AU2014332597A1

AU2014332597A1 - Veterinary formulations and methods

Info

Publication number: AU2014332597A1
Application number: AU2014332597A
Authority: AU
Inventors: Douglas Grant Halligan; Gary Robert Harrison; Mark Colin Vickers
Original assignee: Nexan Ip Holdings Ltd
Current assignee: Nexan Ip Holdings Ltd
Priority date: 2013-10-07
Filing date: 2014-10-07
Publication date: 2016-04-28
Also published as: WO2015053636A1

Abstract

The invention relates to methods and formulations for veterinary treatment. In particular, the invention relates to methods and formulations for improving the treatment of pathogenic and/or resistant worm strains in an animal by administering to the animal a parasitically effective amount of at least two different macrocyclic lactones, wherein at least one of the macrocyclic lactones is a milbemycin and at least one of the macrocyclic lactones is an avermectin. The invention also or alternatively relates to a method of reducing the reinfection by a pathogenic and/or resistant worm strain following treatment.

Description

WO 2015/053636 PCT/NZ2014/000214 1 VETERINARY FORMULATIONS AND METHODS Field of Invention The invention relates to methods and formulations for veterinary treatment. In particular, the 5 invention relates to methods and formulations for treating, reducing or preventing the incidence of a parasite in a non-human animal or for slowing the development of parasite resistance or improving the treatment of pathogenic and/or resistant worm strains in an animal by administering to the animal a parasitically effective amount of at least two different macrocyclic lactones, wherein at least one of the macrocyclic lactones is a milbemycin and at 10 least one of the macrocyclic lactones is an avermectin. The invention also or alternatively relates to methods and formulations for changing the parasitological outcome of a treatment of a non-human animal with a milbemycin by reducing the reinfection by a pathogenic and/or resistant worm strain following treatment. 15 Background The avermectin and related milbemycin compounds are a group of compounds that were first isolated from soil dwelling actinomycetes Streptomyces species. The avermectins and the milbemycins share a common 16 member macrocyclic ring and are commonly known as macrocyclic lactones. The two groups differ in that there is a disaccharide present at the 13 20 position of the macrocyclic ring in the avermectins which is not present in the milbemycins. Therefore the avermectins can be considered glycosylated milbemycins or the milbemycins as deglycosylated avermectins. These macrocyclic lactones have also been described as "endectocides" because they have activity against both "endo" or internal parasite activity, particularly against internal parasitic roundworms, and "ecto" or external parasite activity 25 including against lice, mites, ticks, fleas and some fly stages. Increasingly, formulations of a macrocyclic lactone in combination with other active compounds, particularly those with antiparasitic activity, have been patented and subsequently commercially exploited. These combinations have occurred for two reasons: 30 (1) to widen the spectrum of antiparasitic action to kill different types of parasite, for example macrocyclic lactones are not particularly active against parasites such as liverfluke or tapeworms; and (2) to increase the activity against the same parasite including resistant strains by using different drench families with different modes of action that all act to treat the same parasite, for example parasitic roundworm 35 Drug resistance in anthelmintics, including to the macrocyclic lactone family, is a growing WO 2015/053636 PCT/NZ2014/000214 2 problem in veterinary medicine. To combat resistance and to prolong the life of the anthelmintic drench families, it has become common practice in New Zealand to administer different classes or families of anthelmintic drugs simultaneously. For example see, "P Rattray , Sep 2003, Helminth Parasites in the NZ Meat and Wool Industries. A review of 5 current issues. Meat and Wool Innovation Report". In another example, NZ527961 describes a formulation which combines a macrocyclic lactone with levamisole, benzimidazole and optionally praziquantel. However, the addition of multiple actives from different classes results in formulations which are more difficult and expensive to manufacture. 10 Combinations of two or more macrocyclic lactones using members of the avermectin group are known. W02005037294 describes long acting compositions for controlling parasites which combine ivermectin and abamectin (both avermectins). W02009060063 describes oral or intraruminal compositions including ivermectin, abamectin 15 (both avermectins), albendazole sulfoxide and trichlorfon. An injectable formulation for cattle containing eprinomectin, ivermectin and levamisole phosphate has been registered and commercially launched in New Zealand (Boss Injection, registered in New Zealand 2012). This injection combines ivermectin and eprinomectin (both 20 avermectins) at standard dose rates in conjunction with another active with different activity (levamisole). Barber et al. Vet. Pharmacol. Therap. 26, 343-348, 2003 describes comparative serum disposition kinetics of injectable formulations of doramectin, ivermectin, moxidectin or 25 combinations of two of these actives with each active being administered on either side of the neck of the sheep. The study is a blood study and does not show parasite control rates or administration by any route except injection. It also comments that oral formulations have disadvantages as they are less bioavailable because of their binding to organic matter in the rumen and gastrointestinal tract and that injections overcome this and so do not require the 30 withholding of feed prior to treatment. Resistance can arise by way of selection of resistant parasites. Three main types of selection occur: (1) selection of resistant resident parasites present in the animal immediately after treatment (Head selection - where the resistant parasite is not removed even at the time of 35 treatment); (2) selection of incoming resistant parasite larvae post-treatment (Tail selection where the incoming resistant larvae are able to establish in the persistent activity period WO 2015/053636 PCT/NZ2014/000214 3 following treatment while susceptible larvae cannot); and (3) selection of both resistant parasites present in the animal at the time of treatment and incoming resistant parasite larvae post-treatment (Head and Tail selection). Tail selection with use of moxidectin has been particularly widely investigated and published (for example, see Le Jambre L.F et al., 5 In.J. Parasitology (1999) Jul 29(7) p 1101-11, "Selection for anthelmintic resistance by macrocyclic lactones in Haemonchus contortus" and Ian Sutherland &lan Scott (2009) in "Gastrointestinal Nematodes of Sheep and Cattle. Biology and Control", John Wiley & Sons, page 142). Milbemycins, in particular Moxidectin, have been shown to have very high potency even relative to other macrocyclic lactones's with the ability to remove most resident 10 worms, an attribute that contributes to relatively low head selection. However, milbemycins, in particular moxidectin, also have a particularly long half-life and residence time in the body. The release and elimination following treatment has been suggested to fit with a two compartment model with the ability to redistribute to body fat and then slowly release resulting in low levels of active in the body. This may result in the killing of larvae and 15 prevention of incoming infective worm larvae from re-establishing for a considerable period after administration, therefore conferring persistent activity. However, over time the moxidectin level declines to lower and lower levels until it reaches a point where larvae can establish. More resistant worm larvae can tolerate higher levels and so are able to establish closer and closer to the time of treatment with a consequent loss of the persistent activity. 20 This creates selective advantage where resistant worm larvae establish earlier while sensitive strains cannot. This long residence time and persistent activity is present even in simple formulations of oral moxidectin, particularly in sheep. This differs from the avermectin group of macrocyclic lactones with naturally relatively short half-lives and resident times in the body with little persistent activity or opportunity to allow tail selection to occur. Methods to 25 extend the duration of release of macrocyclic lactones, including the avermectins, by using formulations such as injections, pour-ons, intraruminal devices and implants are known, used commercially and patented. However, a method to change and reduce the selection in the tail of a milbemycin, such as moxidectin, is not known, or at least not widely available, despite the problem of selection being known and researched for some time. Such a method 30 would be highly desirable. In particular, it would be highly desirable to be able to influence changes in re-infestation highly pathogenic and highly fecund (large egg producers, for example Haemonchus spp) after treatment with a milbemycin, such as moxidectin. This could provide the benefit of having very high potency and relatively lower head selection with lower levels of selection of resistance following treatment, including tail selection. 35 It would also be beneficial to be able provide methods of veterinary treatment and/or WO 2015/053636 PCT/NZ2014/000214 4 compositions which provide improved potency against parasites, and, in particular, to slow the development of selection resistance, It would also or alternatively be useful to provide a method of veterinary treatment and/or compositions which provide prolonged treatment against parasites. 5 It is an object of the invention to provide a method of slowing the development of parasite resistance to a macrocyclic lactone in a non-human animal. It is an alternative object of the invention to provide a method of reducing or preventing the incidence of parasites in non human animals. It is a further alternative object of the invention to provide a method of 10 changing the parasitological outcome of a treatment of a non-human animal with a milbemycin. It is a further alternative object of the invention to provide a method of improving the treatment of pathogenic and/or resistant worm strains in an animal. It is a further alternative object of the invention to provide a method of reducing the reinfection by a pathogenic and/or resistant worm strain following treatment. It is a yet further alternative 15 object of the invention to provide a novel veterinary anti-parasitic formulation. It is a still further alternative object of the invention to at least provide a useful choice to the public. Summary of the Invention In broad terms, the invention relates to slowing the development of parasite resistance to a 20 macrocyclic lactone in a non-human animal by providing an improved method or formulation for treating pathogenic and/or resistant worm strains in an animal. The invention is a method or formulation of treating, reducing or preventing the incidence of parasites in non-human animals. However, in the most preferred aspect the invention 25 provides a more beneficial parasitological outcome than with treatment of a non-human animal with a milbemycin alone. The parasitological outcome can be seen as the pattern of reinfection of a parasite following treatment with a milbemycin alone. The methods of the invention change the parasitological outcome to reduce the selection for a resistant parasite strain or the selection for a pathogenic parasite strain, i.e. reducing selective reinfection by 30 the most pathogenic or resistant strains following treatment. In a first aspect the. invention provides a method of improving the treatment of pathogenic and/or resistant worm strains in an animal, the method including administering to the animal a combination of at least two macrocyclic lactones wherein at least one of the macrocyclic 35 lactones is a milbemycin and at least one of the macrocyclic lactones is an avermectin.

WO 2015/053636 PCT/NZ2014/000214 5 In a second aspect the invention provides a method of reducing the reinfection by a pathogenic and/or resistant worm strain following treatment, the method including administering to the animal a parasitically effective amount of at least two macrocyclic lactones, wherein at least one of the macrocyclic lactones is a milbemycin and at least one of 5 the macrocyclic lactones is an avermectin. For the avoidance of doubt, the following preferred embodiments relate to either or both of the first and second aspects. 10 Preferably the milbemycin is selected from any one or more of: moxidectin, milbemycin oxime, nemadectin, milbemectin. Preferably the milbemycin is moxidectin. Preferably the avermectin is selected from any one or more of: abamectin, ivermectin, eprinomectin, doramectin, selamectin, emamectin. 15 Preferably, at least one of the macrocyclic lactones is administered orally. Preferably, at least one of the macrocyclic lactones is administered as an oral liquid drench, tablet, gel or paste. Preferably, at least one of the macrocyclic lactones is administered topically. 20 Preferably, at least one of the macrocyclic lactones is administered as a pour-on. Preferably, at least one of the macrocyclic lactones is administered as a spot-on. 25 Preferably, at least one of the macrocyclic lactones is administered as an injection. Preferably, at least one of the macrocyclic lactones is administered in the form of an intraruminal device. 30 Preferably, at least one of the macrocyclic lactones is administered in the form of a subcutaneous implant. Preferably the macrocyclic lactones are administered within 3 days of each other. Preferably the macrocyclic lactones are administered within 2 days of each other. Preferably the 35 macrocyclic lactones are administered within 1 day of each other.

WO 2015/053636 PCT/NZ2014/000214 6 Preferably the macrocyclic lactones are administered substantially simultaneously. Preferably the macrocyclic lactones are contained in the same formulation. 5 Preferably the macrocyclic lactones are contained in separate formulations delivered sequentially in either order. Preferably the animal is a food producing animal. Preferably the animal is a bovine, deer, sheep, goat and/or buffalo. 10 Preferably the pathogenic worms are any one or more of Haemonchus, Ostertagia, and/or Trichostrongylus species. Preferably the reduction in reinfection by a pathogenic or resistant worm strain is seen at 15 least about 10 days following the administration of the macrocyclic lactones. Preferably the reduction in reinfection by a pathogenic or resistant worm strain is seen at least about 30 days following the administration of the macrocyclic lactones. Preferably the reduction in reinfection by a pathogenic or resistant worm strain is seen at between about 30 days and 100 days following the administration of the macrocyclic lactones. Preferably the reduction in 20 reinfection by a pathogenic or resistant worm strain is seen at least about 40 days following the administration of the macrocyclic lactones. Preferably the reduction in reinfection by a pathogenic or resistant worm strain is seen at between about 40 days and 100 days following the administration of the macrocyclic lactones. Preferably the reduction in reinfection by a pathogenic or resistant worm strain is seen at between about 50 days and 100 days following 25 the administration of the macrocyclic lactones. The reduction in reinfection is in comparison to treatment by a milbemycin alone and includes reduction in overall quantity of infection and/or reduction in ratio of infection by a pathogenic or resistant worm strain to other (less damaging) worm strains. 30 Preferably the method additionally includes administering one or more additional active compound(s) that is/are not a macrocyclic lactone. Preferably one or more additional active compound(s) are administered within 3 days of the macrocyclic lactones. Preferably the one or more additional active compound(s) are administered substantially simultaneously with the 35 macrocyclic lactones. Preferably the one or more additional active compound(s) are contained in the same formulation as the macrocyclic lactones.

WO 2015/053636 PCT/NZ2014/000214 7 Preferably the additional active compound(s) is selected from any one or more of anthelmintics, ectoparasiticides, biologically active extracts, vaccines, insecticides (for example insect growth regulators), minerals, vitamins, biologically active substances (for 5 example vaccines). Preferably, the one or more additional active compound(s) is/are anthelmintics. Preferably the additional anthelmintics are selected from any one or more of anthelmintic nicotinic receptor agonists (for example levamisole, pyrantel and/or morantel), benzimidazoles (for example albendazole, oxfendazole, fendendazole, mebendazole, rycobendazole, parabendazole, triclabendazole, febantel, netobimin, thiabendazole, 10 cambendazole), praziquantel, amino acetonitrile derivatives (AAD) (for example monepantel), and/or closantel. Most preferably, the additional anthelmintic is levamisole. In a third aspect the invention provides is a veterinary anti-parasitic formulation containing a parasitically effective amount of at least one milbemycin and at least one avermectin. 15 Preferably the milbemycin is selected from any one or more of: moxidectin, milbemycin oxime, nemadectin, milbemectin. Preferably the milbemycin is moxidectin. Preferably the avermectin is selected from any one or more of: abamectin, ivermectin, 20 eprinomectin, doramectin, selamectin, emamectin. Preferably the formulation also contains one or more additional active compound(s) that is/are not a macrocyclic lactone. Preferably the additional active compound(s) is selected from any one or more of anthelmintics, ectoparasiticides, biologically active 25 extracts, vaccines, insecticides (for example insect growth regulators), minerals, vitamins, biologically active substances (for example vaccines). Preferably, the one or more additional active compound(s) is/are anthelmintics. Preferably the additional anthelmintics are selected from any one or more of anthelmintic nicotinic receptor agonists (for example levamisole, pyrantel and/or morantel), benzimidazoles (for example albendazole, oxfendazole, 30 fendendazole, mebendazole, rycobendazole, parabendazole, triclabendazole, febantel, netobimin, thiabendazole, cambendazole), praziquantel, amino acetonitrile derivatives (AAD) (for example monepantel), and/or closantel. Most preferably, the additional anthelmintic is levamisole. 35 Preferably the formulation is adapted to be administered orally, parenterally, topically, by way of an intraruminal device, by way of a subcutaneous implant. More preferably, the WO 2015/053636 PCT/NZ2014/000214 8 formulation is adapted to be administered orally or topically. Preferably, the formulation is in the form of a liquid, tablet, gel or paste. 5 As used herein the term "and/or" means "and" or "or", or both. As used herein "(s)" following a noun means the plural and/or singular forms of the noun. The term "comprising" as used in this specification means "consisting at least in part of". When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present, but other features can also be present. Related terms such as 10 "comprise" and "comprised" are to be interpreted in the same manner. It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range 15 (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7). To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and application of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The 20 disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting. Further aspects of the invention, which should be considered in all its novel aspects, will become apparent to those skilled in the art upon reading of the following description which 25 provides at least one example of a practical application of the invention. Brief Description of the Drawings Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which: 30 Figure 1 shows means egg count reduction of Strongyles following treatment orally with known moxidectin formulations compared to treatment orally with a moxidectin and ivermectin combination of the invention. Figure 2 shows means egg count reduction of Nematodirus following treatment orally with known moxidectin formulations compared to treatment orally with a 35 moxidectin with ivermectin combination of the invention. Figure 3 shows percentage of Haemonchus in larval culture following treatment orally WO 2015/053636 PCT/NZ2014/000214 9 with known moxidectin formulations compared to treatment orally with moxidectin and ivermectin combination of the invention. Detailed Description of Preferred Embodiments 5 The invention broadly relates to methods for treating, reducing or preventing the incidence of pathogenic worms in non-human animals. In particular, the methods include the step of administering at least two macrocyclic lactones to the non-human animal at least one of which is a milbemycin and at least one of which is an avermectin. The invention also broadly relates to compositions for use in reducing or preventing the incidence of pathogenic worms 10 in non-human animals which combine at least one milbemycin with at least one avermectin. The invention further broadly relates to methods of slowing the development of parasite resistance to a macrocyclic lactone in a non-human animal. The invention further broadly relates to changing the parasite outcome following treatment 15 such that there is less selection for resistant and/or pathogenic parasite strains than is seen when there is treatment with milbemycin alone. By combining milbemycin treatment with treatment with an avermectin at substantially the same time (or within a few days) the pattern of reinfection of a parasite following the treatment is improved such that there is reduction of the selection for a resistant parasite strains or the selection for a pathogenic parasite strains. 20 The method and compositions of the invention utilise the benefits of the two types of macrocyclic lactones (avermectins and milbemycins) while reducing/modifying the negative impacts of persistent activity and tail selection when using a milbemycin treatment alone. Milbemycins are known to be highly potent, and therefore there use is desirable, particularly against pathogenic worms such as Haemonchus. However, the negative impact of the 25 inherent persistent activity and tail of the milbemycins is a significant disadvantage. Under normal, particularly pasture, farming conditions there will nearly always be reinfection following anthelmintic treatment as the worms (in various life stages) are present in the surroundings. However, a detrimental outcome of anthelmintic treatment can be reinfection 30 with more pathogenic or resistant worm strains than were originally present prior to treatment. This is a common outcome as the stronger (i.e. more pathogenic or resistant) worm are able to re-infect sooner and at a greater rate. This can result in a higher level of pathogenic and resistant worm strains both in the animal and in the local surrounds, which can have a detrimental effect on both the animal treated, other animals which are moved into 35 the same local area and future generations raised in the same area.

WO 2015/053636 PCT/NZ2014/000214 10 Macrocyclic lactones (or ML's) are a class of widely used compounds that have a broad range of activity including against internal and external parasites of animals. It was previously thought that most common macrocyclic lactones used in animals have similar spectrum of activities. The avermectin and milbemycin family are often referred to on labels of registered 5 products as being synonymous and as having the same mode of action. However, recently some differences have been noted between the members of the macrocyclic lactone in relation to resistance by internal parasitic worm strains in sheep and goats (Kieran, P.J Aust Vet J 1994, 71, Issue 1, pages 18-20, Moxidectin against ivermectin-resistant nematodes - a global view). 10 Milbemycins include but are not limited to: moxidectin, milbemycin oxime, nemadectin, milbemectin. In preferred embodiments of the invention the methods and formulations include moxidectin. 15 Avermectins include but are not limited to: abamectin, ivermectin, doramectin, eprinomectin, selamectin, emamectin. The methods of the invention treat, reduce or prevent the incidence of parasites in non human animals (i.e. veterinary treatment) by reducing reinfection by resistant and/or 20 pathogenic worms. Such non-human animals include bovine, deer, sheep, goat and/or buffalo. Treatment of parasites in such animals can be either to prevent illness and/or death and/or for animal welfare, or alternatively can be to improve productively. Preferably the pathogenic worms are the species Haemonchus, Ostertagia and/or Trichostrongylus. 25 Herein reference to "resistant worm strains" or "resistance" or the like, should be taken to mean a worm strain which when treated with an active (in this case macrocyclic lactone) has a kill rate or level of control which is lower than that previously documented for the same at active at the same dose. 30 Pathogenic worms there those that are known to cause significant disease. It is known from tests on resistant worm strains that increasing dose rates of a single active once resistance has developed does not generally improve the control of resistant parasites. Thus is it surprising that combining two macrocyclic lactones in the methods of the invention 35 (an avermectin and a milbemycin) can have an effect on resistant strains of worms. While not wishing to be bound by theory, the inventors believe that while the macrocyclic lactones have WO 2015/053636 PCT/NZ2014/000214 11 common mechanisms of action there are also subtle but significant differences in the action of the milbemycins and the avermectins which the methods and compositions of the invention exploit. The invention in part takes advantage of these apparent differences. The methods and formulations of the invention provide a convenient method of treatment with 5 less need to test for specific resistance in the animal prior to treatment. There are known pharmacokinetic differences between the two groups of macrocyclic lactones, milbemycins and avermectins, including their distribution in animal tissues and their subsequent release and period of persistent activity (for example see, R Prichard et al 10 (2012), International Journal of Parasitology Pages134-153. "Drugs and Drug Interactions. Moxidectin and the Avermectins. Consanguinity but not identity"). While all macrocyclic lactones are lipophilic, milbemycins (particularly moxidectin) are much more lipophilic (high affinity for fat) and have a much longer residence time and slower elimination time in animals than the avermectins (such as ivermectin). This slow release of milbemycin (particularly 15 moxidectin) and it's very high potency against some worm strains results in persistent or prolonged activity. For example, following liquid oral treatment with moxidectin in sheep there can be continued kill of incoming worm larvae from such worms as Haemonchus species for up to 35 days after treatment and Ostertagia species 21 days - if resistance has not built up. The registered product Cydectin oral for sheep (NZ Reg No.A006204) containing moxidectin 20 has label claims of controlling Haemonchus contortus for 35 days and Ostertagia circumcincta for at least 21 days following a single oral dose. In contrast oral avermectins such as ivermectin or abamectin in sheep have no such label claims with less than 7 days activity and likely only 2-5 days activity after treatment against incoming larvae. A summary of the comparison of the half-life and persistent actives of the milbemycins and avermectins 25 is shown in Table 1. Avermectin formulations such as oral ivermectin or abamectin (as shown in Table 1), are more rapidly eliminated from the body and have no significant tail, nor claims for persistent activity, and so do not exert the same tail selection as the milbemycins (particularly moxidectin). 30 Table 1 - Comparison of Half-lives/Residence times of a milbemycin versus avermectins oral formulations in sheep. Macrocyclic Lactone Half-life and/or Residence Persistent Activity times in sheep oral in sheep oral (approximate examples) (label claims) Milbemycins Moxidectin Long 35 days against incoming (approx. 12.5 days) Haemonchus larvae, Low levels detected up to 60 21 days for Ostertagia, WO 2015/053636 PCT/NZ2014/000214 12 ___________________days post treatment 7 days for Trichostrongylus Avermectins Ivermectin Short None (less than 6 days ) Abamectin Short None (less than 6 days ) Doramectin Short None (less than 6 days ) Eprinomectin Short None (less than 6 days ) The persistent activity of the milbemycins (particularly moxidectin), is known to result in a declining "tail" of active when the active is no longer at full therapeutic dose. This tail can result in "tail selection" where the incoming parasite larvae post-treatment are those which 5 have some resistance to the milbemycin and are therefore able to re-infect during this tail period. This tail selection can result in reinfection and proliferation by more pathogenic and resistant worm strains. Over time this favours a more resistant genotype of larvae to develop. These resistant worm larvae are able to withstand higher and higher moxidectin levels and thereby establish earlier and earlier within this tail, while the sensitive genotype remains 10 unable to do so. As such the persistent activity period progressively shortens until it no longer is present. This favours the resistant genotype allowing them to re-establish and produce eggs earlier and therefore contaminate pasture first. Unlike moxidectin oral, the avermectin oral formulations do not have such long residence 15 times or periods of persistent activity and so the opportunity for tail selection is reduced or almost non-existent as susceptible strains can re-establish relatively quickly after treatment. The persistent activity of the avermectins can be enhanced by formulation or administration route including delivery as a pour on or spot on, by injection or implant, or by intraruminal 20 capsule (for example 100 day intraruminal capsules) or controlled release device in ruminants such as cattle, deer, sheep and goats. However, the persistent activity of the milbemycins (particularly moxidectin), is a feature of the active itself and is not formulation based, thus the persistent activity exists even in simple oral formulations. The persistence of milbemycin (for example moxidectin) is quite different from the intraruminal capsules or 25 controlled release devices. In the capsule a consistent continuous low daily dose is released and it does not have the declining tail of active seen with milbemycin oral. As show in Example 1 of this application, conducted on two different farms with resistance to macrocyclic lactones, the use of controlled release devices resulted in very different parasite outcomes after treatment, because they provide a low level of macrocyclic lactone WO 2015/053636 PCT/NZ2014/000214 13 (ivermectin in the case of Example 1) over a long period. Moxidectin oral alone resulted in very high egg output and therefore pasture contamination of the highly pathogenic worm Haemonchus contortus while the combined milbemycin (moxidectin) and avermectin (ivermectin) treatment of the invention, and the ivermectin controlled release capsule 5 appeared far less selective with less egg output and relatively much lower proportions of the particularly pathogenic Haemonchus larvae. Persistent activity is defined as per requirements for label claims, which is that there is demonstrated activity against incoming larvae of a worm species for 7 days or more after 10 treatment and that can kill and prevent establishment of more than 90% or more of susceptible incoming larvae of a worm species relative to untreated control animals. Surprisingly the inventors have shown the combined use of milbemycins (such as moxidectin) and the avermectins (such as ivermectin) appears to have different properties 15 and parasite outcomes to the outcomes when of each active is used alone. This effect is evident, for example, within and after the tail effect of the treatment. This can be seen in changes in a reduction in selection for resistant and/or more pathogenic worm strains in a period of time after treatment by the methods of the invention. The inventors have found the use of an avermectin (for example ivermectin) with a milbemycin (for example moxidectin), 20 make the milbemycin less selective at the tail, reducing or delaying the reinfection by parasites particularly the more pathogenic and resistant parasites. This is particularly surprising when the avermectin used is short acting (for example, less than 7 days), thus it would not be predicted that the avermectin would have any effect on the outcome many days after treatment (for example more than 7 days, but often at 35 days or more). 25 The use of the methods and compositions of the invention potentially also, or alternatively, make the initial treatment more effective (to combat head selection). Use of the formulation of the invention including an avermectin (such as ivermectin) in 30 combination with a milbemycin (such as moxidectin) has applications not only against existing resistance worm strains but also in slowing their development by reducing known forms of resistance selection The macrocyclic lactones used in the invention are preferably administered substantially 35 simultaneously, or alternatively may be administered as separate formulations in quick succession in any order, for example, normally within an hour of each other, but could be WO 2015/053636 PCT/NZ2014/000214 14 delivered up to three days apart, more preferably two days apart, even more preferably one day apart. Most preferably the macrocyclic lactones are combined in a single formulation to provide a fast and efficient means of administration. 5 The avermectin and milbemycin are administered to the animal at parasitically, preferably anthelmintically, effective amounts. Their use rates in animals relative to body weight (the amount of macrocyclic lactone in mg, relative to kg of body weight of the animal, typically expressed as mg/kg) are relatively standard and uniform throughout the world, depending on the method of administration and parasite treated. Typically for administration orally or by 10 injection the standard dose rate for a macrocyclic lactone is 0.2 mg/kg. Preferably the methods of the invention when delivered orally or by injection provide a total milbemycin dose (i.e. total of one or more milbemycins) at about 0.1 mg/kg to about 0.5 mg/kg and a total avermectin dose (i.e. total of one or more avermectins) at about 0.1 mg/kg to about 0.5 mg/kg. Typically for administration as a pour on the standard dose rate for a macrocyclic 15 lactone is 0.5 mg/kg. Preferably the methods of the invention when delivered by pour on provide a total milbemycin dose (i.e. total of one or more milbemycins) at about 0.2 mg/kg to about 1.5 mg/kg and a total avermectin dose (i.e. total of one or more avermectins) at about 0.2 mg/kg to about 1.5 mg/kg. The standard dose rate for a macrocyclic lactone delivered as an intraruminal device or subcutaneous implant ("capsule") is about 0.02 mg/kg/day. 20 Preferably the methods of the invention when delivered by intraruminal device or subcutaneous implant provide a total milbemycin dose (i.e. total of one or more milbemycins) at about 0.01 mg/kg/day to about 0.1 mg/kg/day and a total avermectin dose (i.e. total of one or more avermectins) at about 0.01 mg/kg/day to about 0.1 mg/kg/day. 25 The invention includes formulations which provide a parasitically effective amount of at least one milbemycin and at least one avermectin for use in the treatment of non-human animals. The invention optionally includes the use of more than milbemycin in combination with an avermectin, or alternatively the use of a single milbemycin with more than one avermectin, or alternatively more than one milbemycin with more than one avermectin. Particularly preferred 30 combinations of an avermectin with a milbemycin are: ivermectin with moxidectin, and/or eprinomectin with moxidectin, which the inventors have demonstrated are particularly effective (see Examples 1 and 2). The methods of the invention include administering one or more of the macrocyclic lactones 35 orally, topically, parenterally (injection) and/or in the form of an intraruminal device or a subcutaneous implant. The formulations of the invention are preferably formulated for WO 2015/053636 PCT/NZ2014/000214 15 administration by one of these methods. Where the macrocyclic lactones are administered orally they are preferably in the form of a liquid drench, tablet, gel or paste. Where the macrocyclic lactones are administered topically they are preferably in the form of a pour-on or a spot-on. In a more preferred embodiment, the macrocyclic lactones are administered 5 orally. The inventors have surprisingly shown oral administration to be very effective (see Example 1) despite oral administration being naturally short acting for the avermectins. It is therefore particularly surprising that the avermectin has an effect on the reinfection profile, which included a more mixed less selected worm population with less pathogenic worm eggs such as Haemonchus and more similar to untreated controls following treatment. In an 10 alternative preferred embodiment, the macrocyclic lactones are administered topically (for example pour on). Topical administration naturally has a lower peak and tail off adsorption profile in the animal for all macrocyclic lactones (not just the milbemycins), therefore could be particularly prone to poor reinfection profiles. It is therefore particularly surprising that the inventors have shown the reinfection profile can be improved by use of a combination of an 15 avermectin with the milbemycin. This includes less pathogenic and apparently macrocyclic lactone resistant Ostertagia. In contrast the adsorption profile of injectable can be significantly adjusted using formulation techniques including pattern of release from the injection site (even biphasic release is known) which can change the length or duration of a tail for different macrocyclic lactones. For example, persistent activity for over 100 days has 20 been demonstrated by this means. A person skilled in the art would be capable to formulating macrocyclic lactone formulations, for example, oral, topical, injectable and capsule formulations. For example, solvents which may be used in the formulations of the invention include (but are not limited to) alcohols (for 25 example, C 1

.

8 -alcohols, benzyl alcohol, ethanol, methanol, isopropanol), acetone, acetonitrile, dimethylacetamide, dimethylformamide, glycol ethers (for example, but not limited to, dipropylene glycol n-butyl ether, ethylene glycol monoethyl ether, ethylene glycol, ethylene glycol monomethyl ether, dipropylene glycol monomethyl ether, diethylene glycol monoethyl ether), propylene glycol, pyrrolidones (for example, but not limited to, 2-pyrrolidone, N 30 methylpyrrolidone), vegetable oils (for example, but not limited to castor oil), mineral oil, fatty acid esters (for example, but not limited to, Miglyol 810, 812, 818, 829, 840). A single formulation of a combination of actives according to the invention could be administered sequentially with another formulation as well. Optionally the methods and 35 formations of the invention may include one or more additional active compound(s) which are not macrocyclic lactones. The one or more additional active compound(s) are preferably WO 2015/053636 PCT/NZ2014/000214 16 administered within a few days of the macrocyclic lactones, either before the macrocyclic lactones or after the macrocyclic lactones. Preferably the one or more additional active compound(s) are administered within 3 days of the macrocyclic lactones, more preferably, substantially simultaneously with the macrocyclic lactones. Preferably, the one or more 5 additional active compound(s) are combined in a single formulation or device with the at least one of the macrocyclic lactones, to provide convenience of administration. Most preferably, all the macrocyclic lactones and additional actives are combined in a single formulation or device. 10 Preferably, the one or more additional active compound(s) is/are anthelmintics. Examples of additional anthelmintics which may be used in the invention include anthelmintic nicotinic receptor agonists (for example levamisole, pyrantel and/or morantel), benzimidazoles (for example albendazole, oxfendazole, fendendazole, mebendazole, rycobendazole, parbendazole, triclabendazole, febantel, netobimin, thiabendazole, cambendazole), 15 praziquantel, amino acetonitrile derivatives (AAD) (for example monepantel) and/or closantel. In a preferred embodiment the additional active is levamisole. Other actives include insecticides (including insect growth regulators), minerals, vitamins, and biologically active substances (such as vaccines). It would be within the knowledge of a person skilled in the art to incorporate such additional actives at their standard rates/amounts, with known 20 formulation requirements. Preferably the reduction in reinfection by a pathogenic or resistant worm strain is seen at least about 10 days following the administration of the macrocyclic lactones, more preferably least about 30 days following the administration of the macrocyclic lactones, for example 25 between about 30 days and 100 days following the administration of the macrocyclic lactones. Most preferably the reduction in reinfection by a pathogenic or resistant worm strain is seen at least about 40 days following the administration of the macrocyclic lactones, for example between about 40 days and 100 days following the administration of the macrocyclic lactones. Periods of at least 10 days, or at least 30 days are beneficial where 30 there is reduction in persistence due to resistance. However, periods of greater than 40 days are particularly surprising given the normal persistence periods of oral moxidectin. The "reduction in reinfection" is in comparison to treatment by a milbemycin alone and includes reduction in overall quantity of infection and/or reduction in ratio of infection by a pathogenic or resistant worm strain to other (less damaging) worm strains. 35 WO 2015/053636 PCT/NZ2014/000214 17 Example 1 - Animal Trial (Sheep) Two parasitology trials were conducted in newly weaned 20-40kg lambs all with positive faecal roundworm egg counts at two different farm sites (Farm A and Farm B) in North Auckland , New Zealand in summer (from January to March 2013). 5 The trial was designed to demonstrate the activity of orally administered ivermectin (Ivomec@ liquid for sheep) and orally administered moxidectin (Cydectin@ oral for sheep, or VetMed@ Moxidectin selenium Sheep) when administered individually at the standard dose of 0.200mg/kg. The trial also included an ivermectin capsule (Ivomec Maxmiser) to demonstrate 10 the effect of continuous low dose ivermectin (approximately 1/10 of oral dose, 0.020mg/kg/day) i.e. an avermectin which has been formulated to provide a persistent but constant daily dose. Cydectin @ oral for sheep claims persistent activity to prevent reinfection with Haemonchus 15 contortus for 35 days and Ostertagia circumcincta (Teladorsagia) for at least 21 days following a single oral dose (at recommended dose rate). VetMed@ Moxidectin selenium Sheep also claims persistent activity to prevent reinfection with Haemonchus contortus for 35 days and Teladorsagia circumcincta for at least 21 days following a single oral dose (at recommended dose rate). Ivomec@ liquid for sheep does not have a claim for persistent 20 activity. These were compared to another group where a mixture of ivermectin and moxidectin was orally dosed simultaneously into the same animal at the standard dose rates of 0.200mg/kg for each of the macrocyclic lactones (total 0.400mg/kg of both macrocyclic lactones). 25 The treatment groups were as follows: Farm A 1. Ivermectin oral (Ivomec @sheep liquid, 0.200mg ivermectin /kg bodyweight). 2. Moxidectin oral (Cydectin @, 0.200mg moxidectin/kg bodyweight). 30 3. Moxidectin oral (VetMed @ moxidectin, 0.200mg moxidectin/kg bodyweight). 4. Combined ivermectin oral (Ivomec @sheep liquid, 0.200mg ivermectin /kg bodyweight) and Moxidectin oral (Cydectin @, 0.200mg moxidectin/kg bodyweight). Farm B 1. Ivermectin oral (Ivomec @sheep liquid, 0.200mg ivermectin /kg bodyweight). 35 2. Moxidectin oral (VetMed@ moxidectin, 0.200mg moxidectin/kg bodyweight). 3. Combined ivermectin oral (Ivomec @sheep liquid, 0.200mg ivermectin /kg WO 2015/053636 PCT/NZ2014/000214 18 bodyweight) and Moxidectin oral (Cydectin @, 0.200mg moxidectin/kg bodyweight). Trial Procedure The two farms were selected as they were known to have some roundworm parasites with 5 some resistance to macrocyclic lactones, but were over 30km apart and had different drench management practices. All lambs were individually tagged, weighed using calibrated scales, and faecal sampled and allocated to group (8-10 per group) using egg count to give groups with similar means and distributions of egg count. 10 The lambs were dosed based on individual bodyweight and re-faecal sampled per rectum following treatment at various time intervals using a clean glove per animals to prevent cross contamination of samples. The faecal samples were then examined for egg count (Mc Master technique) and at a sensitivity of 50epg. Each treatment group had the faecal samples pooled evenly by lamb to give a 50 g sample that was cultured for larvae, with the 15 larval numbers determined and identified to worm genera. Those lambs in the combined ivermectin and moxidectin group were dosed 8-10 days later relative to the other groups to act as untreated controls at Day 8-10. All animals were run as one mob on each farm site. 20 Results: The results from Farm A and Farm B are provided below. Results are also shown in Figures 1, 2 and 3. 25 Table 2 - (comparative) Farm A (Group 7) Ivermectin oral (Ivomec @sheep liquid, 0.200mg ivermectin /kg bodyweight). Treated 22 January. Treatment Group Group 7 Group 7 Group 7 Removed (untreated) From study Time after treatment 18-Jan 1-Feb 9-Feb Day -4 Day 10 Day 18 Strongyle 1166.7 550 688.3 (Mean Egg count) epg Reduction (%) NA 52.9% 59% Nematodirus 37.51 0.0 0.0 (Mean Egg count) epg Reduction (%) NA 100% 100% Larval Culture Total larvae/50g 85,0001 28,000 12,000 Haemonchus (%) 80 92 94 Ostertagia (%) 3 8 6 WO 2015/053636 PCT/NZ2014/000214 19 Trichostrongylus % 4 0 0 Cooperia (%) 12 0 0 Oesophagostomuml 1 0 0 Chabertia (%) Nematodirus eggs and larval culture for Group 1, Untreated controls at Day 8 (9-Feb). Table 3 - (comparative) Farm A (Group 5) Moxidectin oral (Cydectin @, 0.200mg moxidectin/kg bodyweight). Treated 22 January. Treatment Group Group 5 Group 5 Group 5 Group 5 Group 5 (untreated) Time after treatment 18-Jan 1-Feb 9-Feb 27-Feb 26-Mar Day -4 Day 8 Day 18 Day 36 Day 63 Strongyle 1240.0 14.3 0.0 2010.0 2056.3 (Mean Egg count) epg Reduction (%) NA 98.8% 100% -62.1% -65.8% Nematodirus 37.51 0.0 0.0 40 81.3 (Mean Egg count) epg Reduction (%) NA 100% 100% -6.7 -116.7% Larval Culture Total larvae/50g 85,000 1 110 0 80,000 85,000 Haemonchus (%) 80 0 0 81 97 Ostertagia (%) 3 94 0 8 1 Trichostrongylus (%) 4 0 0 6 2 Cooperia (%) 12 0 0 5 0 Oesophagostomuml 1 0 0 0 0 Chabertia (%) _ 5 Nematodirus eggs and larval culture for Group 1, Untreated controls at Day 8 (9-Feb). Table 4 - (comparative) Farm A (Group 6) Moxidectin oral (VetMed @ moxidectin, 0.200mg moxidectin/kg bodyweight). Treated 22 January. Treatment Group Group 5 Group 5 Group 5 Group 5 Group 5 (untreated) Time after treatment 18-Jan 1-Feb 9-Feb 27-Feb 26-Mar Day -4 Day 8 Day 18 Day 36 Day 63 Strongyle 1166.7 5.6 11.1 2131.3 2200.0 (Mean Egg count) epg Reduction (%) NA 99.5% 99.0% -82.7% -88.6% Nematodirus 37.51 0 0 0 0 (Mean Egg count) epg Reduction (%) 100% 100% 100% 100% Larval Culture 0 - 018, 07 Total larvae/50g 85,000 1 120 0 18,000 76,000 Haemonchus (%) 80 5 0 90 86 Ostertagia (%) 3 95 0 6 2 Trichostrongylus (%) 4 0 0 0 6 Cooperia (%) 12 0 0 4 6 Oesophagostomuml 1 0 0 0 0 Chabertia (%) WO 2015/053636 PCT/NZ2014/000214 20 Nematodirus eggs and larval culture for Group 1, Untreated controls at Day 8 (9-Feb). Table 5 - (invention) Farm A (Group 1) Ivermectin oral (Ivomec @sheep liquid, 0.200mg ivermectin /kg bodyweight) and Moxidectin oral (Cydectin @, 0.200mg moxidectin/kg 5 bodyweight). Treated 1 Feb Treatment Group Group 1 Group 1 Group 1 Group 1 Group 1 (untreated) lv+Mox lv+Mox lv+Mox lv+Mox Time after treatment 18-Jan 1-Feb 9-Feb 27-Feb 26-Mar Day -15 Day 0 Day 8 Day 26 Day 53 Strongyle 1045.0 1118 0 200 1100 (Mean Egg count) epg Reduction (%) NA NA 100% 80.9% -5.0% Nematodirus 37.5 37.5 0.0 0.0 0.0 (Mean Egg count) epg Reduction (%) NA NA 100% 100% 100% Larval Culture Total larvae/50g 85,0001 85 5 0 140 40,000 Haemonchus (%) 80 80 0 0 53 Ostertagia (%) 3 3 0 18 0 Trichostrongylus (%) 4 4 0 72 8 Cooperia (%) 12 12 0 10 39 Oesophagostomuml 1 1 0 0 0 Chabertia (%) I I Nematodirus eggs and larval culture for Group 1, Untreated controls at Day 8 (9-Feb). Table 6 - (comparative) Farm A (Group 9) Ivomec @Maximiser Lamb 100 day capsule (20 40kg), 0.02mg/kg/day ivermectin for 40kg sheep. Treated 22 January. Treatment Group Group 5 Group 5 Group 5 Group 5 Group 5 (untreated) Time after treatment 18-Jan 1-Feb 9-Feb 27-Feb 26-Mar Day -4 Day 8 Day 18 Day 36 Day 63 Strongyle 1240.0 475.0 278.0 80.0 308.3 (Mean Egg count) epg Reduction (%) NA 58.5% 75.7% 93.0% 73.1% Nematodirus 37.51 0 0 0 0 (Mean Egg count) epg Reduction (%) NA 100% 100% 100% 100% Larval Culture Total larvae/50g 85,000 1 110 0 80,000 85,000 Haemonchus (%) 80 0 0 81 97 Ostertagia (%) 3 94 0 8 1 Trichostrongylus (%) 4 0 0 6 2 Cooperia (%) 12 0 6 5 0 Oesophagostomuml 1 0 0 0 0 Chabertia (%) _L 10 T Nematodirus eggs and larval culture for Group 1, Untreated controls at Day 8 (9-Feb).

WO 2015/053636 PCT/NZ2014/000214 21 Table 7 - (comparative) Farm B (Group 10) Ivermectin oral (Ivomec @sheep liquid, 0.200mg ivermectin /kg bodyweight). Treated 24 January. Treatment Group Group 1 Group 10 Group 10 Removed Removed (untreated) From From study study Time after treatment 21-Jan 3-Feb 11-Feb Day -3 Day 10 Day 18 Strongyle 605.0 210.0 120.0 (Mean Egg count) epg Reduction (%) NA 65.0% 80.0% Nematodirus 0 0.0 0.0 (Mean Egg count) epg Reduction (%) NA NA NA Larval Culture Total larvae/50g 85,0001 4,800 2,880 Haemonchus (%) 36 0 18 Ostertagia (%) 12 74 44 Trichostrongylus (%) 18 14 10 Cooperia (%) 31 12 28 Oesophagostomuml 3 0 0 Chabertia (%) T Larval culture for Group 1, Untreated controls at Day 10 (3-Feb). 5 Table 8 - (comparative) Farm B (Group 5) Moxidectin oral (VetMed@ moxidectin, 0.200mg moxidectin/kg bodyweight). Treated 24 January. Treatment Group Group 5 Group 5 Group 5 Group 5 Group 5 (untreated) Time after treatment 21-Jan 3-Feb 11-Feb 28-Feb 26-Mar Day -3 Day 10 Day 18 Day 35 Day 61 Strongyle 620 0 35 250 2025.0 (Mean Egg count) epg Reduction (%) NA 100% 94.0% 60.0% -227.0% Nematodirus 0 0 0 0 0 (Mean Egg count) epg Reduction (%) NA NA NA NA NA Larval Culture Total larvae/50g 85,0001 1,400 1,650 7,700 30,000 Haemonchus (%) 36 0 0 70 78 Ostertagia (%) 12 95 83 8 0 Trichostrongylus (%) 18 2 17 2 3 Cooperia (%) 31 3 0 18 18 Oesophagostomuml 3 0 0 2 1 Chabertia (%) 1 Larval culture for Group 1, Untreated controls at Day 10 (3-Feb) 10 WO 2015/053636 PCT/NZ2014/000214 22 Table 9 - (comparative) Farm B (Group 12) Ivomec @Maximiser Lamb (20-40kg), 0.02mg/kg/day ivermectin for 40kg sheep. 100 day capsule. Treated 24 January. Treatment Group Group 1 Group 12 Group 12 Group 12 Group 12 (untreated) Time after treatment 21-Jan 3-Feb 11-Feb 28-Feb 26-Mar Day -3 Day 10 Day 18 Day 35 Day 61 Strongyle 620.0 545.0 185.0 135 195 (Mean Egg count) epg Reduction (%) NA 12.1% 70.2% 78.2% 69.0% Nematodirus 0 0 0 0 0 (Mean Egg count) epg Reduction (%) NA NA NA NA NA Larval Culture Total larvae/50g 85,000 1 5,530 2,100 1,750 22,000 Haemonchus (%) 36 26 45 16 8 Ostertagia (%) 12 40 18 24 22 Trichostrongylus (%) 18 30 29 40 50 Cooperia (%) 31 4 8 20 20 Oesophagostomuml 3 0 0 0 0 Chabertia (%) 1 Larval culture for Group 1, Untreated controls at Day 10 (3-Feb) 5 Table 10 - (invention) Farm B (Group 1) Ivermectin oral (Ivomec @sheep liquid, 0.200mg ivermectin /kg bodyweight) and Moxidectin oral (Cydectin @, 0.200mg moxidectin/kg bodyweight). Treated 1 February. Treatment Group Group 5 Group 5 Group 5 Group 5 Group 5 (untreated) Time after treatment 21-Jan 3-Feb 11-Feb 28-Feb 26-Mar Day -3 Day 0 Day 8 Day 25 Day 51 Strongyle 705.0 2350 12.5 250 550 (Mean Egg count) epg Reduction (%) NA NA 98.5% 60.0% 22.12% Nematodirus 0 0 0 0 0 (Mean Egg count) epg Reduction (%) NA NA NA NA NA Larval Culture Total larvae/50g 85,0001 85,00 7 50 2,800 1,350 Haemonchus (%) 36 36 0 0 58 Ostertagia (%) 12 12 100 28 3 Trichostrongylus (%) 18 18 0 11 8 Cooperia (%) 31 31 0 61 30 Oesophagostomuml 3 3 0 0 1 Chabertia (%) 1 Larval culture for Group 1, Untreated controls at Day 10 (3-Feb). 10 Findings: Both farms demonstrated different patterns of macrocyclic lactone resistance but on both WO 2015/053636 PCT/NZ2014/000214 23 farms ivermectin oral alone was ineffective with only a 50-80% reduction in egg count 8-18 days after treatment (due to the short acting nature of the avermectin). Moxidectin on both farms appeared effective or highly effective (94% and greater) at Days 8-18 post treatment. The ivermectin and moxidectin mix of the invention also appeared highly effective but not 5 fully effective at Day 8-10 with 98.5-100% reduction. However 25-26 days after treatment the ivermectin and moxidectin mix of the invention was 60-81% effective. There was evidence of a mixed infection of Trichostrongylus, Ostertagia and Cooperia but no Haemonchus at this time. Surprisingly even 51 to 53 days later the egg 10 counts in these groups on both farms were still below pre-treatment levels and contained a mix of most worm species with only 53 to 58% of Haemonchus larvae and with quite high numbers of Cooperia (30-39%). This effect was observed on both farms. In contrast by 36-63 days on both farms the lambs treated with moxidectin only showed 15 increases in both egg count and larval numbers, with mean egg counts rising in both farms up to 2,000 epg and higher, and larval cultures Haemonchus dominant at 81-97% (Farm A) and 70-78% (Farm B). Nematodirus eggs were also found at these times in only the moxidectin treated group (Cydectin 6 out of 8 animals) and not in any other group including those treated with combined moxidectin and ivermectin of the invention. 20 In Farm A there was evidence of a Haemonchus species and Ostertagia with resistance to ivermectin, which moxidectin appeared to control initially. In Farm B there appeared a more general lower level of resistance that appeared to involve multiple worm genera including Ostertagia, Trichostrongylus and Cooperia and possibly also Haemonchus. 25 Figure 1 shows the means egg count reduction of Strongyle following treatment with known moxidectin orals and combination moxidectin with ivermectin oral of the invention. Of particular note is the re-infestation of Strongyle (large egg output which is usually strongly correlated to worm numbers in young sheep) in the known moxidectin orals around 40-60 30 days following treatment. This is in contrast to the mean egg counts for Strongyle in the moxidectin with ivermectin combination of the invention, which continues to show some effectiveness, or at least not a large re-infestation or large egg output, at 50-60 days following treatment. This is particularly surprising given that ivermectin is not considered to have persistent activity (which is supported by the finding for ivermectin oral in this study), so 35 would not be expected to have any effect on efficacy 40-60 days following treatment. Figure 2 shows mean egg count reduction of Nematodirus following treatment with known WO 2015/053636 PCT/NZ2014/000214 24 moxidectin orals and combination moxidectin with ivermectin oral of the invention. While the effect is not as pronounced as for Strongyle, at least one of the known moxidectin orals shows large re-infestation at around 40 days following treatment, whereas the moxidectin with ivermectin combination of the invention and one of the known moxidectin orals 5 continues to show efficacy 60 days following treatment. The percentage of Haemonchus in larval culture for the moxidectin orals compared to the combined moxidectin with ivermectin oral is shown in Figure 3. This shows how the percentage of Haemonchus in the larvae following treatment with known moxidectin orals is 10 significantly higher than following treatment with the combination moxidectin with ivermectin of the invention. Haemonchus is a particularly pathogenic worm which can be damaging to the growth of lambs. Moxidectin used alone appeared to have lost most of its persistent activity in both farms, 15 likely due to resistance macrocyclic lactones. If it is assumed that it takes at least 21 days from infective larvae to patency (egg production), even for Haemonchus, then it appears that moxidectin has less than 14 days persistent activity. Further once the persistent activity ceases there is massive egg laying by re-establishing Haemonchus which contaminates pasture with what is a highly pathogenic worm. On both Farms, when moxidectin and 20 ivermectin were dosed simultaneously (according to the invention), it appeared that this effect was much reduced. The egg output is not only lower, but the output also shifts to a higher percentage of less pathogenic worms, such as Cooperia. While on Farm A lambs treated with moxidectin (Cydectin@) alone had Nemadirus eggs detected at several time points in most animals while, those treated with moxidectin (Cydectin@) and ivermectin 25 (Ivomec@) as per the invention, these were not detected in any animal at any time after treatment. The results for Ivomec@ Maximiser Lamb are provided for comparison with the moxidectin orals. Ivomec@ Maximiser Lamb is an intraruminal capsule that provides a persistent and 30 constant pay out of low dose ivermectin over 100 days. Despite the ivermectin (an avermectin) being in a persistent form, the results for the Ivomec@ Maximiser Lamb did not show selection for re-infection by Haemonchus nor high egg output seen in the naturally persistent moxidectin oral. Thus it appears it may not merely be the persistent action of moxidectin which causes selection for reinfection by Haemonchus. 35 It can therefore be concluded that combining the macrocyclic lactones ivermectin and WO 2015/053636 PCT/NZ2014/000214 25 moxidectin improves the anthelmintic activity and parasite treatment outcome of both compounds. Anthelmintic activity and also changes in the outcome of treatment can be seen at a time when a macrocyclic lactone, such as ivermectin, would not be considered to be exerting any effect. The resultant effect is highly beneficial as it reduces the numbers of 5 pathogenic worms on pasture and the animal better responds to treatment and subsequent reinfection. Example 2 - Animal Trial (Cattle) A parasitology trial using faecal egg counts and larval cultures was conducted in young cattle 10 on a farm where there was evidence of Ostertagia surviving combination abamectin and levamisole pour-ons. Ostertagia is one of the most pathogenic roundworm of cattle. In this trial 20 young female Angus cattle approximately one year of age with a mean body weight of 215 kg (177-238kg range) were first treated on the 26 July with one of two 15 registered combination abamectin and levamisole pour-ons, Eclipse@ Pour on (NZ Registration A009270) or Boss@ Pour-on (NZ Registration A01 0817). A total of 20 animals were treated, 10 animals were treated with Eclipse@ Pour on and 10 animals with Boss@ Pour-on ("first treatment"). The animals treated on the basis of individual 20 bodyweight. The animals were re-faecal sampled 16 days after treatment for egg count and larval culture. Seven of the twenty animals were found to still have positive egg counts (4 in the Eclipse Pour on group [animal tag numbers 104, 105, 107, 110] 3 in Boss Pour on group [animal tag numbers 151, 152, 159] with the pooled faecal larval culture at this time consisting of 100% Ostertagia larvae. This provided seven animals that had evidence of 25 Ostertagia with resistance to combination abamectin and levamisole treatment (animal tag numbers 104, 105, 107, 110, 151, 152, 159). Thirty seven days after the first treatment with the registered abamectin and levamisole pour ons, two of these seven animals received the "second treatment" on the first of September as 30 follows: " Animal tag number 159 was retreated with Boss Pour on (for comparison), receiving 0.50mg/kg abamectin and 10mg/kg of levamisole, and * Animal tag number 105 was simultaneously treated using the method of the invention with 0.50mg/kg moxidectin pour on (Cydectin Pour on, NZ Registration A006203) and 35 0.50mg/kg eprinomectin (Ivomec@ Eprinex Pour on, A009270) (no further levamisole applied).

WO 2015/053636 PCT/NZ2014/000214 26 The remaining 5 animals were not assessed in this part of the trial and no second treatment is presented. 10 days after the second treatment (11 September) the animals were faecal sampled, with the treatment effect measured both by egg count and the total number of worm larvae and 5 worm genera detected in faecal culture. Reductions were compared with those egg counts obtained from the animals just prior to the second treatment. The results are shown in Tables 11 and 12. Table 11 - showing Faecal Egg Count (FEC) reduction(%) or efficacy after initial macrocyclic 10 lactone and levamisole pour on treatment and then repeated with both a macrocyclic lactone and levamisole pour on treatment and a combination milbemycin and avermectin (moxidectin+eprinomectin) pour on treatment of the invention. Tag Faecal Egg Faecal Egg Count FaecalEgg Faecal Egg Count - 10 Count - prior - 16 days Count. Prior days after second to first following first to second treatment (11 Sep) treatment treatment treatment (% Reduction in 26 July (11 Aug) (1 Sep) brackets) (% Reduction in brackets) 104 100 100 (0%) 105 250 50 (80%) 200 25 (87.5%) - following method of invention 107 50 50(0%) 110 150 200 (-33%) 151 200 50 (75%) 152 150 50 (67%) 159 150 100(33%) 150 100(33%) Comparison (treatment with abamectin and levamisole) Table 12 - comparing larval culture of two treatments (abamectin with levamisole pour-on 15 Tag 159 and moxidectin with eprinomectin pour on - Tag 105), comparing larval numbers and composition of larval culture 10 days after second treatment. Tag Faecal Total Ost Ost Coop Coop Others Others Egg Larvae % Total % Total % Total Count /50g 105 200 2100 21 441 79 1650 0 0 (Just prior to second treatment) 105 25 160 9 14.4 91 145.6 0 0 (10 days after 2nd WO 2015/053636 PCT/NZ2014/000214 27 treatment) % 87.5% 92.4% NA 96.7% NA 91.22% NA NA Reduction 159 150 320 1 3.2 99 99 0 0 (Just prior to second treatment) 159 100 23 100 23 0 0 0 0 (10 days after 2nd treatment) % 33.3% 92.8% NA -6.19% NA 100% NA NA Reduction Ost = Ostertagia, NA= Not appicable, Coop = Cooperia % Reduction expressed as a % of the initial pre-treatment count. Comment: 5 Against this resistant Ostertagia strain the combinations of abamectin with levamisole gave reductions between -30% (i.e. increase) to 80% in egg count which is considered ineffective. Additionally the faecal larval cultures post treatment consisted of a monoculture (100%) of Ostertagia larvae. These findings are consistent with Ostertagia with resistance to both actives abamectin and levamisole. This observation was repeatable. Tag 159 treated on two 10 successive occasions with such a combination (Boss Pour on) gave a reduction of 33.3 % (ineffective on both occasions). However, Tag 105 treated with the combined moxidectin pour on with eprinomectin pour on of the invention gave a higher reduction of 87.5%. This was higher than the reduction of 80% 15 achieved when previously treated with an abamectin with levamisole combination pour on (Eclipse Pour on). However, of greater significance, the larval cultures showed that after combined abamectin with levamisole treatment (Boss Pour on or Eclipse Pour on) the surviving worm larvae were totally Ostertagia (100%). While with the combined macrocyclic lactone treatment of moxidectin with eprinomectin as a pour on (as per the method of the 20 invention), there was good control of the macrocyclic lactone resistant Ostertagia (96.7% reduction in numbers in larval culture). Effective control is considered greater than 90%. There was slightly less but still effective control of Cooperia larvae at 91.2%, which again is considered quite high by New Zealand standards for a Cooperia worm strain. 25 By way of contrast the abamectin with levamisole formulation (Boss Pour on) appeared to fully control Cooperia larvae at 100% but failed to control this resistant Ostertagia strain, with Ostertagia numbers actually rising after treatment (-6.19% reduction). As such this combined WO 2015/053636 PCT/NZ2014/000214 28 abamectin with levamisole pour on appears highly selective for the resistant Ostertagia strain. The prior art treatment (abamectin with levamisole such as Eclipse pour on or Boss Pour on) 5 confers selective advantage favouring the survival of resistant Ostertagia initially after treatment (head selection) with subsequent contamination of pasture which results in a shift in the new infective stages on pasture to this resistant worm strain. The Ostertagia worm genera are ones that can cause serious disease in cattle. This effect is 10 made additionally worse because such pour on products are often able to prevent reestablishment of susceptible strains of Ostertagia for some time so continue to offer selective advantage to resistant Ostertagia for as much as 14-21 days after treatment (tail selection). 15 Cooperia is a less pathogenic worm of cattle, affecting young cattle up to around 12 months of age and is typically not as well controlled by the macrocyclic lactone family in New Zealand. Cooperia also re-infects more rapidly after treatment so there is generally less tail selection. Cooperia is normally well controlled by levamisole, as demonstrated in this abamectin with levamisole pour on trial. 20 Although in this example standard pour on doses of moxidectin (0.5mg/kg) and eprinomectin (0.5mg/kg) were applied (so total macrocyclic lactone dose 1 mg/kg), it is known that with resistant worm strains that dosing with more active once a strain is resistant does not generally give significantly greater worm kill or control. The positive effects shown by the 25 invention are therefore unlikely to be merely as a result of a higher dose of active. This trial supports the view that there is a positive benefit and parasitological outcome in the combining of the macrocyclic lactones (milbemycin and avermectin) in cattle and that in fact it was more effective than combining two different action families (abamectin with levamisole) 30 at full dose rates This is particularly evident with the Ostertagia strain that appears to have quite marked resistance to the macrocyclic lactone family already when used alone (such as abamectin in the abamectin with levamisole combinations) but appears still relatively sensitive to the combination of two types of macrocyclic lactone (an avermectin with a milbemycin) of the invention. 35 The mix of macrocyclic lactones of the invention (avermectin with a milbemycin) appears WO 2015/053636 PCT/NZ2014/000214 29 more potent together than when they are used alone. While not wishing to be bound by theory, it is possible this is because they have similar but also different mechanisms of action, or they may be able to potentiate the action of the other macrocyclic lactone when used in such combinations. This has application in the control of both these resistant worm 5 strains, but could also be exploited to prevent these strains from developing. Example 3 - Formulation examples As previously noted, a person skilled in the art would be capable to formulating macrocyclic lactone formulations for various administration routes, for example, oral, topical and 10 injectable. The following are examples of such formulations. Example 3.1 - Formulations for oral administration Table 13: Oral Formulation Example One (Dose: 1 ml per 5kg) Ingredient Function Quantity (g/L) Moxidectin ML Active 1 Abamectin ML Active 1 Benzyl alcohol Preservative About 20 to 40 BHT Preservative About 0.1 to 2.5 Polysorbate 80 Solubilizing agent About 100 to 200 Monopropylene Glycol Solubilizing agent About 100 to 200 Disodium hydrogen Buffer As required phosphate Monosodium phosphate Buffer As required Hydrochloric Acid pH adjuster As required Purified Water Up to 1L 15 Table 14: Oral Formulation Example Two (Dose: 1 ml per 5kg) Ingredient Function Quantity (g/L) Moxidectin ML Active 1 Eprinomectin ML Active 1 Benzyl alcohol Preservative About 20 to 40 BHT Preservative About 0.1 to 2.5 Polysorbate 80 Solubilizing agent About 100 to 200 Monopropylene Glycol Solubilizing agent About 100 to 200 Disodium hydrogen Buffer As required phosphate Monosodium phosphate Buffer As required Hydrochloric Acid pH adjuster As required Purified Water Up to 1L WO 2015/053636 PCT/NZ2014/000214 30 Table 15: Oral Formulation Example Three (Dose: 1ml per 5kg) Ingredient Function Quantity (g/L) Moxidectin ML Active 1 Ivermectin ML Active 1 Benzyl alcohol Preservative About 20 to 40 BHT Preservative About 0.1 to 2.5 Polysorbate 80 Solubilizing agent About 100 to 200 Monopropylene Glycol Solubilizing agent About 100 to 200 Disodium hydrogen Buffer As required phosphate Monosodium phosphate Buffer As required Hydrochloric Acid pH adjuster As required Purified Water Up to 1L Example 3.2 - Formulations for administration by injection Table 16: Injection Formulation Example One (Dose: 1ml per 50kg) Ingredient Function Quantity (g/L) Moxidectin ML Active 10 Eprinomectin ML Active 10 Benzyl alcohol Preservative About 30 to 50 BHT Preservative About 0.1 to 2.5 Polysorbate 80 Solubilizing agent About 100 to 200 Monopropylene Glycol Solubilizing agent About 100 to 200 Disodium hydrogen Buffer As required phosphate Monosodium phosphate Buffer As required Hydrochloric Acid pH adjuster As required Purified Water Up to 1L 5 Table 17: Injection Formulation Example Two (Dose: 1ml per 35kg). Ingredient Function Quantity (g/L) Moxidectin ML Active 7 Ivermectin ML Active 7 Levamisole phosphate Other Active 233 Benzyl alcohol Preservative About 20 to 40 BHT Preservative About 0.1 to 2.5 Polysorbate 80 Solubilizing agent About 80 to 180 Monopropylene Glycol Solubilizing agent About 80 to 180 Disodium hydrogen Buffer As required phosphate Monosodium phosphate Buffer As required Hydrochloric Acid pH adjuster As required Purified Water Up to 1L WO 2015/053636 PCT/NZ2014/000214 31 Example 3.3 - Formulations for pour-on administration Table 18: Pour On Formulation Example One (Dose: 1ml per 10kg) Ingredient Function Quantity (g/L) Moxidectin ML Active 5 Eprinomectin ML Active 5 BHT Preservative About 0.1 to 2.5 Miglyol 812 Solvent About 200 to 400 Miglyol 840 Solvent About 200 to 400 Isopropyl Alcohol Solvent Up to 1 L 5 Table 19: Pour On Formulation Example Two (Dose: 1 ml per 10kg) Ingredient Function Quantity (g/L) Moxidectin ML Active 5 Abamectin ML Active 5 BHT Preservative About 0.1 to 2.5 Miglyol 812 Solvent About 200 to 400 Miglyol 840 Solvent About 200 to 400 Isopropyl Alcohol Solvent Up to 1 L Table 20: Pour On Formulation Example Three (Dose: 1 ml per 10kg) Ingredient Function Quantity (g/L) Moxidectin ML Active 5 Ivermectin ML Active 5 BHT Preservative About 0.1 to 2.5 Miglyol 812 Solvent About 200 to 400 Miglyol 840 Solvent About 200 to 400 Isopropyl Alcohol Solvent Up to 1L Example 3.4 - Formulations for administration by intraruminal device 10 Table 21: Intraruminal Capsule : Example One (Dose: One 100 day capsule, 40-80kg. Each capsule has 370 mg of total ML, 4620mg (4.62g) of Albendazole and delivers abamectin and moxidectin dosed at 0.02mg/kg/day (or 0.04mg total ML) in 80kg animal.

WO 2015/053636 PCT/NZ2014/000214 32 Ingredient Function Quantity (g/kg) Moxidectin ML Active 16.66 Abamectin ML Active 16.66 Albendazole Other Anthelmintic 361.3 Selenium selenate Mineral 4.5 Cobalt Sulfate Mineral 44 5 Sucrose fatty ester Gel forming About 350 to 500 Lactose Binder/Filler Up to 1000 BHA Antioxidant About 0.1 to 0.2 Magnesium stearate Tableting aid About 3-5.0 10 Table 22: Intraruminal Capsule Example Two (Dose: 1 100 day capsule, 40-80kg. Each capsule has 370 mg of total ML. 4620mg (4.62g) of Albendazole Delivers both moxidectin and eprinomectin at 0.02mg/kg/day (Total ML of 0.04mg/kg/day) in 80kg animal. Ingredient Function Quantity (g/kg) Moxidectin ML Active 16.66 Eprinomectin ML Active 16.66 Selenium selenate Mineral 4.5 Cobalt Sulfate Mineral 44 Sucrose fatty ester Gel forming About 350 to 500 Lactose Binder/Filler Up to 1000 BHA Antioxidant About 0.1 to 0.2 Magnesium stearate Tableting aid About 3-5.0 15 General The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference. Reference to any prior art in this specification is not, and should not be taken as, an 20 acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour in any country in the world. The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in 25 any or all combinations of two or more of said parts, elements or features. Wherein the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set WO 2015/053636 PCT/NZ2014/000214 33 forth. It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and 5 modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the scope of the invention.

Claims

1. A method of improving the treatment of pathogenic and/or resistant worm strains in an animal, the method including administering to the animal a combination of at least 5 two macrocyclic lactones wherein at least one of the macrocyclic lactones is a milbemycin and at least one of the macrocyclic lactones is an avermectin.

2. A method of reducing the reinfection by a pathogenic and/or resistant worm strain following treatment, the method including administering to the animal a parasitically effective amount of at least two macrocyclic lactones, wherein at least one of the 10 macrocyclic lactones is a milbemycin and at least one of the macrocyclic lactones is an avermectin.

3. The method of claim 1 or 2 wherein the milbemycin is selected from any one or more of: moxidectin, milbemycin oxime, nemadectin, milbemectin.

4. The method of claim 3 wherein the milbemycin is moxidectin. 15

5. The method of any one of the previous claims wherein the avermectin is selected from any one or more of: abamectin, ivermectin, eprinomectin, doramectin, selamectin, emamectin.

6. The method of any one of the previous claims wherein at least one of the macrocyclic lactones is administered orally. 20

7. The method of any one of the previous claims wherein at least one of the macrocyclic lactones is administered topically.

8. The method of any one of the previous claims wherein the macrocyclic lactones are administered within 3 days of each other.

9. The method of any one of the previous claims wherein the macrocyclic lactones are 25 administered substantially simultaneously.

10. The method of any one of claims 1 to 7 wherein the macrocyclic lactones are contained in the same formulation.

11. The method of any one of the previous claims wherein the pathogenic worms are any one or more of Haemonchus, Ostertagia, and/or Trichostrongylus species. 30

12. The method of any one of the previous claims wherein the reduction in reinfection by a pathogenic or resistant worm strain is seen at least about 10 days following the administration of the macrocyclic lactones.

13. The method of any one of the previous claims wherein the reduction in reinfection by a pathogenic or resistant worm strain is seen at least about 30 days following the 35 administration of the macrocyclic lactones.

14. The method of any one of the previous claims wherein the method additionally WO 2015/053636 PCT/NZ2014/000214 35 includes administering one or more additional active compound(s) that is/are not a macrocyclic lactone.

15. The method of claim 14 wherein the one or more additional active compound(s) are administered within 3 days of the macrocyclic lactones. 5

16. The method of claim 14 wherein the one or more additional active compound(s) are contained in the same formulation as the macrocyclic lactones.

17. A veterinary anti-parasitic formulation containing a parasitically effective amount of at least one milbemycin and at least one avermectin.

18. The formulation of claim 17 wherein the milbemycin is selected from any one or more 10 of: moxidectin, milbemycin oxime, nemadectin, milbemectin.

19. The formulation of claim 17 or 18 wherein the milbemycin is moxidectin.

20. The formulation of any one of claims 17 to 19 wherein the avermectin is selected from any one or more of: abamectin, ivermectin, eprinomectin, doramectin, selamectin, emamectin. 15

21. The formulation of any one of claims 17 to 20 wherein the formulation is adapted to be administered orally.

22. The formulation of any one of claims 17 to 20 wherein the formulation is adapted to be administered topically.