Fluralaner molecules which could be potential breakdown products. By comparing the peaks of the hypochlorite incubated samples, to those of the standards, actual breakdown products could be identified.

[0284] Interestingly, two major breakdown products by hypochlorite of the original Fluralaner molecule were identified; each was cleaved at one of the amide groups that are in the side chain of the substituted benzamide moiety.

[0285] In tact Fluralaner molecule:

##STR00012##

Hypochlorite breakdown products detected:

##STR00013##

2-[[4-[5-(3,5-dichlorophenyl)-5-(trifluoromethyl)-4H-isoxazol-3-yl]-2-methyl-benzoyl]amino]acetic acid, and

##STR00014##

4-[5-(3,5-dichlorophenyl)-5-(trifluoromethyl)-4H-isoxazol-3-yl]-2-methyl-benzoic acid

Example 4: Prevention of Isoxazoline Degradation in Sanitized Medicated Drinking Water

[0286] The inventors tested several ways to prevent the degradation of isoxazolines in drinking water of different qualities, especially in drinking water containing an oxidising sanitizer, such as hypochlorite.

[0287] 4.1. Sample Preparation

[0288] To the base of a pharmaceutical composition as described herein, comprising isoxazoline, solvent, and surfactant, different compounds were added as protectors to prevent breakdown of isoxazoline when diluted in water with a water sanitizer such as hypochlorite.

[0289] The base composition was: 0.95% w/w Fluralaner in a mixture of Transcutol V and Tween 80 in 25:75 ratio, whereby the Transcutol and Tween are at a % w/w that was 99.05 minus the % w/w of the protector used. Where the protector amount was zero, this sample served as unprotected control sample. The Fluralaner level was determined using UHPLC. The alpha-tocopherol used was alpha-tocopherol EP (Fluka).

TABLE-US-00006 TABLE 4 Tested protectors and amounts: Amount of protector Mw in the pharmaceutical Protector (g/mol) composition (in % w/w) Lauryl gallate (dodecyl gallate) 338.44 6.4 BHA (butylated hydroxyanisole) 180.25 3.4 BHT (butylated hydroxytoluene) 220.35 4.2 α-Tocopherol (vitamin E) 430.71 8.2 Sodium metabisulfite 190.10 3.6 Sodium sulphite (anhydrous 126.04 2.4 pure) EDTA (acid pure) 292.24 5.5

[0290] To prepare the test samples, the required amounts of Transcutol and Tween were measured, combined in a beaker at room temperature, and mixed magnetically. Next, when no protector was added, the required amount of Fluralaner was weighed, added, and mixed until complete dissolution, which usually required about 10 minutes, at 70% speed. The prepared concentrated solution of Fluralaner at 0.95% w/w, had a clear and yellow appearance. The complete dissolution was verified visually. Next the density of the resulting solution was checked with a Densimeter, at 20° C.

[0291] For samples containing a protector, Fluralaner was added to a mixture of Transcutol V, Tween 80 and protector, and then mixed to dissolve.

[0292] BHT, BHA and lauryl gallate, which are solids, were dissolved in the base composition, which took up to 60 minutes; the alpha-tocopherol, which is a viscous liquid started to mix quickly but required up to 30 minutes for mixing to be complete.

[0293] 4.2. Test of Prevention of Degradation by Oxidizing Sanitizer

[0294] To test the capacity of the different protectors to prevent degradation of Fluralaner in medicated drinking water by an oxidising sanitizer, samples of the pharmaceutical composition as prepared above were diluted to different concentrations, and incubated with different amounts of hypochlorite. Concentrations of Fluralaner in medicated drinking water used were: 1, 5, and 10 μg/ml. One negative control sample was also included, which did not contain Fluralaner.

[0295] The hypochlorite used was from a 13% sodium hypochlorite solution (Acros Organics), which was prepared into samples with a concentration of hypochlorite of: 0, 0.5, 2, 5, or 10 ppm, wherein 1 ppm equals 20 μM. Samples without hypochlorite served as positive controls. While the dilutions of hypochlorite were made in purified water, one additional sample at 5 ppm hypochlorite was prepared in standard tap water (Angers, France). Control samples were tested at day zero, all others were tested after an incubation for 7 days at 40° C./75% RH.

[0296] The concentration of protector in the test sample was dependent of the dilution factor of the concentrated Fluralaner composition applied: 1:10,000, 1:2000, or 1:1000, respectively.

[0297] 4.3. Results of Prevention of Degradation by Oxidizing Sanitizer

[0298] The compounds Na-metabisulfite, Na-sulphite, and EDTA were not soluble in the base pharmaceutical composition, therefore they could not be used as protector added to the concentrated solution. The compound Lauryl gallate was soluble, however this presented an orange coloration after mixing. As this was suspected to represent some type of interaction or degradation, this compound was not tested further. The compounds BHA and BHT were soluble, and did not show an immediate colour-change, but a colour change did develop a few days into the incubation in sanitized water. Therefore only alpha-tocopherol was suitable as protector.

TABLE-US-00007 TABLE 5 Effect of incubation in sanitized medicated drinking water on Fluralaner content; no protector Hypochlorite concentration in medicated Conc. Fluralaner drinking water (ppm) (μg/ml); 0 0 0.5 2 5 5 (tapw.) 10 no protector T = 0 T = 7 d at 40° C./75% RH 0 — — — — — — n.d. 1 0.93 0.92 0.91 0.91 0.29 0.13 <LoQ 5 5.50 5.51 5.52 5.50 5.31 4.43 1.54 10 10.10 10.07 10.03 10.03 9.97 8.69 5.05 — = not done n.d. = not detectable LoQ = 50 ng/ml

TABLE-US-00008 TABLE 6 Effect of incubation in sanitized medicated drinking water on Fluralaner content; alpha- tocopherol as protector Hypochlorite concentration in medicated Conc. Fluralaner drinking water (ppm) (μg/ml); 0 0 0.5 2 5 5 (tapw.) 10 with α-tocopherol T = 0 T = 7 d at 40° C./75% RH 0 — — — — — — n.d. 1 0.91 0.89 0.91 0.92 0.89 0.86 0.05 5 5.52 5.45 5.52 5.52 5.49 5.42 5.56 10 10.14 10.01 10.10 10.10 9.99 9.94 10.05

[0299] From these results it was apparent that without any protector, Fluralaner becomes degradated in sanitized drinking water: after 7 days at 40° C., in 5 or 10 ppm hypochlorite, this significantly degradated the Fluralaner, especially when tested in tap water.

[0300] However, as is evident from Table 6, alpha-tocopherol could completely protect Fluralaner at 5 or 10 μg/ml from degradation in sanitized drinking water, even up to 7 days, even when at 40° C., and even with up to 10 ppm hypochlorite. Only for the lowest concentration Fluralaner and the highest concentration hypochlorite tested (1 μg/ml and 10 ppm), the alpha tocopherol could not protect. However this is not a problem, because in practice medicated water will not be used after 7 days, but rather within 1 day. Also, the concentration of Fluralaner in the sanitized medicated drinking water will usually be higher, namely above 5, 10, or even 15 μg/ml.

[0301] Again, the concentration of vitamin E used depended on the dilution factor of the concentrated Fluralaner composition applied.

Example 5: Variation of Concentration of Vitamin E

[0302] In a follow-up experiment, it was demonstrated that vitamin E, even at a reduced concentration can still effectively protect against degradation of isoxazoline by an oxidising sanitizer. While in Example 4.3 alpha-tocopherol was used in the pharmaceutical composition at 8.2% w/w, it was now tested at 80% of that value, namely at 6.6% w/w.

[0303] Pharmaceutical compositions according to the invention were prepared, containing either 8.2 or 6.6% w/w alpha-tocopherol. Also one base composition was prepared without protector. These were diluted 1:10.000 to prepare medicated drinking waters with 1 μg/ml Fluralaner. Next hypochlorite was added to 5 ppm, and 20 ml samples were filled-out in 50 ml glass vials, sealed with a rubber stopper, and stored for 7 days at 40° C./75% RH.

[0304] The amount of Fluralaner of the three samples was measured at t=0 and at t=7 days using UHPLC. The concentration of the vitamin E in the final test sample was 1:10,000 of 6.6 or 8.2% w/w.

TABLE-US-00009 TABLE 7 Effect of reduced amount of vitamin E Vitamin E conc. (% w/w in pharm. Fluralaner conc. (μg/ml) after incubation Reduction comp.) used at in 5 ppm ClO— at 40° C./75% RH, for t = 0 to 1:10.000 t = 0 d t = 7 d t = 7 in % 0 0.80 0.15 81 6.6 0.91 0.49 46 8.2 0.90 0.49 46

[0305] As can be seen from Table 7, the Fluralaner in dilutions without alpha-tocopherol was considerably degradated by 5 ppm hypochlorite after 7 days at 40° C.: the reduction in Fluralaner amount was 81%.

[0306] However, with alpha-tocopherol this reduction was almost halved, from 81% to 46%. Interestingly, this level of protection was obtained with sample prepared from pharmaceutical composition comprising 8.2% w/w vitamin E, but also with the sample from pharmaceutical composition comprising 6.6% w/w vitamin E. This indicates that both these amounts of vitamin E in a pharmaceutical composition according to the invention can provide an equally effective protection to Fluralaner when diluted in drinking water comprising an oxidizing sanitizer.

Example 6: Stability of Pharmaceutical Composition Comprising a Protector

[0307] Similar to the experiments described in Example 3 above, where the effect of water quality on Fluralaner-containing medicated drinking water was tested; here samples of the pharmaceutical composition were tested for an effect of the addition of a protector, on their stability during prolonged storage.

[0308] 6.1. Accelerated Stability Testing of Pharmaceutical Composition

[0309] To test the long term stability of the pharmaceutical composition with a protector, some samples were stored under conditions of accelerated ageing. Samples of the pharmaceutical composition with or without one of the protectors, were filled out in portions of 7 gram each, in 10 ml glass vials, sealed with rubber stoppers, and stored under different conditions: 2-8° C.; 30° C./65% RH; 40° C./75% RH, and 50° C. Samples were taken for analysis at t=0, 1, 2, 4, and 8 months; the Fluralaner level was determined using UHPLC; the decrease over time, as compared to the t=0 samples was investigated.

[0310] 6.2. Results of Accelerated Stability Testing of the Pharmaceutical Composition [0311] Placebo: No change was detected. [0312] Lauryl gallate: Shortly after preparation of the pharmaceutical composition, an orange coloration appeared, similar to what was apparent in sanitized medicated drinking water, described in Example 4. As this indicated some type of interaction or degradation, this protector was not used further. [0313] BHT and BHA: Stability results up to 4 months were obtained, when the tests were terminated because of the detection of a discoloration, similar to what was apparent in sanitized medicated drinking water tests. This is not acceptable for the commercial product, therefore these were not used further. [0314] Alpha-tocopherol: Stability was tested up to 8 months: no significant reduction of the level of Fluralaner, or of the level of alpha-tocopherol was observed, under any of the storage conditions. Currently stability data up to 18 months are available, and these show the same positive results of a lack of interaction by alpha-tocopherol in the pharmaceutical composition according to the invention.

Example 7: Examples of Dosing Calculations for Preparation of Medicated Drinking Water

[0315] Some examples are provided for the calculation of ways to prepare a medicated drinking water according to the invention, for administration to e.g. a group of laying hen chickens, either by way of a dosing pump, or by way of a medication tank.

[0316] 7.1. Use of a Dosing Pump:

Exemplary Starting Values:

[0317] Number of chickens to be treated: 3500 (when not known exactly, best guess the number) [0318] Average weight per hen: 1.7 kg (measured shortly before administration, on a number of animals that allows a significant measurement) [0319] Concentration of Fluralaner in pharmaceutical composition: 10 mg/ml [0320] Total Fluralaner dose to be administered this treatment period: 0.5 mg/kg BW [0321] Average water consumption over 4 hr treatment period: 200 litres (measured shortly before treatment) [0322] Dosing pump injection rate: 5% [0323] Concentration of vitamin E in pharmaceutical composition: 8.2% w/w

Calculated Values:

[0324] Total BW to be treated: 3500×1.7 kg=5950 kg [0325] Total amount of Fluralaner required: 5950 kg×0.5 mg/kg=2975 mg [0326] Total amount of pharmaceutical composition required for making the dilution: 2975 mg: 10 mg/ml=297.5 ml [0327] Volume of stock solution required for dosing pump: 5% of 200 l=10 litres [0328] Preparation of 10 l dosing pump stock solution: 9.7025 l water+297.5 ml pharmaceutical composition (mix adequately) [0329] Concentration of Fluralaner in stock solution for dosing pump: 2975 mg in 10 l=297.5 mg/I [0330] Concentration of Fluralaner in final medicated drinking water: 2975 mg in 200 l=14.88 mg/I [0331] Dilution factor of pharmaceutical composition in final medicated drinking water: 297.5 ml in 200 l=1:672 [0332] Amount of vitamin E in final medicated drinking water: 1:672 of 8.2% w/w=0.0122% w/w=122 μg/g

[0333] 7.2. Use of a Medication Tank

Exemplary Starting Values:

[0334] Number of chickens to be treated: 3500 (when not known exactly, best guess the number) [0335] Average weight per hen: 1.7 kg (measured shortly before administration, on a number of animals that allows a significant measurement) [0336] Concentration of Fluralaner in pharmaceutical composition: 10 mg/ml [0337] Total Fluralaner dose to be administered this treatment period: 0.5 mg/kg BW [0338] Average water consumption over 4 hr treatment period: 200 litres (measured shortly before treatment) [0339] Concentration of vitamin E in pharmaceutical composition: 8.2% w/w [0340] Medication tank volume to be used: 175 litres

Calculated Values:

[0341] Total BW to be treated: 3500×1.7 kg=5950 kg [0342] Total amount of Fluralaner required: 5950 kg×0.5 mg/kg=2975 mg [0343] Total amount of pharmaceutical composition required for making the dilution: 2975 mg: 10 mg/ml=297.5 ml [0344] Preparation of medication tank water: 174.7 l water with 297.5 ml of pharmaceutical composition (mix adequately) [0345] Concentration of Fluralaner in medication tank water: 2975 mg in 175 l=17 mg/I [0346] Concentration of Fluralaner in final medicated drinking water: same as medication tank water [0347] Dilution factor of pharmaceutical composition in final medicated drinking water: 297.5 ml in 175 l=1:588 [0348] Amount of vitamin E in final medicated drinking water: 1:588 of 8.2% w/w=0.0139% w/w=139 μg/g