AU2003236560A1

AU2003236560A1 - Worming formulation

Info

Publication number: AU2003236560A1
Application number: AU2003236560A
Authority: AU
Inventors: Paul John Martin
Original assignee: Virbac SA
Current assignee: Virbac SA
Priority date: 2002-06-26
Filing date: 2003-06-26
Publication date: 2004-01-19
Also published as: NZ537760A; AUPS323002A0; WO2004002537A1

Description

WO 2004/002537 PCT/AU2003/000830 1 WORMING FORMULATION The present invention relates to a veterinary formulation to effect broad spectrum worming in mammals and in particular to a synergistic 5 combination of a macrocyclic lactone and piperonyl butoxide. For many years it has been appreciated that macrocyclic lactones endectocides constitute reasonably effective antiparasitic formulations for a number of species including companion animals such as horses, cats and dogs and food animals such as sheep, cattle, pigs and goats. These 10 compounds are also known to combat parasites in humans. The dual activity of this class of compounds against endoparasites (anthelmintic) and ectoparasites (acaricide and insecticide) gave rise to the term endectocide. Macrocyclic lactones endectocides include two closely related groups of molecules: avermectins (e.g., ivennectin, 15 abamectin, and eprinomectin) and milbemycins (e.g., milbemycin oxime and moxidectin). Such compounds are broad spectrum in the sense that they are active for example against large strongyles, small strongyles (cyathostomes), pinworms, roundworms (ascarids), hairwonrs, large mouth stomach worms, neck threadwonns, heartworms, bots, lungworms 20 and intestinal threadworms. Relatively quickly over time however macrocyclic lactones in existing formulations reach peak concentration levels in the animal's blood and are then metabolised by the treated animals and cease to have activity against the target parasites. Relatively heavy dosage rates quickly WO 2004/002537 PCT/AU2003/000830 2 metabolise so that within one week of oral administration plasma sample analysis often reveals ineffective concentrations. There is a need, therefore, for a macrocyclic lactone based worming formulation for mammals which facilitates elevated plasma 5 concentrations of the macrocyclic lactone in the treated mammal. Piperonyl butoxide, (The Merck Index, thirteenth Edition, Abstract 7557), is known to inhibit microsomal enzymes of insects and to extend, therefore, the activity of several insecticide preparations, especially the formulations which contain pyrethroids and rotenone. 10 The European patent EP 0 125 155 described the use of piperonyl butoxide as an insect metabolism inhibitor, in agricultural insecticidal, pesticidal and acaricidal combinations, together with certain naturally occuring avermectin product compounds or certain semi-synthetic derivatives thereof. The pesticidal formulations are used for controlling 15 agricultural insect and mite pests of stored grain and agricultural plants in crops. The combination is useful in that the avennectin component has a long term efficacy at very low doses and the piperonyl butoxide has very rapid effects. It has now surprisingly been found that a macrocyclic lactone compound 20 exhibits an unexpected efficacy against helminthes parasites in mammals when administered simultaneously with or when formulated with piperonyl butoxide. We have found that this increased efficacy coincides with a more elevated plasma concentration of the macrocyclic lactone in WO 2004/002537 PCT/AU2003/000830 3 the treated animal when the macrocyclic lactone is administered together with piperonyl butoxide. It is consequently an object of the present invention to provide a wonring formulation which achieves longer lasting elevated plasma 5 concentrations of macrocyclic lactones in mammals or at least provides the market with an alternative to existing formulations. In one aspect, the invention provides a broad spectrum mammal worming formulation comprising an effective amount of a macrocyclic lactone, together with an amount of piperonyl butoxide as a synergist. 10 The term "worming formulation" as used herein means a formulation or a composition or a preparation for the prevention or the treatment of anthelmintic infestations in mammals. In another aspect, the invention provides a method of prevention or treatment of anthelnintic infections in mammals consisting in 15 administering substantially simultaneously to said mammals one macrocyclic lactone and piperonyl butoxide. The macrocyclic lactone and the piperonyl butoxide are administered either in the same composition or in two separate compositions which may be in different forms. 20 In still another aspect, the invention discloses the use of a formulation comprising a macrocyclic lactone, together with piperonyl butoxide in the manufacture of a medicament for the prevention or treatment of anthelmintic infections in mammals.

WO 2004/002537 PCT/AU2003/000830 4 Macrocyclic lactone compounds include, for example, the avennectins and the milbemycins, or derivatives thereof, such as ivermectin, abamectin, eprinomectin, doramectin, milbemycin oxime, moxidectin. In a preferred embodiment, the macrocyclic lactone is abamectin. In another 5 preferred embodiment, the macrocyclic lactone is ivermeetin. In still another embodiment, the macrocyclic lactone is milbemycin oxime. The present invention is suitable for treating the hehninthes which occur in productive or breeding animals and pets. Illustrative but non-limiting examples of the animals are equines, bovines, felines, canines, swines, 10 ovines. In a preferred embodiment, the worming preparation according to the present invention is administered to equidae and the invention provides a method of prevention or treatment of anthelmintic infections in equidae consisting in administering simultaneously to said mammals one macrocyclic lactone and piperonyl butoxide. In another preferred 15 embodiment, the wonning preparation is administered to dogs and the invention provides a method of prevention or treatment of anthelmintic infections in dogs consisting in administering simultaneously to said mammals one macrocyclic lactone and piperonyl butoxide. In still another preferred embodiment, the worming preparation is administered 20 to sheep and the invention provides a method of prevention or treatment of anthelmintic infections in sheep consisting in administering simultaneously to said mammals one macrocyclic lactone and piperonyl butoxide.

WO 2004/002537 PCT/AU2003/000830 5 Administration can be effected prophylactically as well as therapeutically. In a preferred embodiment, the invention provides a method of prevention or treatment of anthelmintic infections in mammals consisting 5 in administering simultaneously to said mammals abamectin and piperonyl butoxide. In another preferred embodiment, the invention provides a method of prevention or treatment of anthelmintic infections in mammals consisting in administering simultaneously to said mammals ivermectin and piperonyl butoxide. In still another embodiment, the 10 invention provides a method of prevention or treatment of anthelmintic infections in mammals consisting in administering simultaneously to said mammals milbemycin oxime and piperonyl butoxide. The active compounds are formulated and administered in the form of suitable preparations, enterally, parenterally, transdermally, nasally, or 15 with the aid of articles such as, for example, strips, plates, bands, collars, ear marks, limb bands, marking devices including any controlled release devices. The active compounds are administered enterally, for example orally, in the form of oral pastes, powders, tablets, capsules, drinks, granules, or 20 solutions, suspensions and emulsions which can be administered orally, or as a bolus, medicated feed or drinking water. Oral pastes are particularly suitable forms for the administration of the formulations according to the invention to horses. Drenches are well suited for the administration of the formulations according to the invention to sheep.

WO 2004/002537 PCT/AU2003/000830 6 Dermal administration is effected, for example, in the form of dipping, spraying or pouring-on and spotting-on. Parenteral administration is effected, for example, in the form of an injection (intramuscular, subcutaneous, intravenous, intraperitoneal) or by implants. Injectable 5 compositions are suitable for the administration of the formulation according to the invention to dogs. Suitable solvents, carriers, adjuvants and/or excipients depend of the chosen formulation and are well known by the skilled persons. Optionally, the formulation may contain at least one other parasiticidal 10 compound, including, but not limited to, anthelmintics, such as beonzimidazoles, piperazine, levamisole, pyrantel, praziquantel and the like; endectocides such as avermectins, milbemycins and the like; ectoparasiticides such as arylpyrroles including chlorfenapyr, organophosphates, carbamates, gainabutyric acid inhibitors including 15 fipronil, pyrethroids, spinosads, imidacloprid and the like; insect growth regulators such as pyriproxyfen, cyromazine and the like; and chitin synthase inhibitors such as benzoylureas including flufenoxuron, in order for example to further extend the spectrum of activity against other parasite species. In a preferred embodiment, the formulation according to 20 the invention contains also an effective amount of praziquantel, a compound active against tapeworms. The addition of other anthelmintic agents such as pyrantel, oxfendazole or the like would also provide a means of 'redundant' killing which is WO 2004/002537 PCT/AU2003/000830 7 known to limit the onset of resistance to a single action anthelmintic compound. The preferred range of concentration for the macrocyclic lactone is such that the effective dose is from 0.02 to 2 mg per kg (mg/kg) of animal 5 body weight. More preferably the dose is from 0.1 to 1.0 mg/kg of animal body weight. The preferred dose range of piperonyl butoxide is from 0.2 - 20 mg/kg of animal body weight, preferably from 1 to 10 mg/kg of animal body weight and more preferably from 1 to 5 mg/kg of animal body weight. 10 The worming formulation of the present invention has been found to achieve elevated plasma concentrations of the macrocyclic lactone in the treated animal over time. The fonnulation according to the invention, has, therefore, the advantage of extending the activity of the macrocyclic lactone compound in the treated mammal. It prolongs the duration of the 15 activity of the macrocyclic lactone and facilitates formulation of long acting veterinary compositions for the prevention or the treatment of anthelmintic infestations in mammals. It may also enable the use of reduced amounts of macrocyclic lactones, as active ingredients, while still providing effective anthelmintic prevention -or control. The reduction 20 in the amount of chemical agents introduced into the environment is an additional advantageous element of the subject invention. Specific examples of formulations in accordance with the present invention will be described hereafter without limiting the generality of the invention as above described.

WO 2004/002537 PCT/AU2003/000830 8 Example 1: Compound Quantity g/L Purpose Macrocyclic Lactone* 6 Active Ingredient Piperonyl Butoxide 30 Synergist Praziquantel 45 Active Ingredient Pyrantel Embonate 390 Active Ingredient Excipient** Qs to 1 litre Vehicle * The Macrocyclic Lactone can consist of abamectin, ivenectin, moxidectin, doramectin, eprinomectin or other members of the class of avermectin or milbemycin. 5 ** The excipient base will consist of aqueous or organic solvent, preservative system, thickners, fillers, flavours colours or the like. Example 2: Injectable formulations for cattle, sheep, cats or dogs. Compound Quantity g/L Purpose Macrocyclic Lactone* 6 Active Ingredient Piperonyl Butoxide 25-175 Synergist Thixcin R 1 Thickener Sesame Oil 400 Vehicle/solvent Myvacet 9-45 K Qs to 1 litre Vehicle/co-solvent * The macrocylclic lactone can consist of abamectin, ivennectin, 10 moxidectin, doramectin, eprinomectin or other members of the class of avermectin or milbemycin. Example 3: Liquid oral formulation for sheep, goats, cattle, dogs and cats. Compound Quantity g/L Purpose WO 2004/002537 PCT/AU2003/000830 9 Macrocyclic Lactone 0.5-5.0 Active Ingredient Piperonyl Butoxide 2.5-25.0 Synergist Other active ingredient Optional Active ingredient Excipients Qs to 1 litre Vehicle/solvent/ stabilisers/thickner /etc. Example 4: Tablet oral formulation for sheep, goats, dogs and cats. Compound Quantity g/L Purpose Macrocyclic Lactone 0.1-1.0 Active Ingredient Piperonyl Butoxide 0.5-5.0 Synergist Other active ingredient Optional Active ingredient Excipients Qs to 1 litre Solvent/fillers/ stabilisers/etc.. 5 Based upon the theory that the inclusion of piperonyl butoxide may increase the bioavailability of the macrocyclic lactone by inhibiting the hepatic metabolism of these compounds in the treated manmnal, studies have been undertaken to evaluate the pharmacokinetic behaviour of a macrocyclic lactone based oral paste formulation in horses both with and 10 without the addition of piperonyl butoxide. The observed dual result is an elevation in the peak plasma concentration (Cmax) of the macrocyclic lactone and an increase in the area under the plasma curve (AUC) over time.

WO 2004/002537 PCT/AU2003/000830 10 The increase in Cmax and AUC lead to a greater efficiency of the anthelmintic paste and allow lower doses to be administered and still be effective in removing parasites. Trial 1: 5 An existing oral paste formulation for control of worms in horses branded "Equimax" substantially comprising praziquantel and abamectin as actives was administered to six horses in the prescribed dosage. Six similar horses were dosed with the same Equimax formulation at the same dosage rate but in addition were treated with 10mg per kilogram of 10 animal body weight of piperonyl butoxide. The standard Equimax product was ascertained to contain the abamectin active at 3.7 g per litre (g/1) and praziquantel at 46.2 g/l and the dosage rate of the formulation was 1 ml per 20kg of animal body weight. The piperonyl butoxide formulation contained piperonyl butoxide at a 15 concentration of 500g/l and was administered at a rate of 2 ml per 100 kg of animal body weight. The animals selected were horses being female thoroughbred or thoroughbred cross horses having a bodyweight between 320 and 465 kg and having no history of treatment with any macrocyclic lactone 20 compounds within two months prior to the test. The horses were adults between the ages of 3 and 10 years. All trial horses were grazed as a single group on a mix of native and improved pastures including rye grass, phalaris, fescue and white clover WO 2004/002537 PCT/AU2003/000830 11 in open grazing paddocks with constant access to water from dams and/or concrete troughs. The trial horses were weighed on Day-5 and ranked in order of decreasing body weight and sequentially blocked into pairs. Horses were 5 then randomly allocated from within each pair to the two treatment groups using numbered plastic tags randomly drawn from a plastic container. Allocation was such that each group had a similar group mean body weight and range of body weights within the group. The group mean body weight of the six horses treated with a formulation in 10 accordance with the present invention was 376.5 kg whereas the group mean bodyweight of the group treated with standard Equimax product was 375,8 kg. Duplicate 9 ml heparinized blood samples were taken from all horses prior to treatment and at 2, 4, 6, 8 and 12 hours post treatment and 15 plasma harvested by high speed centrifugation and stored frozen on the day of administration. The same sampling and harvesting method was repeated at 24 and 36 hours post treatment as well as at 48, 60, 72, 84, 96, 120, 144 and 168 hours post treatment. 20 The Group 1 horses comprising horses numbered 2, 3, 17, 78, 198 and 305 were administered with the Equimax and piperonyl butoxide formulations whereas the Group 2 horses were treated with standard Equimax. Group 2 comprised horses numbered 1, 4, 5, 109, 188 and 211.

WO 2004/002537 PCT/AU2003/000830 12 The concentration of the active abamectin in the plasma samples at the abovementioned plasma sampling intervals is set out in tables 1 and 2 hereafter with table 1 being referable to Group 1 horses and table 2 referable to the Group 2 horses. Tables 1 and 2 show not only the 5 concentration of abamectin in the plasma sample at the time of the particular sample but also the area under the curve generated by plotting the abamectin concentrations over the sampling times. Plasma samples for all horses were analysed for their plasma abamectin levels by high performance liquid chromatography (HPLC) following 10 extraction with ethyl acetate. The limit of detection with the analytical methodology used was 0,05 mg/mL and the limit of quantitation was 0.39 mg/mL. Figure 1 appearing hereafter shows the group mean plasma concentrations in nanograms per millilitre of plasma for both Group 1 15 and Group 2 over the 168 hours of sampling.

WO 2004/002537 PCT/AU2003/000830 13 VA1208/TDB 5210 35.00 j 30.00 25.00 20.00X -+-Aba+ PBO S 1 0 --. Equimax E 10.00 S5.00 0.00 0 50 100 150 200 Time (h) Figure 1: Abamectin Plasma-time response curves showing the AUC and Cmax for Equimax (Group 2) and Abamectin plus piperonyl butoxide (group 1) 5 Trial horses were observed for adverse reactions to either treatment for one hour post treatment and at each sampling point post treatment. No adverse reactions to treatment were noted. From tables 1 and 2 appearing hereafter as well as figure 1 it will be 10 observed that the Group 1 horses having been treated in accordance with the present invention exhibit plasma concentrations of the abamectin active significantly higher than those horses treated with Equimax alone and indeed the concentrations are approximately 100% higher at most sampling time intervals resulting in the area under the curves of Group 15 mean plasma abamectin levels over time being of a similar superior order. of magnitude as depicted in Figure 1.

WO 2004/002537 PCT/AU2003/000830 14 Table 1: Concentration of abamectin from horses treated with abamectin plus piperonyl butoxide (PBO) 2 3 17 78 198 305 Time (h) Cone Cone Cone Cone Conc Cono (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) 0.0 0.00 0.00 0.00 0.00 0.00 0.00 2.0 0.56 19.03 44.69 33.63 10.82 26.67 4.0 2.06 33.04 55.28 29.85 23.29 53.06 6.0 2.63 24.95 45.17 19.11 34.59 60.20 8.0 1.62 29.13 49.96 23.12 33.58 59.55 12.0 4.32 18.54 36.69 11.91 31.47 57.00 24.0 32.86 9.06 21.13 5.95 22.74 42.97 36.0 26.66 3.24 15.73 3.77 14.01 34.00 48.0 15.15 1.21 11.29 2.11 8.67 23.93 60.0 10.33 0.56 8.80 1.48 8.44 17.22 72.0 1.15 0.28 6.74 0.99 3.26 12.37 84.0 3.64 0.16 5.34 0.62 2.09 9.29 96.0 2.16 0.14 4.49 0.51 1.36 6.67 120.0 0.82 0.00 2.66 0.32 0.98 3.34 144.0 0.32 0.00 1.88 0.23 0.42 2.36 168.0 0.18 0.00 1.37 0.19 0.17 1.26 5 Table 2: Concentration of abamectin in horses treated with Equimax. G OU P 2 Equimax 1 4 5 109 188 211 Time (h) Cone Cone Cone Cone Cone Conc (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) 0.00 0.00 0.00 0.00 0.00 0.00 0,00 2.0 3.49 12.14 23.28 24.81 8.47 1,28 4.0 9.84 16.44 20.29 20.58 21.16 4.60 6.0 14.88 14.33 13.82 14.71 27.16 17.84 8.0 14.89 15.87 17.59 17.50 29.26 7.10 12.0 14.98 21.01 9.78 11.66 23.42 23.46 24,0 12.66 10.54 6.18 8.65 -13.63 12.15 36.0 7.93 5.86 4.23 7.19 8.10 8.21 48.0 4.67 3.22 3.08 5.74 5.50 5.21 60.0 2.33 2.32 2.34 4.43 3.75 3.10 72.0 6.12 1.20 1.76 3.45 1.99 1.89 84.0 0.55 0.85 1.28 2.53 1.25 1.06 96.0 0.36 0.52 1.06 2.20 0.72 0.78 120.0 0.00 0.25 0.74 1.40 0.37 0.30 144.0 0.00 0.13 0.58 0.94 0.13 0.14 168.0 0.00 0.00 0.40 0.80 0.07 0.07 WO 2004/002537 PCT/AU2003/000830 15 Trial 2: In another study (VA073/TDB3360), groups of horses were treated with either Equimax as described earlier or with a liquid formulation 5 containing abamectin and piperonyl butoxide as tabulated below. Table 3: Groups and treatments in the study VA073/TDB3360 Group Treatment Abamectin Piperonyl Praziquantel Number Number dose pgg butoxide mg&g of mgkg animals 1 Untreated 0 0 0 4 2 Equimax |200 0 2.5 4 3 Abamectin 200 10 0 4 + pip but 10 Each selected horse was weighed and treated in accordance with a dosage rate tied to the individual animal's body weight. Treatments were administered orally. Throughout the study, animals were managed similarly to those described above. Thirteen or fourteen days after treatment the animals were slaughtered 15 for recovery of any parasitic worms in the gastrointestinal tract to evaluate the efficacy of the chosen formulation. Before slaughter, blood samples were taken for later evaluation of the plasma abamectin concentrations over time. The results of the percentage efficiency are tabulated below.

WO 2004/002537 PCT/AU2003/000830 16 Table 4: Percentage efficiency based on the estimated number of worms recovered from the lumen of the large intestine. Group Treatment Number of Percentage Number small efficiency cyathostomes 1 Untreated 51,500 2 Equimax 4,000 92 3 Abamectin 0 100 + pip but 5 Table 5: Percentage efficiency based on the estimated number of worns recovered from the wall of large intestine following pepsin digestion. Group Treatment Number of Percentage Number small efficiency cyathostomes* 1 Untreated 29.8 2 Equimax 0.46 98 3 Abamectin 0.60 98 + pip but * Estimated by weighing total organ and subsampling a unit weight for digestion and counting. 10 Plasma samples were analysed for the concentration of abamectin similar to those described above. The results for AUC and Cmax are tabulated below. 15 Table 6: Area under the curve (AUC) and maximum concentration (Cmax) Group Treatment Mean AUC Cmax Number ngmL(br Ag/mL 2 Equimax 346 14.2 3 Abamectin 953 40.2 + pip but The abamectin plasma-time response curves are illustrated below. 20 WO 2004/002537 PCT/AU2003/000830 17 Aba Synergy VA073/TDB3360 45 30 P 25 E 15 10 5 0 0 20 40 60 80 100 120 Time (h) Figure 2: Abamectin Plasma-time response curves showing the AUC and Cmax for Equimax (Group 2) and Abamectin plus piperonyl butoxide (Group 3) 5 From Table 6 and figure 2, it will be observed that the mean of the Group treated with abamectin plus piperonyl butoxide had a higher mean AUC and Cmax than the group treated with Equimax alone. Statistical analysis was performed on the data and it revealed a statistically significant difference between these two groups with respect 10 to both AUC and Cmax. Trial 3 : This study aims to define the pharmacokinetic behaviour of the ivermectin component of an oral paste formulation containing different concentrations of piperonyl butoxide. This trials investigated oral paste 15 formulations containing ivennectin, with varying quantities of piperonyl WO 2004/002537 PCT/AU2003/000830 18 butoxide in order to deliver to the animals respectively 0, 1 and 5 mg/kg of animal bodyweight. This study comprises three groups of horses containing each six horses, which were treated with one of the three oral paste fonnulation. 5 Allocation was such that each group had a similar group mean body weight and range of body weights within the group. Each selected horse was weighed and treated in accordance with a dosage rate tied to the individual animal's body weight. Treatments were administered orally. Throughout the study, animals were managed 10 similarly to those described in the trial 1. Moreover, We use the sampling and harvesting method described in the trial 1. The group B horses were administered with the oral paste formulation delivering 0.2 mg/kg of ivermectin and 5 mg/kg of piperonyl butoxide. The group C horses were administered with the oral paste formulation 15 delivering 0.2 mg/kg of ivermectin and 1 mg/kg of piperonyl butoxide. Finally, the group D horses were administered with the oral paste formulation delivering 0.2 mg/kg of ivernectin but no piperonyl butoxide. The mean maximum concentration of the active ivennectin (Cmax) for 20 each groups is set out in table 7. This table show not only the mean maximum concentration of ivernectin but also the mean area under the curve (AUC) generated by plotting the ivermectin concentrations over the sampling times.

WO 2004/002537 PCT/AU2003/000830 19 Table 7 : Mean maximum concentration of the ivermectin and mean AUC for each group of horses. Group of Dose of PBO Peak Time AUC n horses (mg/kg) B 5 54.46 6.33 3806 6 C 1 63.08 5.33 3898 6 D 0 49.11 8.33 3216 6 Notes : Time represents time to reach peak plasma concentration (hours) 5 Peak represents the peak plasma concentration in ng/mL A UC represents area under the plot of concentration versus time using the linear trapezoidal approximation (ng/mL/h) Trial horses were observed for adverse reactions to either treatment for 10 one hour post treatment and at each sampling point post treatment. No adverse reactions to treatment were noted. From table 7, it will be observed that the group B and C horses having been treated with a formulation in accordance with the present invention containing piperonyl butoxide exhibit mean AUC and mean Cmax higher 15 than the group D horses treated with ivermectin alone.

Claims

1. A veterinary worming formulation comprising an effective amount of a macrocyclic lactone, together with an amount of piperonyl butoxide. 5

2. A veterinary worming formulation according to claim 1 wherein the macrocyclic lactone active is present at a concentration such that it may be administered to an animal at a dose of from 0.02 to 2 mg per kg of animal body weight.

3. A veterinary worming formulation according to claim 1 wherein the 10 macrocyclic lactone active is present at a concentration such that it may be administered to an animal at a dose of from 0.1 to 1.0 mg per kg of animal body weight.

4. A veterinary worming formulation according to claim 1 wherein the macrocyclic lactone is ivermectin or abamectin or milbemycin oxime. 15

5. A veterinary worming formulation according to claim 1 wherein the piperonyl butoxide dosage is from 0.2 to 20 mg per kg of animal body weight.

6. A veterinary worming formulation according to claim 1 wherein the piperonyl butoxide dosage is from 1 to 10 mg per kg of animal body 20 weight.

7. A veterinary worming formulation according to claim 1 wherein the piperonyl butoxide dosage is from 1 to 5 mg per kg of animal body weight. WO 2004/002537 PCT/AU2003/000830 21

8. A veterinary worming formulation according to claim 1 characterized in that said veterinary worming formulation is for oral use.

9. A method for preventing or treating anthelnintic infections in mammals, comprising administering substantially simultaneously to 5 said mammals, a macrocyclic lactone and piperonyl butoxide.

10. A method according to claim 9 wherein the macrocyclic lactone and the piperonyl butoxide are in the same composition.

11. A method according to claim 9 wherein the macrocyclic lactone and the piperonyl butoxide are in separate compositions. 10

12. A method according to claim 9 wherein the macrocyclic lactone is administered at a dose of 0.02 to 2 mg per kg of animal body weight.

13. A method according to claim 9 wherein the macrocyclic lactone is administered at a dose of 0.1 to 1.0 mg per kg of animal body weight.

14. A method according to claim 9 wherein the macrocyclic lactone is 15 selected from the* avermectins and milbemyciis or derivatives thereof.

15. A method according to claim 9 wherein the macrocyclic lactone is ivermectin or abamectin or milbemycin oxime.

16. A method according to claim 9 wherein the piperonyl butoxide is 20 administered at a dose of 0.2 to 20 mg per kg of animal body weight.

17. A method according to claim 9 wherein the piperonyl butoxide is administered at a dose of 1 to 10 mg per kg of animal body weight.

18. A method according to claim 9 wherein the piperonyl butoxide is administered at a dose of 1 to 5 mg per kg of animal body weight. WO 2004/002537 PCT/AU2003/000830 22

19. A method according to claim 9 wherein the macrocyclic lactone and piperonyl butoxide are administered orally.

20. A method according to claim 9 wherein the macrocyclic lactone and piperonyl butoxide are administered in the form of an oral paste to 5 equine animals.

21. A method according to claim 9 wherein the macrocyclic lactone and piperonyl butoxide are administered in the fonn of a drench to sheep.

22. A method according to claim 9 wherein the macrocyclic lactone and piperonyl butoxide are administered parenteraly. 10

23. A method according to claim 9, wherein the macrocyclic lactone and piperonyl butoxide are administered in the form of an injectable composition to dogs.

24. The use of a formulation according to claim 1, in the manufacture of a medicament for the prevention or treatment of anthelmintic 15 infections in mammals.

25. The use of a formulation according to claim 24 in the manufacture of an oral paste for equine animals suitable to administered to said equine animal a dose of 0.1 to 10 mg per kg of animal body weight of the macrocyclic lactone and a dose of 1 to 5 mg per kg of animal 20 body weight of piperonyl butoxide.

26. The use of a formulation according to claim 24 in the manufacture of an injectable composition for dogs suitable to administered to said dogs a dose of 0.1 to 10 mg per kg of animal body weight of the WO 2004/002537 PCT/AU2003/000830 23 macrocyclic lactone and a dose of 1 to 5 mg per kg of animal body weight of piperonyl butoxide.

27. The use of a formulation according to claim 24 in the manufacture of an oral drench for sheep suitable to administered to said sheep a dose 5 of 0. 1 to 10 mg per kg of animal body weight of the macrocyclic lactone and a dose of 1 to 5 mg per kg of animal body weight of piperonyl butoxide. 10 15 20