AU2004210461B2

AU2004210461B2 - Anthelmintic composition

Info

Publication number: AU2004210461B2
Application number: AU2004210461A
Authority: AU
Inventors: Brian Desmond Ford; Marcus Holdsworth; Kai Kin Lau; John James O'brien; Edward Lionel Bruce Whittem
Original assignee: Jurox Pty Ltd
Current assignee: Jurox Pty Ltd
Priority date: 2003-02-05
Filing date: 2004-02-04
Publication date: 2009-12-03
Anticipated expiration: 2024-02-04
Also published as: AU2009201942C1; AU2009201942A1; GB2413764B; GB2413764A8; GB2413764A; AR043044A1; AU2009201942B2; BRPI0407271B1; GB2413764B8; WO2004069242A1; BRPI0407271A; AU2004210461C1; NZ540391A; AU2003900505A0; GB0516881D0; AU2004210461A1

Description

WO 2004/069242 PCT/AU2004/000126 ANTHELMINTIC COMPOSITION Field of the Invention 5 This invention relates to the treatment of anthelmintic infections in animals, and more particularly to compositions that are effective against parasites that are resistant to a wide variety of drug treatments, particularly in non-human animals.. Background to the Invention 10 Farm animals such as lambs, weaners and sheep may typically be infected by a wide variety of parasites. Such parasites include Haenonconchus spp., Ostertagia spp., Trichostrongylus spp., Cooperia spp., Nematodirus spp., Chabertia spp., Oesophagostomum spp., Trichuris spp., Strongyloides spp., Bunostomum spp., Oestrus 15 spp., Dictyocaulus spp., Fasciola spp. and Monezia spp. Specific examples of these parasites are set out in Table 1. For a variety of reasons, there is an increasing number of such parasites that have developed resistance to available drug treatments. Moreover, because of the infective 20 nature and ready transmission from animal to animal, the presence of resistant parasites will rapidly spread to infect a substantial number, if not all, of the animals in a flock or herd. One means by which such infection will rapidly spread is where new animals in which the presence of drug resistance is known or suspected are to be introduced onto a property. 25 There are a variety of drug substances that are used to treat parasitic infections. Amongst these broad groups of substances are macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Unfortunately, many of the parasites mentioned in Table 1 have developed resistance to these substances. 30 Although the problem of resistance has been tackled through the development of new substances, the time to develop, evaluate and demonstrate efficacy of such substances is substantial and expensive. Moreover for the reasons that resistance has developed against existing substances it is very likely that resistance will occur in relation to these 35 new substances.

2 Summary of the Invention Rather then tackling the problem of resistance through the development of new 5 substances, the present inventors have found that it is possible to circumvent resistance by combining specific classes of anthelmintics. The efficacy of this combination arises out of the finding that the combination is synergistic. Accordingly, the present invention provides in a first aspect, a synergistic 10 anthelmintically effective composition comprising at least four anthelmintic compounds and a therapeutically acceptable carrier, wherein at least one anthelmintic compound is selected from the group of macrocyclic lactones, at least one anthelmintic compound is selected from the group of benzimidazoles, one anthelmintic compound is closantel, and at least one anthelmintic compound is selected from the group of 15 imidazothiazoles. In a further aspect there is provided a method of treating parasitic infections in an animal comprising administering to the animal, a synergistic anthelmintically effective amount of a composition comprising at least four anthelmintic compounds and a 20 therapeautically acceptable carrier, wherein at least one anthelmintic compound is selected from the group of macrocyclic lactones, at least one anthelmintic compound is selected from the group of benzimidazoles, one anthelmintic compound is closantel, and at least one anthelmintic compound is selected from the group of imidazothiazoles. 25 In a further aspect there is provided the use of a synergistic anthelmintically effective amount of a composition comprising at least four anthelmintic compounds and a therapeautically acceptable carrier, wherein at least one anthelmintic compound is selected from the group of macrocyclic lactones, at least one anthelmintic compound is selected from the group of benzimidazoles, one anthelmintic compound is closantel, 30 and at least one anthelmintic compound is selected from the group of imidazothiazoles, in the treatment of a parasitic infection in an animal.

3 Table 1 Parasite Species Species Common Name Comments Haemonchus contortus Barber's pole worm includes inhibited L4 stage Haemonchus placei Large stomach worm Ostertagia circumcincta Small brown stomach includes inhibited L4 stage worm Trichostrongylus axei Stomach hair worm Trichostrongylus colubriformis Trichostrongyus vitrinus Black scour worm Cooperia curticel Cooperia oncophera Small intestinal worm Nematodirus spathiger Nematodirusfilicollis Thin-necked intestinal worm Chabertia ovina Large mouthed bowel worm Oesophagostomum Nodule worm columbianum Oesophagostomum Large bowel worm venulosum Trichuris ovis Whip worm Strongyloides papillosus Intestinal threadworm Bunostomum spp Hookworm Oestrus ovis Dictyocaulus viviparus Large lungworm Fasciola hepatica Monezia Includes head and segments In a further aspect, the present invention provides a method for treating parasitic infections in an animal, comprising administering to the animal, a synergistic anthelmintically effective amount of a composition which consists of at least one 4 compound selected from each of the following groups: macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles and a therapeutically acceptable carrier. 5 In a further aspect, the present invention further provides the use of a synergistic anthelmintically effective amount of a composition which consists of at least one compound selected from each of the following groups: macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles and a therapeutically acceptable carrier in the treatment of a parasitic infection in an animal. 10 Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps, 15 The aforementioned treatments may be desirably administered to animals prior to introduction to a land area so as to prevent the land area from becoming infested with parasites which may or may not be resistant to one or more compounds selected from the groups consisting of macrocylic lactones, benzimidazoles, salicylanilides and 20 imidazothiazoles. Typically, animals such as sheep, will be isolated for at least 2 days after treatment before being placed on pasture. Alternatively, animals may be treated at any time, as appropriate, particularly when it is suspected that the animal may be carrying at least one parasite which is resistant to at 25 least one of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. The compositions of this invention have application where the parasites are resistant to known drug treatments. In particular, the compositions are effective in situations 30 where parasites are resistant to at least one of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Preferably, the compositions are effective in situations where parasites are resistant to at least two of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. More preferably, the compositions are effective in situations where parasites are resistant to 35 at least three of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Most preferably, the compositions are effective in situations 5 where parasites are resistant to all of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. In use, a preferred indication is the treatment of stock to eliminate adult gastro 5 intestinal worms and liver fluke. Typically, treatment results in the clearance of >95% of total worm count including worms resistant to at least one of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Compositions of this invention include at least one compound selected from each of the 10 groups: macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. Representative examples of compounds from each of these group are set out in Table 2. Table 2 Compounds Macrocyclic lactone Benzimidazole Salicylanilide Imidazothiazole abamectin albendazole closantel levamisole ivermectin fenbendazole pyrantel pamoate doramectin thiabendazole butamisole moxidectin oxfenbendazole milbemycin fenbantel mebendazole parbendazole flubendazole oxibendazole carbendazole Of these combinations which include at least abamectin from the macrocylic lactone group together with one compound from each of the other three groups; at least 15 albendazole from the benzimidazole group together with one compound from each of the other three groups; closantel together with one compound from each of the other three groups and levamisole together with one compound from each of the other three groups are each preferred. Particularly preferred is the specific combination of abamectin, albendazole, closantel and levamisole. Most preferably, the levamisole is 20 used in the form of a water soluble salt such as the hydrochloride.

6 The therapeutically active compounds used in the invention are preferably incorporated into formulations in the range of concentrations as follows (g/L) macrocylic lactones: 0.1-20.0 g/L, preferably 0.5-1.5 g/L 5 benzimidazoles: 1-100 g/L, preferably 18-30 g/L salicylanilides: 1-100 g/L, preferably 30-50 g/L imidazothiazoles: 1-100 g/L, preferably 3 0-50 g/L Although drenches are preferred dosage forms for the compositions of this invention, a 10 number of alternative compositions may be used. These pour-on transdermals, slow release boluses for rumenal deposition and injectable formulations. Each dosage form requires a therapeutically effective carrier. In the case of drenches, typically a formulation will include a solvent system for the macrocylic lactones, one or 15 more dispersing and suspending agents for the benzimidazoles and salicylanilides , one or more surfactants, one or more preservatives, a buffering system and water as a carrier, The solvent system for the macrocyclic lactones includes at least one solvent selected 20 from the group consisting of: polyethylene glycol, tetraglycol, ethanol, benzyl alcohol and propylene glycol. The dispersing and suspending agents for the benzimidazoles and salicylanilides include at least one selected from the group consissting of: glyceryl palmitostearate, 25 bentonite, colloidal silica, xanthan gum and polymeric pyrrolidones. Surfactants that may be used include polysorbate 80 and ethoxylated castor oil. A variety of buffer systems may be used, particularly phosphate buffers based on 30 combinations of varying amounts of monobasic and dibasic sodium phosphate to achieve the desired pH. The compositions of the invention are effective when used in a variety of animals. For example, sheep, goats, ruminants (including cattle) and camelids.

7 Modes for Carrying Out the Invention In order to better understand the nature of the invention, a number of examples will now be described as follows: Example 1 Material Supplier Grade Amount (g) Abamectin Haiman Technical 94% 1.0 Albendazole Pacific Resource USP24 25.0 Closantel Pacific Resource Technical 98% 37.5 Levamisole Hydrochloride Pacific Resource BP 1998 40.0 Tetraglycol AGRAR Food Grade 400.0 Benzyl alcohol APS BP1998 80.0 Keltrol F (Xanthan Gum) USP24 10.0 Rhone-Poulene Phenonip (Phenoxyethanol) Food 10.0 Bronson& Jacobs Glyceryl Palmitostearate (Precirol ATO 5) USP24 5.0 Bronson & Jacobs Veegum Regular (Magnesium USP24 Aluminium Silicate) RT Vanderbilt Co. PVP29/30 (Ethenyl-2- USP24 pyrrolidinone hornopolymer) ISP PVP C15 ISP USP24 Polyethylene glycol (PEG 2000) BASF USP24 Cremophor (ethoxylated Castor USP24 Oil) BASF Propylene Glycol USP24 Sodium phosphate monobasic Bronson & Jacobs Technical Sodium phosphate dibasic Bronson & Jacobs Technical Polysorbate 80 Bronson & Jacobs USP24 Cab-O-Sil M5 (Colloidal Silicon USP24 Dioxide) Cabot Corp Water qs I litre 8 Example 2 Material Supplier Grade Amount (g) Abamectin Haiman Technical 94% 0.8 Albendazole Pacific Resource USP24 19.0 Closantel Pacific Resource Technical 98% 30.0 Levamisole Hydrochloride Pacific Resource BP 1998 35.5 Glycerol formal 30.0 Tetraglycol AGRAR Food Grade Ethanol 20.0 Benzyl alcohol APS BP1998 80.0 Keltrol F (Xanthan Gum) USP24 10.0 Rhone-Poulenc Phenonip (Phenoxyethanol) Food Bronson& Jacobs Potassium sorbate 10.0 Glyceryl Palmitostearate (Precirol ATO 5) USP24 Bronson & Jacobls Veegum Regular (Magnesium USP24 50.0 Aluminium Silicate) RT Vanderbilt Co PVP29/30 (Ethenyl-2- USP24 50.0 pyrrolidinone homopolymer) ISP PVP C15 ISP USP24 Polyethylene glycol (PEG 2000) BASF USP24 Cremophor (ethoxylated Castor USP24 Oil) BASF Propylene Glycol USP24 Sodium phosphate monobasic Bronson & Jacobs Technical Sodium phosphate dibasic Bronson & Jacobs Technical Polysorbate 80 Bronson & Jacobs USP24 Cab-O-Sil M5 (Colloidal Silicon USP24 Dioxide) Cabot Corp qs I litre Water q____________ __________ s litre 9 Example 3 Material Supplier Grade Amount (g) Abamectin Haiman Technical 94% 1.0 Albendazole Pacific Resource USP24 25.0 Closantel Pacific Resource Technical 98% 37.5 Levamisole Hydrochloride Pacific Resource BP 1998 40.0 Tetraglycol AGRAR Food Grade Benzyl alcohol APS BP1998 20.0 Keltrol F (Xanthan Gum) USP24 Rhone-Poulene Phenonip (Phenoxyethanol) Food 20.0 Bronson& Jacobs Glyceryl Palmitostearate (Precirol ATO 5) USP24 Bronson & Jacobs Bentonite 20.0 Veegum Regular (Magnesium USP24 Aluminium Silicate) RT Vanderbilt Co. PVP29/30 (Ethenyl-2- USP24 pyrrolidinone homopolymer) ISP PVP C15 ISP USP24 100.0 Polyethylene glycol (PEG 2000) BASF USP24 Cremophor (ethoxylated Castor USP24 80.0 Oil) BASE Propylene Glycol USP24 Sodium phosphate monobasic Bronson & Jacobs Technical Sodium phosphate dibasic Bronson & Jacobs Technical Polysorbate 80 Bronson & Jacobs USP24 Cab-O-Sil M5 (Colloidal Silicon USP24 Dioxide) Cabot Corp Water qs Ilitre 10 Example 4 Material Supplier Grade Amount (g) Abamectin Haiman Technical 94% 1.0 Albendazole Pacific Resource USP24 25.0 Closantel Pacific Resource Technical 98% 37.5 Levamisole Hydrochloride Pacific Resource BP 1998 40.0 Tetraglycol AGRAR Food Grade Benzyl alcohol APS BP1 998 80.0 Keltrol F (Xanthan Gum) USP24 Rhone-Poulenc Phenonip (Phenoxyethanol) Food -Bronson& Jacobs Glyceryl Palmitostearate (Precirol ATO 5) USP24 Bronson & Jacobs Veegum Regular (Magnesium USP24 Aluminium Silicate) RT Vanderbilt Co. PVP29/30 (Ethenyl-2- USP24 pyrrolidinone homopolymer) ISP PVP C15 ISP USP24 Polyethylene glycol (PEG 2000) BASF USP24 Cremophor (ethoxylated Castor USP24 Oil) BASF Propylene Glycol USP24 300.0 Sodium phosphate Bronson & Jacobs Technical 9.0 monobasic Sodium phosphate dibasic Bronson & Jacobs Technical 1.0 Polysorbate 80 Bronson & Jacobs USP24 200.0 Cab-O-Sil M5 (Colloidal Silicon USP24 200.0 Dioxide) Cabot Corp Water qs llitre WO 2004/069242 PCT/AU2004/000126 11 Example 4 was prepared as follows: 1. Dissolve avermectin in benzyl alcohol and propylene glycol. 2. Add polysorbate 80 to step 1. 5 3. Add water to the solution from step 2 and mix until homogeneous. 4. Dissolve sodium phosphate dibasic and sodium phosphate monobasic in the solution from step 3. 5. Add closantel, albendazole and levamisole hydrochloride. Mix until fully dispersed. 10 6. Add Cab-O-Sil M5 to the suspension and homogenise until the thicken agent fully hydrated. Based on this disclosure, the person skilled in the art would appreciate the general approach to be taken in preparing the compositions of this invention. 15 In order evaluate the efficacy of the compositions of the invention, a number of trials were conducted using Example 4 as above. Trial RD0201-H002: A critical pen sacrifice study evaluating the therapeutic efficacy 20 of a combination abamectin, levamisole hydrochloride, albendazole and closantel anthelmintic formulation against resistant strains of Haemonchus contortus, Trichostrongvlus colubriformis and Teladorsagia circumcincta in sheep. This study was conducted from the 2 5 th of February to the 29th of August, 2002, with 25 the animal phase conducted from the 7 th May to the 2 7 th June 2002. Suitable sheep (18) were relocated to the University of New England Animal House Facility on the 7 th May 2002 were weighed, identified with individually numbered ear tags and treated with twice the recommended dose rate of Ivomec (Liquid for Sheep, Merial Australia Pty Ltd), to remove any residual worm burden. 30 On 22 "d May 2002 (Day -26) faecal samples were collected from each trial animal to confirm individual zero faecal egg counts. Later that day trial sheep were infected with approximately 5000 Haemonchus contortus (macrocyclic lactone and closantel resistant strains), 6000 Trichostrongylus colubriformis (levamisole hydrochloride and 35 albendazole resistant strains) and 5000 Osteratagia circumcincta (macrocyclic and albendazole resistant strains) infective larvae.

WO 2004/069242 PCT/AU2004/000126 12 Faecal samples were collected from each sheep on 14th June 2002 (Day -3) and individual faecal egg counts were conducted. Animals were ranked on the basis of decreasing faecal egg counts and blocked into eight blocks each of two animals and 5 randomly allocated to the treatment groups from these blocks. The 16 animals with the highest counts were selected for inclusion in the trial and the two animals with the lowest faecal.egg counts were selected as spare animals. On 17 th June 2002 (Day 0) all trial animals were weighed, faecal sampled and animals 10 in Group 2 were treated as follows. Animals were weighed and dosed according to individual live weight as outlined in Table 3. Table 3: Dosage regime Treatment Dose rate Active jhgredient Group 1 Untreated control - Group 2 Example 4 1 mL/5 kg 37.5 g/L closantel g/L abamectin 40 g/L levamisole hydrochloride 25 g/L albendazole The 18 trial sheep (including the 2 spare animals) were sacrificed on 2 7 th June 2002 (Day 10) for collection of faecal samples, abomasal and small intestine contents. Individual faecal egg counts, treatment group coprocultures and total worm counts 15 were conducted for calculation of treatment efficacies. Drenchrites (CSIRO Research - Horizon Technology 1996) was performed between the 10 th July and the 2 3 rd August 2002 to clarify that strains of Trichostrongylus colubriformis used were resistant to levamisole hydrochloride and albendazole and, 20 Osteratagia circuicincta were resistant to albendazole. Faecal samples were collected according to standard procedures and submitted to the Veterinary Health Research parasitological laboratory. Individual strongyle faccal egg counts and group bulk coproculture for larval differentiation were carried out. 25 Gastrointestinal tracts were recovered according to standard procedures and following gut washing were submitted to the parasitological laboratory. Individual total worm counts were conducted and results are summarised in the accompanying tables, 4-10 and figures 1-4.

WO 2004/069242 PCT/AU2004/000126 13 Table 4: Group mean strongyle faecal egg counts. Group Treatment Day 0 Day 10 Arithmetic Means 1 Control 9320.0' 8568.9 2 Example 4 8177.8' 22.2 Geometric Means 1 Control 6754.5 6754.51 2 Example 4 1.8 1.82 'Means with different superscripts within the same column are significantly different at p<0.05 Table 5: Percentage reduction of strongyle species (based on group mean strongyle faecal egg count data) Group Treatment Day 10 Arithi etic Effcacy 2 Example 4 99.7% Geometric Efficacy 2 Example 4 >99.9% WO 2004/069242 PCT/AU2004/000126 14 c"1q ,C) 0 '~'n 00 C'0 C.) -Z,-S Q.) on WO 2004/069242 PCT/AU2004/000126 15 41V A0 A A 00 050 0066 ON t4 F- L WO 2004/069242 PCT/AU2004/000126 16 .4 4--, A Z 80 cNJ A '-4, oCIO -e 0<L WO 2004/069242 PCT/AU2004/000126 17 Conclusion: Excellent control (>99.9% reduction) of a mixed gastrointestinal strongyle burden as assessed by geometric faecal egg counts was achieved by the use of the Example 4 formulation at the conclusion of the trial (Day 10),. 5 Excellent control (>99.9% reduction) was achieved by the Example 4 formulation against the major nematodes, macrocyclic lactone and closantel resistant strains of Haemonchus spp. (adult and immature stages - geometric -means), macrocyclic and albendazole resistant strains Ostertagia spp. (adult, immature and L4 stages - geometric means) and levamisole hydrochloride and albendazole resistant strains of 10 Trichostrongylus spp (adult and immature stages - geometric means) as assessed by geometric total worm counts. Trial JUA1240r: A property faecal egg count reduction study evaluating the therapeutic efficacy of the Example 4 formulation against field strains of mixed nematode 15 population of either Haemonchus contortus, Trichostrongylus colubriformis and/or Teladorsagia circumcincta in sheep. This study was conducted from the 16th May 2002 to the 20t" August 2002 with the animal phase conducted between the lt July 2002 to the 17 th July 2002 and animal 20 phase 2 between 5ff September 2002 to the 4 th October 2002. A trial site was sought, containing a mob of Merino sheep that were known to be harbouring resistant strains of nematodes (including either closantel resistant and/or macrocyclic lactone resistant Haemonchus species, as well as either benzimidazole resistant and/or levamisole resistant Trichostrongylus colubriformis and/or Teladorsagia circumcincta.) Pre-trial 25 monitoring of the site confirmed that the intended trial animals carried a nematode burden of greater than 400 eggs/gram. A group coproculture was performed on these prospective trial sheep to establish the genera present. On Day -3 of the trial, a mob of approximately 300 Merino ewes was mustered into a 30 set of sheep yards. Eighty ewes were identified with uniquely numbered eartags and faecal sampled as they presented in the race. The faecal samples were returned to Veterinary Health Research for individual faecal egg counts and a bulk coproculture.

WO 2004/069242 PCT/AU2004/000126 18 The sixty animals with the highest strongyle faecal egg counts, as determined by the Day -3 faecal egg counts, were selected for inclusion in the trial. These sheep were allocated to one (1) of six (6) treatment groups, on the basis of their faecal egg counts, such that each group had a similar arithmetic group mean faecal egg count. 5 On Day 0 (treatment day), each animal was weighed and treated according to the treatment schedule outlined in Table 11. Clinical observations were conducted one hour post-treatment to determine whether any adverse reactions had occurred in relation to treatments. None were detected. 10 Table 11: Treatment table (phase 1) Treatment FEomlation Dosage regimen Number of Group sheep 1 Untreated control - 10 2 Example 4 1 mL/5 kg 10 3 Ivomec (Liquid for Sheep, 1 mL/4 kg 10 Merial Pty Ltd) 4 Sustain (Dover Laboratories 1 mL/5 kg 10 Pty Ltd) 5 Youngs Levamisole (Youngs 1 mL/4 kg 10 Animal Health Pty Ltd) 6 Valbazen (Coopers Animal 1 mL/4 kg 10 Health) The trial concluded on Day 13 when faecal samples were collected and returned to the 15 Veterinary Health Research Laboratory for individual faecal egg counts and group coprocultures. The entire mob was administered an effective broad-spectrum anthelmintic to remove any existing worm burden. The aim of this field study was to study and evaluate under field conditions, the 20 therapeutic efficacy of Example 4 when administered to sheep that are known to be harbouring resistant strains of nematodes. The selected trial site was known to harbour closantel resistant Haemonchus contortus. This however was not confirmed during the initial phase of the study as a full dose of closantel was administered (as stated in the WO 2004/069242 PCT/AU2004/000126 19 protocol). Standard industry practice for diagnosis of closantel resistance in the field involves either the administration of a full dose of closantel and sequential sampling of treated sheep over three to six weeks post treatment, or alternatively administration of a 1/3 dose and sampling at 10 tol4 days post treatment. 5 A second faecal egg count reduction study was conducted after consultation with the Study Sponsor to confirm the closantel resistance status at the trial site, "Kelvin East". The second phase of the study involved two groups of sheep each consisting of ten animals. Ten random faecal samples were collected prior to treatment from the mob of 10 wethers to confirm a nematode burden of greater than 400 eggs/gram, and a group coproculture that confirmed a very high percentage (91%) of Haenonchus contortus were present. On Treatment Day (Day 0), individual faecal samples were collected from twenty animals as they presented in the race. These animals were weighed and weights recorded and treatments administered in accordance to the treatment regime 15 (detailed in Table 12). Faecal samples were returned to Veterinary Health Research for individual faecal egg counts and group coprocultures. Animals were observed post treatment for adverse reactions. None were detected. Table 12: Treatment table (phase 2) 20 Treatment Formulation Dosage Regim n Number of Group Sheep 1 Untreated control -10 2 Sustain (Dover Laboratories 1 mL/15 kg 10 Pty Ltd) The second phase of this trial concluded on Day 11, with the collection of individual faecal samples from all animals. These samples were returned to Veterinary Health Research for individual faecal egg counts and group coprocultures. 25 Faecal samples for phase 1 were collected during pre trial monitoring, (Day -3), at treatment (Day 0) and at the conclusion of the trial (Day 13) and for phase 2 at treatment (Day 0) and at the conclusion of the trial (Day 11). Results from faecal egg counts, larval differentiation and calculated treatment efficacies are summarised in the accompanying tables 13-24 and figures 5 and 6. Note that in figures 5 and 6, "Jurox" 30 refers to example 4.

WO 2004/069242 PCT/AU2004/000126 20 Table 13: Pre trial monitoring results. Date Group mean faebal egg Range of faecal egg count (epg) counts (epg) 01 July 2002(Day-3) 900 320-1880 Table 14: Group arithmetic mean faecal egg counts and body weights at Day 0. 5 Group Treatment Grp mean ec egg roup mean body weight count( (ep) (kg) 1 Untreated control 984' 49.301 2 Example 4 5841 45.251 3 Ivomec 588,46.05' 4 Sustain 584 49.451 5 Levamisole 452 46.35' 6 Valbazen 328 48.251 1 Means in the same column with different superscripts are significantly different at p< 0

.

0 5 Table 15: Group arithmetic and geometric mean faecal egg counts (epg). Pre-trial (Day Day 0 Day 13 'Group Treatment Arithmetic Means 1 Untreated controls 892 984 6641 2 Example 4 872 584' 44 3 Ivomec 940 5881 04 4 Sustain 924 584' 1601,2 5 Levamisole 900 4521 123,4 6 Valbazen 872 328' 682,3 WO 2004/069242 PCT/AU2004/000126 21 Geometric Meian~s.

I Untreated controls 823.0 691.5 340.3 2 Example 4 768.9 508.7 0.5 3 Ivomec 869.9 416.4 0 4 Sustain 860.1 450.1 97.4 5 Levamisole 834.1 379.7 2.1 6 Valbazen 818.9 179.7 14.9 1 Means in the same column with different superscripts are significantly different at p< 0

.

0 5 Table 16: Overall percentage efficacy calculated using arithmetic and geometric group mean faecal egg counts. [Group = Treatment Effcy(% Arithmetic _Effcacy 2 Example 4 99.4 3 Ivomec >99.9 4 Sustain 75.9 5 Levamisole 98.19 6 Valbazen 89.76 Greoinzetrlj Efficacy 2 Example 4 99.87 3 Ivomec >99.9 4 Sustain 71.37 5 Levamisole 99.4 6 Valbazen 95.61 WO 2004/069242 PCT/AU2004/000126 22 00 o -'~~~C as CS C CCd i I I I I I In 0 ID > 0 0~ CO 4-4 uokA A A A 0 e~0 cK ~ 0 -_ rn~ In LL - - WO 2004/069242 PCT/AU2004/000126 23 Phase 2 Table 19: Treatment day monitoring results. Date of Sampling Group Mean Faecal Egg Range of Faecal Egg Count (eggsa/grn) Count (eggs/gram) 23 Sept 2002 (Day 0) 1428 80-3000 Table 20: Group arithmetic mean faecal egg counts and body weights at Day 0. Group Treatment Group Mean Faecal Egg Group Mean Body Count (eggs/gram) Weight (kg) 1 Untreated controls 16281 51.61 2 Sustain 1228' 50.2' 1 Means in the same column with different superscripts are significantly different at 5 p<0.05 Table 21: Group arithmetic and geometric mean faecal egg counts. Group Treatment Day 0 Day 11 Arithmetic Means 1 Untreated controls 1628.01 3008.01 2 Sustain 1228.01 2088.01 Geometrc Means 1 ] Untreated controls 1237.7 2041.1 2 Sustain 940.0 1511.9 Means in the same column with different superscripts are significantly different at p<0.05 WO 2004/069242 PCT/AU2004/000126 24 Table 22: Overall percentage efficacy (arithmetic and geometric means) Group T'eatment Percentage Efficacy (%) rithmetic E acy 2 1/3 Sustain 30.6 Geometric 2 1/3 Sustain 5.9 WO 2004/069242 PCT/AU2004/000126 25 0 1~ 0 0.4 0 0 I I ~ 00000 ~ 0 0 0 0 0 0 0 ,-4 ,- .. c~) o 0 0 ~.) <~ 0 0 ~4 -( 0.4 o 0 o 0 0 o.J C~) C..

2 0 0 *~

.,

I ~ C\ 0~ ~ 0~ 0~ ~ o 0 S 0 0 0 -~ 0 0 &-~ ~ - - ~ ~ o -~ 0 .4-. 0 0 0 -Cl -~ Cl z Cl I Cl 0 ~ 0 z -~ 0 WO 2004/069242 PCT/AU2004/000126 26 Conclusion: The second faecal egg count reduction test was to confirm the presence closantel resistant Haemonchus at the trial site. This was achieved by administering a one third dose of closantel to a group of ten (10) animals and the addition of another group of ten (10) animals retained as untreated controls. The use of a one third dose of closantel is standard industry practice for diagnosis of closantel resistance in the field. Reduced efficacy of closantel was observed against Haemonchus, confirming. The presence of closantel resistant Haemonchus at the trial site. The inclusion of the levamisole and benzimidazole groups confirmed the resistance status of Trichostrongylus. Excellent efficacy (> 99.0%) was attained by the Example 4 formulation against a mixed gastrointestinal population including closantel resistant Haemonclus as well as levamisole and benzimidazole resistant Trichostrongylus.

WO 2004/069242 PCT/AU2004/000126 27 Trial JUA1273r: A property faccal egg count reduction study evaluating the therapeutic efficacy of the Example 4 formulation against field strains of mixed nematode populations, including closantel resistant strains of Haemonchus contortus in sheep. 5 This study was conducted from the 5* of September 2002 to the 2 3 rd of October 2002, with the animal phase conducted between 10t of September 2002 and the 1 st of October 2002. Routine monitoring of a trial site known to harbour closantel resistant strains of Haemonchus contortus was conducted to identify a suitably infected group of sheep. Pre-trial monitoring confirmed that one group of sheep (270 Merino hoggets) 10 was suitably infected with a high burden of Haemonchus contortus. On Day -2 of the trial individual faecal samples were collected from ninety (90) potential trial sheep and individual strongyle faecal egg counts performed. Trial sheep had already been identified using uniquely numbered ear tags as part of standard 15 farming practice at the trial site. From the ninety (90) potential trial sheep sixty (60) sheep were selected and allocated (according to individual strongyle faecal egg counts) to six (6) groups of ten (10) sheep each, such that each group had a similar group arithmetic mean strongyle faecal egg count and range of faecal egg counts within the group. 20 On Day 0 of the trial (18th September 2002) selected trial sheep were weighed (see figure 7 for arithmetic mean body weights and note that the treatment "Jurox" refers to the treatment with Example 4), the weights recorded and individual faecal samples collected for individual strongyle faecal egg counts. Trial sheep in Group 2 were 25 treated according to individual body weight with the test formulation, trial sheep in Groups 3-6 were treated with the respective reference formulation and trial sheep in Group 1 were retained untreated as negative controls. Groups 2, 3, 5 and 6 were treated at the recommended dose rate for each active, while sheep in Group 4 were treated at one third the normal closantel dose rate, to determine and demonstrate the presence of 30 closantel resistance (Reference: Rolfe PF; Fourth International Congress for Sheep Veterinarians 1997, pg 55). Sheep were observed in the immediate post-treatment period for adverse reactions (none were observed). Individual strongyle faecal egg counts and group bulk coprocultures for larval differentiation were subsequently performed on the samples collected.

WO 2004/069242 PCT/AU2004/000126 28 Trial sheep were returned to the sheep yards on Day 13 of the trial ( 1 t October 2002) and individual faecal samples again collected. All trial sheep received a single therapeutic dose of Rycozole@ due to animal welfare concerns. Individual strongyle faecal egg counts and group bulk coprocultures for larval differentiation were 5 subsequently performed on the samples collected. Treatment efficacies were then calculated using group arithmetic and geometric strongyle faecal egg counts for the major strongyle species present (see figures 8 and 9 and note that the treatment "Jurox" refers to treatment with Example 4). Rycozole Oral Anthelmintic for Sheep and Cattle, Novartis Animal Health Australasia Pty Ltd WO 2004/069242 PCT/AU2004/000126 29 00 00 CN 00 -0 C.,r 0. r4~ CC 0f \-m~ 0000~i tfr) _ _ _ V;- \,O I' k -C 00 0 7 0 0 0~C ;1 00n~I0 'D- r-0 o000 1 C:,~ c0 C) c_'0 0 z 0 1 0 , 0v C) C)C) C)O en 't tn 01m zl*kn\0 WO 2004/069242 PCT/AU2004/000126 30 1 a~ cl v rrd oD i~ cn -4 m :)0Q t co00 V -4 v~ 0 O~ ~00 ~0 t4 I . rj cl WO 2004/069242 PCT/AU2004/000126 31 rIN ~~IQ 0) 0 000 0000 hiN m N 0= 0 00N 00 N 00 ~ ~ t .- 0 CO - . ~ ,-. o 0o > IS (0 CI Ion I It c I I I c l ) Icl WO 2004/069242 PCT/AU2004/000126 32 0 00 00 000000 0 00000 0o 0c-0 000 00 00 I . ~ 0 _ 0 C? 9 cili WO 2004/069242 PCT/AU2004/000126 33 Table 31: Overall treatment efficacies, against all strongyle species (apart from Nematodirus spp.) Group Treatment Batch No. Efficacy at Day 13 Arithmetic Means 2 Example 4 FS489 >99.9% 3 Ivomec@ 51983 95.6% 4 1/3 Sustain@ 13146 82.6% 5 Levamisole@ 6053 99.7% 6 Valbazen@ V03790/2 85.0% Geometric Means 2 Example 4 FS489 >99.9% 3 Ivomec® 51983 97.2% 4 1/3 Sustain® 13146 97.2% 5 Levamisole@ 6053 >99.9% 6 Valbazen@ V03790/2 84.4% Table 32: Treatment efficacies against Haemonchus contortus. Group Treatmenit Batch No. IEfficac at Day 13 Arithmetic Means ______ 2 Example 4 FS489 >99.9% 3 Ivomec® 51983 96.1% 4 1/3 Sustain@ 13146 83.0% 5 Levamisole@ 6053 99.8% 6 Valbazen@ V03790/2 85.5% 2 Example 4. FS489 >99.9% 3 Ivomec@ 51983 97.5% 4 1/3 Sustain@ 13146 86.8% 5 Levamisole® 6053 >99.9% 6 Valbazen@ V03790/2 84.9% WO 2004/069242 PCT/AU2004/000126 34 Conclusion: Excellent efficacy (greater than 99.9% based on group arithmetic and geometric means and larval differentiation results) was attained by the Example 4 formulation against a gastrointestinal strongyle population consisting almost exclusively of Haemonchus contortus. 5 Efficacies attained by the comparison formulations against this strain of Haemonchus contortus ranged from 85.5% for the albendazole formulation (Valbazen@) through 96.1% for the ivermectin formulation (Ivomec@) to 99.8% for the levamisole formulation (Levamisole@), based on group arithmetic mean faecal egg counts and 10 larval differentiation. Efficacies attained against this strain based on geometric mean faecal egg counts and larval differentiation were 84.9%, 97.5% and >99.9% for these formulations respectively. These results indicate that this strain is moderately resistant to white drenches (benzimidazoles) and fully susceptible to levamisoles, with a slight but non-significant reduction in efficacy for ivermectin. Ivermectin was 96.1% 15 (arithmetic) and 97.5% (geometric) efficacious, which establishes that this strain could not be defined as macrocyclic lactone resistant at present. Treatment with a 1/3 dose of closantel resulted in a treatment efficacy of 83.0% based on arithmetic group mean faecal egg counts and a treatment efficacy of 86.8% based on 20 geometric group mean faecal egg counts, confirming the presence of moderate closantel resistance by this Haemonchus strain. Insufficient numbers of other gastrointestinal strongyles (Nematodirus, Teolodorsagia and Trichostrongylus species) were present to draw any conclusions about efficacy of the test formulation against these strains.

WO 2004/069242 PCT/AU2004/000126 35 Trial JUAl270r: A property faecal egg count reduction study evaluating the therapeutic efficacy of the Example 4 formulation against field strains of mixed nematode populations, including macrocyclic lactone resistant strains of Haemonchus contortus in sheep in sheep. 5 This study was conducted from the 5* of September 2002 to the 7t of November 2002, with the animal phase conducted between 11 th and 2 5 th of October 2002. Routine monitoring of a trial site known to harbour macrocyclic lactone resistant strains of Haemonchus contortus was conducted to identify a suitably infected group of sheep. 10 Pre-trial monitoring confirmed that one group of sheep (approximately 200 Merino wether hoggets) was suitably infected with a high burden of Haenonchus contortus. On Day 0 of the trial, ninety six (96) sheep were randomly selected from a larger mob as they appeared in the sheep handling facility, weighed (see figure 10 for arithmetic 15 mean body weights and note that the treatment "Jurox" refers to treatment with example 4) and individual faecal samples collected for subsequent individual strongyle faecal egg counts and group bulk coprocultures. Sheep had been previously allocated to six (6) treatment groups, one (1) of eleven (11) sheep to act as untreated (negative) controls and five (5) groups of seventeen (17) sheep, to be treated with the test 20 formulation and a range of registered reference formulations. Trial sheep in Group 1 were retained untreated, while sheep in Groups 2-6 were treated according to individual body weight with the test and reference formulations. Sheep were observed in the immediate post-treatment period for adverse reactions (none were observed). Trial sheep were then returned to their parent flock and maintained in open grazing 25 paddocks. On Day 13 of the trial sheep were returned to the sheep handling facilities. Individual faecal samples were collected from trial sheep and individual strongyle faecal egg counts and group bulk coprocultures for larval differentiation were subsequently 30 performed on the samples collected. Treatment efficacies were then calculated using group arithmetic and geometric strongyle faecal egg counts for the major strongyle species present (see figures 11 and 12 and note that the treatment "Jurox" refers to treatment with Example 4).

C C) 0 t ES0 bO g0 oE ~ o o >>a , S -5.bn25 ,Sx >~ eN -e N -,4 3 Cc2 LN ) C)) in t - o o o *e -~~ C - CN CO C> o-o o S-4 000 00 ) 6~ -: 4 t 00 a) Cll - " o 0f8 - > 00 C) ___-____d___d d-d_ C)~~~ V)5 ~ 0 00 V gC 2 m666oo 6 Co C) o0o oo) toc o -e C)d V oS S 9 C0~4) a,, Cl) O - m0' - C) C N -0 C)It 0 1 - e -_-~n co ~~ tf C)C)C -- c - = ,73 fl - ~ ~ ~ / Cl 2nS C ~ ~ r') WO 2004/069242 PCT/AU2004/000126 37 vi r- - -n -c 0 ov~o v 0 0_ W_ _q0 M 00 W 0 C) 00 tn Q 4)o 71tz ~~~~o ~ ~ C Ioc1\% ~ ~ 04 PI 4 LO ) WO 2004/069242 PCT/AU2004/000126 38 00 m 000 _o -o m 000 -. 4 rrtr o)c. 8r . rAl C0 CD C> 0 0 lv g 0

CI

WO 2004/069242 PCT/AU2004/000126 39 C0 0 0 0 0D C) 000 0 0 0000 0> C0 00 000 0 O 0 00 01 0 0 0 0 0 o C,_ -= C: 0 (: -tog 000 Z o1 1 WO 2004/069242 PCT/AU2004/000126 40 -- - o6 - - - 00 0 \o t.- W ;!00 Z) G~_o~ Z) mot C0 0 0 l > 0) CR 00 Ct") -lot C-11 0 41 Conclusion: Efficacies attained by the formulations against this strain of Haemonchus contortus for the ivermectin formulation (Ivomec®), the albendazole formulation (Valbazen®), the closantel formulation (full dose Sustain®), the levamisole formulation (Levamisole Gold@) and for the test formulation, based on group 5 geometric mean faecal egg counts and larval differentiation were negative, 3.0%, 65.3%, 78.9% and >95% respectively. While moderate efficacies were attained by the reference closantel formulation, a full (label) dose rate of this formulation was used in this case, In instances of moderate 10 closantel resistance efficacies are still usually >95% for a full dose, with a reduction in initial efficacy only evident at a 1/3 normal dose rate. This particular strain is therefore severely resistant to closantel. These results indicate that this strain also has severe resistance to white drenches (benzimidazoles) and macrocyclic lactones and moderate resistance to levamisole. This strain of Haemonchus contortus is, therefore, moderately 15 to severely resistant to all four drug families. Unexpectedly, the Example 4 formulation containing an example of all four of these families resulted in effective treatment of the infestation. It will be appreciated by persons skilled in the art that numerous variations and/or 20 modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. 25 Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date 30 of each claim of this application.

Claims

1. A synergistic anthelmintically effective composition comprising at least four anthelmintic compounds and a therapeutically acceptable carrier, wherein at least one anthelmintic compound is selected from the group of macrocyclic lactones, at least one 5 anthelmintic compound is selected from the group of benzimidazoles, one anthelmintic compound is closantel, and at least one anthelmintic compound is selected from the group of imidazothiazoles.

2. The composition of claim 1 wherein the at least one benzimidazole and closantel 10 are suspended in the composition.

3. The composition of claim 1 or 2 wherein the macrocyclic lactone compound is at least one selected from the group consisting of abamectin, ivermectin, doramectin, moxidectin, and milbemycin. 15

4. The composition as claimed in any preceding claim wherein the benzimidazole compound is at least one selected from the group consisting of albendazole, fenbendazole, thiabendazole, oxfenbendazole, fenbantel, mebendazole, parbendazole, flubendazole, oxibendazole and carbendazole. 20

5. The composition as claimed in any preceding claim wherein the imidazothiazole compound is at least one selected from the group consisting of levamisole, pyrantel pamoate and butamisole. 25

6. The composition as claimed in any preceding claim wherein the selected macrocyclic lactone compound is at least abamectin.

7. The composition as claimed in any preceding claim wherein the benzimidazole compound is at least albendazole. 30

8. The composition as claimed in any preceding claim wherein the imidazothiazole compound as at least levamisole;

9. The composition as claimed in any preceding claim wherein the composition 35 consists of at least abamectin, albendazole, closantel and levamisole. 43

10. The composition of claim 8 or 9 wherein the levamisole is included in the form of a water soluble salt.

11. The composition of claim 10 wherein the water soluble salt is a hydrochloride 5 salt.

12. The composition of any one of claims 1 to 11 wherein the composition comprises: macrocyclic lactone compounds in an amount of from 0.1-20.0 g/L; 10 benzimidazole compounds in an amount of from 1-1 OOg/L; closantel in an amount of from 1-100 g/L; and imidazothiazole compounds in an amount of from 1-100 g/L.

13. The composition of any one of claims 1 to 12 wherein the composition 15 comprises: macrocyclic lactone compounds in an amount of from 0.5- 1.5 g/L; benzimidazole compounds in an amount of from 18-30 g/L; closantel in an amount of from 30-50 g/L; and imidazothiazole compounds in an amount of from 30-50 g/L. 20

14. The composition of any one of claims 1 to 14 wherein the composition is in the form of a drench, a pour-on transdermal formulation, a slow release bolus or an injectable formulation. 25

15. The composition of any one of claims 1 to 14 wherein the composition is in the form of a drench including a solvent system for the macrocylic lactones, one or more dispersing and suspending agents for the benzimidazoles and closantel, one or more surfactants, one or more preservatives, a buffering system and water as a carrier. 30

16. The composition of claim 16 wherein the solvent system for the macrocyclic lactones includes at least one solvent selected from the group consisting of: polyethylene glycol, tetraglycol, ethanol, benzyl alcohol and propylene glycol.

17. The composition of claim 16 wherein the dispersing and suspending agents for 35 the benzimidazoles and closantel include at least one selected from the group consisting 44 of: glyceryl palmitostearate, bentonite, colloidal silica, xanthan gum and polymeric pyrrolidones.

18. The composition of claim 16 wherein the surfactant is polysorbate 80 and/or 5 ethoxylated castor oil.

19. The composition of claim 16 wherein the buffering system includes monobasic and dibasic sodium phosphate. 10

20. A method of treating parasitic infections in an animal comprising administering to the animal a synergistic anthelmintically effective amount of a composition comprising at least four anthelmintic compounds and a therapeautically acceptable carrier, wherein at least one anthelmintic compound is selected from the group of macrocyclic lactones, at least one anthelmintic compound is selected from the group of 15 benzimidazoles, one anthelmintic compound is closantel, and at least one anthelmintic compound is selected from the group of imidazothiazoles.

21. The method of claim 20 wherein the at least one benzimidazole and closantel are suspended in the composition. 20

22. The method of any one of claims 20 to 21 wherein the macrocyclic lactone compound is at least one selected from the group consisting of abamectin, ivermectin, doramectin, moxidectin, and milbemycin. 25

23. The method of any one of claims 20 to 22 wherein the benzimidazole compound is at least one selected from the group consisting of albendazole, fenbendazole, thiabendazole, oxfenbendazole, fenbantel, mebendazole, parbendazole, flubendazole, oxibendazole and carbendazole. 30

24. The method of any one of claims 20 to 23 wherein the imidazothiazole compound is at least one selected from the group consisting of levamisole, pyrantel pamoate and butamisole.

25. The method of any one of claims 20 to 24 wherein the selected macrocyclic 35 lactone compound is at least abamectin. 45

26. The method of any one of claims 20 to 25 wherein the benzimidazole compound is at least albendazole.

27. The method of any one of claims 20 to 26 wherein the imidazothiazole 5 compound as at least levamisole.

28. The method of any one of claims 20 to 27 wherein the composition consists of at least abamectin, albendazole, closantel and levamisole. 10

29. The method of any one of claims 27 to 28 wherein the levamisole is included in the form of a water soluble salt.

30. The method of claim 29 wherein the water soluble salt is a hydrochloride salt. 15

31. The method of any one of claims 20 to 30 wherein the composition comprises: macrocyclic lactone compounds in an amount of from 0.1-20.0 g/L; benzimidazole compounds in an amount of from 1-I OOg/L; closantel in an amount of from 1-100 g/L; and imidazothiazole compounds in an amount of from 1-100 g/L. 20

32. The method of any one of claims 20 to 31 wherein the composition comprises: macrocyclic lactone compounds in an amount of from 0.5- 1.5 g/L; benzimidazole compounds in an amount of from 18-30 g/L; elU-4Ite- in an amount of fom 30-50 g; and 25 imidazothiazole compounds in an amount of from 30-50 g/L.

33. The method of any one of claims 20 to 32 wherein the method is a method of treating infection in an animal by at least one species of parasite selected from the group consisting of Haemonchus contortus, Haemonchus place, Ostertagia 30 circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Cooperia curticel, Cooperia oncophera, Nematodirus spathiger, Nematodirus filicollis, Chabertia ovina, Oesophagostomum columbianum, Oesophagostomum venulosum, Trichuris ovis, Strongyloides papillosus, Bunostomum spp, Oestrus ovis, Dictyocaulus viviparus, Fasciola hepatica, and Monezia. 35 46

34. The method of any one of claims 20 to 33 wherein it is suspected that the animal may be carrying at least one parasite which is resistant to at least one of the groups of macrocyclic lactones, benzimidazoles, imidazothiazoles and salicylanilides. 5

35. The method of any one of claims 20 to 34 wherein the method is a method of treating infection in an animal by parasites resistant to at least one of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

36. The method of any one of claims 20 to 35 wherein the method is a method of 10 treating infection in an animal by parasites resistant to at least two of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

37. The method of any one of claims 20 to 36 wherein the method is a method of treating infection in an animal by parasites resistant to at least three of each of the 15 groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

38. The method of any one of claims 20 to 37 wherein the method is a method of treating infection in an animal by parasites resistant to all of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. 20

39. The method of any one of claims 20 to 38 wherein the method is a method of treating infection in an animal by gastro-intestinal worms and liver fluke.

40. The method of any one of claims 20 to 39 wherein the composition is 25 administered to an animal prior to introduction to a land area so as to prevent the land area from becoming infested with parasites which may or may not be resistant to one or more compounds selected from the groups consisting of macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. 30

41. The method of any one of claims 20 to 40 wherein the composition is used in the treatment of a parasitic infection in an animal selected from the group consisting of sheep, goats, ruminants and camelids.

42. The use of a synergistic anthelmintically effective amount of a composition 35 comprising at least four anthelmintic compounds and a therapeautically acceptable carrier, wherein at least one anthelmintic compound is selected from the group of 47 macrocyclic lactones, at least one anthelmintic compound is selected from the group of benzimidazoles, one anthelmintic compound is closantel, and at least one anthelmintic compound is selected from the group of imidazothiazoles, in the treatment of a parasitic infection in an animal. 5

43. The use of claim 42 wherein the at least benzimidazole and closantel are suspended in the composition.

44. The use of any one of claims 42 to 43 wherein the macrocyclic lactone 10 compound is at least one selected from the group consisting of abamectin, ivermectin, doramectin, moxidectin, and milbemycin.

45. The use of any one of claims 42 to 44 wherein the benzimidazole compound is at least one selected from the group consisting of albendazole, fenbendazole, 15 thiabendazole, oxfenbendazole, fenbantel, mebendazole, parbendazole, flubendazole, oxibendazole and carbendazole.

46. The use of any one of claims 42 to 45 wherein the imidazothiazole compound is at least one selected from the group consisting of levamisole, pyrantel pamoate and 20 butamisole.

47. The use of any one of claims 42 to 46 wherein the selected macrocyclic lactone compound is at least abamectin. 25

48. The use of any one of claims 42 to 47 wherein the benzimidazole compound is at least albendazole.

49. The use of any one of claims 42 to 48 wherein the imidazothiazole compound as at least levamisole. 30

50. The use of any one of claims 42 to 49 wherein the composition consists of at least abamectin, albendazole, closantel and levamisole.

51. The use of any one of claims 49 to 50 wherein the levamisole is included in the 35 form of a water soluble salt. 48

52. The use of claim 51 wherein the water soluble salt is a hydrochloride salt.

53. The use of any one of claims 42 to 52 wherein the composition comprises: macrocyclic lactone compounds in an amount of from 0.1-20.0 g/L; 5 benzimidazole compounds in an amount of from 1-100g/L; closantel in an amount of from 1-100 g/L; and imidazothiazole compounds in an amount of from 1-100 g/L.

54. The use of any one of claims 42 to 53 wherein the composition comprises: 10 macrocyclic lactone compounds in an amount of from 0.5- 1.5 g/L; benzimidazole compounds in an amount of from 18-30 g/L; closantel in an amount of from 30-50 g/L; and imidazothiazole compounds in an amount of from 30-50 g/L. 15

55. The use of any one of claims 42 to 54 wherein the parasitic infection in the animal to be treated is infection by at least one species of parasite selected from the group consisting of Haemonchus contortus, Haemonchus placei, Ostertagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Cooperia curticel, Cooperia oncophera, Nematodirus spathiger, Nematodirus 20 filicollis, Chabertia ovina, Oesophagostomum columbianum, Oesophagostomum venulosum, Trichuris ovis, Strongyloides papillosus, Bunostomum spp, Oestrus ovis, Dictyocaulus viviparus, Fasciola hepatica, and Monezia.

56. The use of any one of claims 42 to 55 wherein the parasitic infection in the 25 animal to be treated is infection by parasites resistant to at least one of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

57. The use of any one of claims 42 to 56 wherein the parasitic infection in the animal to be treated is infection by parasites resistant to at least two of each of the 30 groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles.

58. The use of any one of claims 42 to 57 wherein the parasitic infection in the animal to be treated is infection by parasites resistant to at least three of each of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. 35 49

59. The use of any one of claims 42 to 58 wherein the parasitic infection in the animal to be treated is infection by parasites resistant to all of the groups macrocylic lactones, benzimidazoles, salicylanilides and imidazothiazoles. 5

60. The use of any one of claims 42 to 59 wherein the parasitic infection in the animal to be treated is infection by gastro-intestinal worms and liver fluke.

61. The use of any one of claims 42 to 60 wherein the composition is used in the treatment of a parasitic infection in an animal selected from the group consisting of 10 sheep, goats, ruminants and camelids.

62. A method, composition or use substantially as hereinbefore described with reference to the accompanying examples.