AU2013257335B2

AU2013257335B2 - Anthelmintic compositions

Info

Publication number: AU2013257335B2
Application number: AU2013257335A
Authority: AU
Inventors: Douglas Grant Halligan; Gary Robert Harrison
Original assignee: Nexan Corp Ltd
Current assignee: NEXAN Corp Ltd
Priority date: 2012-05-01
Filing date: 2013-05-01
Publication date: 2017-03-02
Anticipated expiration: 2033-05-01
Also published as: WO2013165264A1; AU2013257335A1

Abstract

The invention relates to anthelmintic compositions, their preparation and their use. More particularly the invention relates to an anthelmintic composition including micronized macrocyclic lactone particles and water. The invention also more particularly relates to method of manufacturing an anthelmintic composition including the step of suspending micronized macrocyclic lactone particles in water.

Description

WO 2013/165264 PCT/NZ2013/000078 1

ANTHELMINTIC COMPOSITIONS

Field of Invention

The invention relates to anthelmintic compositions, their preparation and their use. More particularly the invention relates to anthelmintic compositions including macrocyclic lactones.

Background

Macrocyclic lactones are a class of anthelmintic compounds which are widely used for veterinary applications due to their broad antiparasitic spectrum and low dose levels.

The macrocyclic lactones are lipophilic and are generally formulated in oil solutions for administration. When macrocyclic lactones are formulated in an aqueous composition, it is widely believed a neutral pH is required for stability of the compounds.

It is also known to be beneficial to combine anthelmintic drugs which have different modes of action in order to reduce the incidence of parasites developing drug resistance. To conveniently administer multiple anthelmintic drugs at the same time they may be formulated into a single composition. However, when macrocyclic lactones are combined in a composition with other anthelmintics the formulation requirements of the macrocyclic lactone (lipophilic, neutral pH) are often at odds with the requirements of the other anthelmintics. For example, levamisole, a commonly used anthelmintic, is generally formulated as an aqueous solution with a pH of approximately 3-4. Benzimidazoles (another commonly used anthelmintic) are sparingly soluble in most physiologically acceptable solvent systems. US4,389,397 discloses formulations of ivermectin. The document notes the insolubility and unstability of the avermectins in water. In order to provide a largely aqueous formulation of ivermectin, the document teaches dissolving the ivermectin in solvents with surface active agents to form colloidal particles (micelles) of the ivermectin. NZ527961 discloses anthelmintic compositions with a macrocyclic lactone dissolved in oil (for example soybean oil) which then forms an emulsion in an aqueous solution of levamisole at pH 3.5. Benzimidazoles may also be present in the form of particles suspended in the aqueous phase. It Is noted in the discussion; the formulations of the invention are re-suspended and re- PCT/NZ2013/000078 WO 2013/165264 2 emulsified with minimal agitation. NZ523128 discloses anthelmintic formulations in which macrocyclic lactones are dissolved in Capmul MCM (a medium chain mon-/di-glyceride) and sorbed (absorption and/or adsorption) onto/into silica (generally Aerosil R972 - colloidal silicon dioxide). The macrocyclic lactone/silica is dispersed in an aqueous solution of levamisole. The preferred formulations are described as having some sediment formed upon standing at room temperature. However, the sediment portion was easily redispersed upon shaking. NZ584629 discloses anthelmintic compositions in which avermectin is dissolved in the organic solvents benzyl alcohol and propylene glycol (both referred to as water miscible solvents). A surfactant (polysorbate 80) is added to the macrocyclic solution prior to the solution being dispersed in water to form a micellar solution. Levamisole is present in solution in the aqueous phase and micronized albendazole particles are suspended in the aqueous phase. The micellar solution is described as providing “a product wherein the actives can be resuspended readily and remain in suspension for a long time during use without it being necessary to further shake the formulation”. WO2010021555 discloses anthelmintic compositions in which a macrocyclic lactone is stabilised using a “protecting agent”. In all examples the protecting agent is hydroxypropyl starch phosphate. The “protected” macrocyclic lactone is then suspended in water. It is stated “the ML is normally present in a separate phase at least during preparation of the formulation, such as being suspended in water or dissolved or partially dissolved in a suitable organic liquid. When suspended in water the particle size of the abamectin is desirably substantially uniform. In a typical embodiment all particles are less than 150 micron. It is indicated “some milling may be needed to achieve the desired size”. No further indication is given of the particle size. It is further stated “Generally the composition if formed as a suspension desirably remains as such but it is within the scope of the invention for such suspensions where settling out may have occurred to be reformed at the time of application by vigorous shaking of the suitable container”.

It is an object of the invention to provide a novel anthelmintic composition including a macrocyclic lactone. PCT/N Z2013/000078 WO 2013/165264 3

Alternatively, it is an object to provide a novel method of manufacturing a composition including a macrocyclic lactone.

Alternatively, it is an object of the invention to at least provide a useful choice to the public. Summary of the Invention

According to a first aspect of the invention, there is provided an anthelmintic composition including: micronized macrocyclic lactone particles; and water.

Preferably, the particles in the composition are less than 100pm.

Preferably, the particles in the composition are less than 70pm.

Preferably, the particles in the composition are less than 50pm.

Preferably, the composition includes at least one suspension aid, capable of suspending the macrocyclic lactone particles in the water.

Preferably, the composition is substantially homogenous.

Preferably, the at least one suspension aid is a dispersant.

Preferably, the dispersant is a polyethylene glycol with average molecular weight of 6000-8000. Preferably, the dispersant is polyethylene glycol 6000 Preferably, the suspension aid is a wetting agent.

Preferably, the wetting agent is a polyethylene glycol stearate.

Preferably, the wetting agent is polyethylene glycol 40 stearate. PCT/NZ2013/000078 WO 2013/165264 4

Preferably, the suspension aid is a thickening agent.

Preferably, the thickening agent is xantham gum.

Preferably, the suspension aid is a suspension stabiliser.

Preferably, the suspension stabiliser is colloidal silicon dioxide.

Preferably, the composition includes any one or more of: a dispersant, a wetting agent, a thickening agent, a suspension stabiliser.

Preferably, the composition includes a dispersant, a wetting agent, a thickening agent, and a suspension stabiliser.

Preferably, the composition remains homogenous after 6 months of storage in a suitable container.

Preferably, the composition includes at least one benzimidazole.

Preferably, the benzimidazole is oxfendazole.

Preferably, the composition includes at least one anthelmintic nicotinic receptor agonist. Preferably, the anthelmintic nicotinic receptor agonist is an imidazothiazole.

Preferably, the anthelmintic nicotinic receptor agonist is levamisole.

Preferably, the pH of the composition is between about 2 and about 5.

Preferably, the pH of the composition is between about 3 and about 4.

Preferably, the pH of the composition is between about 3.2 and about 3.7. 5 2013257335 09 Feb 2017

Preferably, the composition includes at least one preservative.

Preferably, the composition includes at least one buffer system.

Preferably, the composition further includes at least one mineral.

Preferably, the composition is a drench, oral or injection composition. Most preferably, the composition is a drench composition.

In a preferred embodiment, the invention provides an anthelmintic composition including: micronized macrocyclic lactone particles; polyethylene glycol with average molecular weight of 6000-8000 as a suspension aid; and water.

According to a second aspect of the invention, there is provided a method of manufacturing an anthelmintic composition including the step of suspending micronized macrocyclic lactone particles in water.

Preferably, the macrocyclic lactone is micronized by milling following addition of the macrocyclic lactone to the water.

Alternatively, the macrocyclic lactone is in micronized form prior to addition to the water. 25 Preferably, the micronized macrocyclic lactone particles are less than 100μηι diameter in the composition.

Preferably, the micronized macrocyclic lactone particles are less than 70μηι diameter in the composition. 30

Preferably, the micronized macrocyclic lactone particles are less than 50μηι diameter in the composition.

Preferably, the method further includes addition of at least one suspension aid to the 35 composition, capable of suspending the micronized macrocyclic lactone particles in the water. 6 2013257335 09 Feb 2017

Preferably, the composition is substantially homogenous.

Preferably, the at least one suspension aid is a dispersant.

Preferably, the dispersant is a polyethylene glycol with average molecular weight of 6000-8000.

Preferably, the dispersant is polyethylene glycol 6000

Preferably, the suspension aid is a wetting agent.

Preferably, the wetting agent is a polyethylene glycol stearate.

Preferably, the wetting agent is polyethylene glycol 40 stearate.

Preferably, the suspension aid is a thickening agent.

Preferably, the thickening agent is xantham gum.

Preferably, the suspension aid is a suspension stabiliser.

Preferably, the method includes addition of at least one or more of: a dispersant, a wetting agent, a thickening agent, 25 a suspension stabiliser.

Preferably, the macrocyclic lactone is micronized by milling following addition of the macrocyclic lactone to a mixture of the water and a dispersant. 30 Preferably, the macrocyclic lactone is micronized by milling following addition of the macrocyclic lactone to a mixture of the water and a wetting agent.

Preferably, the macrocyclic lactone is micronized by milling following addition of the macrocyclic lactone to a mixture of the water, a dispersant and a wetting agent.

Preferably, the method includes addition of least one benzimidazole to the composition. 7 2013257335 09 Feb 2017

Preferably, the benzimidazole is oxfendazole.

Preferably, the benzimidazole is in the form of micronized particles.

Preferably, the benzimidazole is dispersed in water with at least one suspension aid prior to addition to the composition.

Preferably, the method includes addition of least one anthelmintic nicotinic receptor agonist to the composition.

Preferably, the anthelmintic nicotinic receptor agonist is dissolved in water, either prior to addition to the composition or on addition to the composition.

Preferably, the anthelmintic nicotinic receptor agonist is dissolved in water prior to addition to the composition.

Preferably, the anthelmintic nicotinic receptor agonist an imidazothiazole.

Preferably, the anthelmintic nicotinic receptor agonist is levamisole.

Preferably, the method includes addition of at least one preservative.

Preferably, the method includes addition of at least one buffer system. 25

Preferably, the at least one buffer system, buffers the composition at a pH of about 2 and about 5, preferably, a pH of between about 3 and about 4, preferably, a pH of between about 3.2 and about 3.7. 30 Preferably, the method includes addition of at least one mineral.

In a preferred embodiment, the invention provides a method of manufacturing an anthelmintic composition including the step of: 2013257335 09 Feb 2017 KAE508926NZPR 303040207

7A suspending micronized macrocyclic lactone particles in water using polyethylene glycol with average molecular weight of 6000-8000 as a suspension aid.

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 provides at least one example of a practical application of the invention.

Detailed Description of Preferred Embodiments

The invention provides an anthelmintic composition including micronized particles of WO 2013/165264 PCT/NZ2013/000078 8 macrocyclic lactone and water.

Macrocyclic lactones for use in the invention may be selected from any or more of the avermectins or milbemycins, for example, but not limited to, abamectin, ivermectin, doramectin, eprinomectin, selamectin, moxidectin, milbemycinoxime.

The macrocyclic lactone is present in the composition at between about 1-3 g/L, more preferably at about 1.5-2.5 g/L, most preferably at about 2-2.2 g/L.

Reference to “micronized” in the context of the invention should be taken to mean a particle size where at least about 80% and more preferably at least about 90% of particles are less than about 10pm diameter, more preferably less than about 8μιτι, more preferably less than about 5μιτι, more preferably less than about 4pm, even more preferably less than about 2pm. However, it has been found by the inventors that particle sizes may be used in the invention when in the aqueous carrier where at least about 80% and more preferably at least about 90% of particles are less than about 100pm diameter, more preferably less than about 70pm, more preferably less than about 50pm, more preferably less than about 45pm, even more preferably less than about 40pm can be used in the invention. However, such particles will have a minimum diameter of at least 10nm, more preferably at least 5nm, even more preferably at least 1nm. So a preferred range would be between about 1 nm to 100pm. Any intermediate ranges such as about 5nm to 70pm or about 10nm to 50pm, for example. The particle sizes referred in the application, unless the context requires otherwise, are mean particle sizes. Unless the context clearly requires otherwise, reference to “particles” should be taken to mean particles in the solid state, i.e. not dissolved, and can refer to particles suspended in the composition made up of agglomerations of smaller particles. Without wishing to be bound by theory the macrocyclic lactones in the aqueous solution may clump or agglomerate together to form larger particles.

As noted in the introduction, there are significant problems with formulating macrocyclic lactones in compositions including water (aqueous formulations). It was previously thought that macrocyclic lactones had poor stability in aqueous conditions, hence the need for protection of the macrocyclic lactone in such compositions (e.g. “protecting agent” in WO2010021555). However, aqueous compositions can have advantages over oil based/organic solvent based compositions, as they are useful when combining other anthelmintic compounds into the composition which require an aqueous composition. In addition, aqueous compositions can be cheaper to produce and are physiologically compatible (e.g. they are less likely to cause burns PCT/NZ2013/000078 WO 2013/165264 9 or irritation to skin, mouth or injection site or be toxic).

Formulations disclosed in the prior art generally approach the problem of formulating macrocyclic lactones in formulations including water, by dissolving the macrocyclic lactone in an organic solvent/oil to protect the macrocyclic lactone from decomposition in the aqueous solution. The organic solution of macrocyclic lactone is then dispersed in an aqueous phase as an emulsion or micellar solution (particularly where the aqueous phase has a high pH). However, as such compositions include organic solvents/oil they are more expensive to make, can have issues with physiological compatibility and may be less bioavailable. A further disadvantage of the prior art approach of forming an emulsion/micellar solution, is that the formulations can settle out and/or split, so that the formulation must be shaken prior to use in order disperse the active ingredients evenly through the composition. If the active ingredient(s) are not evenly dispersed throughout the composition there is a risk of either under-dosing or over-dosing of the active when the composition is administered. In a veterinary or farm setting, the formulations are often supplied in large drums which can be difficult to handle and shake. This can result in insufficient shaking, and thus insufficient redispersal when attempting to re-disperse compositions which have settled.

The composition of the invention comprises micronized particles of macrocyclic lactone in water i.e. particles in an aqueous composition, not dissolved in oil/organic solvent. The compositions of the invention do not include oils and other solvents in sufficient quantities which will substantially dissolve the macrocyclic lactone. The inventors have surprisingly found micronized particles of macrocyclic lactone in water form a stable composition. Further, surprisingly, it has been found the compositions of the invention, despite being in a solid particulate form in suspension (rather than in solution) are able to provide effective levels of macrocyclic lactone to a subject on administration.

The invention in its broadest form therefore provides a composition of micronized macrocyclic lactone in water without suspension aids. Such a composition overcomes some of the above discussed disadvantages of the prior art compositions which include organic solvents. For example the compositions of the present invention are cheaper to produce, are physiological compatible and surprisingly have good bioavailability. However, such a composition will tend to settle out if stored for prolonged periods of time. Where settling of the composition is not an issue, for example if it is easy to shake the container to re-suspend the particles, or there is PCT/NZ2013/000078 WO 2013/165264 10 minimal storage time, such a composition will be useful and provide advantages to the user.

However, in a more preferred embodiment the composition will include at least one suspension aid. The inventors have found the micronized macrocyclic lactones particles can be readily suspended in the aqueous solution using a standard suspension aid to give a stable, substantially homogeneous composition. Preferably, the composition of the invention remains substantially homogenous after 6 months of storage in a suitable container, more preferably 8 months, more preferably 12 months, more preferably 18 months, even more preferably 24 months. It is standard practice in the art to shake such compositions prior to use. However, as the preferred compositions of the invention are stable and substantially homogeneous the need for this is very much reduced if not negated. It may still be desirable to shake the composition of the present invention prior to use particularly where the composition has been stored for extended periods of time. However, the inventors have shown the compositions of the invention (including suitable suspension aids) are stable and homogenous for a period of at least 6 months (see Example 5). Further, the compositions are very readily re-suspendable, if any settling has taken place. A person skilled in the art would be aware of containers suitable for the storage of anthelmintic compositions. Examples of such containers include HDPE (High Density Polyethylene) containers with induction seals, cone seals or o-rings.

Reference to “homogenous” should be taken to mean in the context of the present invention that the composition does not split into phases, or that particles present in the composition do not settle into a layer. Particles present in the composition are substantially evenly distributed throughout the composition.

Suspension aids for use in the invention include wetting agents, dispersants, thickening agents, and suspension stabilisers. The suspension aids of use in the invention will not substantially dissolve the macrocyclic lactone particles when used in the quantities required to aid in the suspension of the solid macrocyclic lactone particles.

Suitable wetting agents for use in the invention will be known to people skilled in the art, and will include wetting agents available from Huntsman Corporation, for example. However, the polyethylene glycol stearates, in particular polyethylene glycol 40 stearate, have been found to be most effective. WO 2013/165264 PCT/NZ2013/000078 11

Suitable dispersants for use in the invention include but are not limited to polyethylene glycols, ethoxylated polyarylphenol phosphate amine salt (for example Soprophor FL), sodium polynaphthalene sulphonate (for example Atlox 4862), Alkyl naphthalene sulphonate formaldehyde condensate sodium salt, (for example Tersperse 2425) and Naphthalene sulfonate condensate sodium salt (for example Morwet D-425). In particular polyethylene glycols with an average molecular weight of 6000-8000 have been found by the inventors to be effective as a suspension aid. Most preferably polyethylene glycol 6000 has been found by the inventors to be effective as a suspension aid. In a particularly preferred embodiment the invention provides an anthelmintic composition including micronized macrocyclic lactone particles and water and a dispersant, wherein the dispersant is preferably polyethylene glycol 6000-8000, even more preferably polyethylene glycol 6000. In a further particularly preferred embodiment of the invention the invention provides a method of manufacturing an anthelmintic composition including the step of suspending micronized macrocyclic lactone particles in water, and further including the addition of at least one dispersant to the composition, capable of suspending the micronized macrocyclic lactone particles in the water, wherein the dispersant is a polyethylene glycol with average molecular weight of 6000-8000, even more preferably polyethylene glycol 6000. The inventors have found the use of such a dispersant (particularly but not limited to, polyethylene glycol with average molecular weight of 6000-8000), allows for an aqueous composition of micronized particulate macrocyclic lactone which is substantially homogeneous, with good stability, without the need for protecting agents or bi-phase compositions. Such aqueous compositions being able to additionally include other anthelmintics such as levamisole (as will be discussed in more depth later in this specification).

The composition may optionally contain at least one thickening agent as a suspension aid and additionally or alternatively to help to obtain the required thickness of composition, for example a thickness which is suitable for handling. For example when the composition is a drench composition, it is required to be of suitable viscosity for use in a drench gun. Examples of suitable thickening agents for use in the invention include xantham gum and and/or carbipol. Preferably the thickening agent is xantham gum.

Suitable suspension stabilisers for use in the invention include colloidal silicon dioxide, for example Aerosil 200.

The Inventors have found that a preferred composition according to the invention includes a PCT/NZ2013/000078 WO 2013/165264 12 combination of one more wetting agents, one or more dispersants, one or more thickening agents, and one or more suspension stabilisers, to provide a stable substantially homogenous composition.

The composition may advantageously but optionally include a preservative. Suitable preservatives include, but are not limited to formalin/formaldehyde solution, potassium sorbate, parabens, and BHA (Butylatedhydroxyanisole) and/or BHT (Butylatedhydroxytoluene).

The composition may advantageously but optionally include a buffer or buffers. Suitable buffers include, but at not limited to citric acid, sodium citrate, and other citrate salts (such as trisodium citrate dehydrate), phosphate buffer systems, and sodium hydroxide/hydrochloric acid buffer systems.

The composition optionally contains at least one mineral. Suitable minerals for use in the invention include, but are not limited to, cobalt, sodium, zinc, iodine and copper. Preferably, the mineral is added to the composition in the form of a salt and/or a complex. Examples of salts include, but are not limited to sodium selenate. Examples of complexes include, but are not limited to, cobalt EDTA. Such minerals are optionally beneficially added to the composition to enable administration of mineral supplements to a subject at the same time as treatment with a macrocyclic lactone.

The composition is preferably a drench composition. The aqueous composition of the invention are particularly suitable for drench application as the aqueous base is more palatable than oil-based formulations (for example taste). The aqueous compositions of the invention are less likely to spat out than oil-based compositions and thus are more easily and effectively administered. Further, if oil-based compositions are accidentally inhaled they can cause respiratory irritation and lung disease. However, compositions of the invention may also be formulated to be compositions for injection. Aqueous compositions are well tolerated at injection sites. The compositions of the invention also show good syringability which provides ease to administration by either injection or with a drench gun.

In particularly preferred forms of the invention the composition further include the use of an anthelmintic nicotinic receptor agonists. These include, for example, levamisole, pyrantel and/or morantel, most preferably levamisole. Preferably, the anthelmintic nicotinic receptor agonists is an imidazothiazole, most preferably levamisole. The anthelmintic nicotinic receptor agonists are

304143060 KAE509890AUPR 13 2013257335 11 Aug 2016 in solution in the water that contains the micronized macrocyclic lactone particles. In such compositions, the pH of the composition is preferably between about 2 and about 5, more preferably between about 3 and about 4, even more preferably between about 3.2 and about 3.7. Such pH ranges are known to be preferred to form a stable solution of a nicotinic receptor agonist. Where levamisole is present in the composition it is preferably at a concentration between about 50-100 g/L, more preferably at about 70-90 g/L, most preferably at about 80 g/L. Surprisingly, compositions of the invention including an anthelmintic nicotinic receptor agonists (for example levamisole) have been found to a very stable, with little deterioration of the suspended particulate micronized macrocyclic lactone despite the acidic pH of the water. As previously discussed, it was previously thought macrocyclic lactones had poor stability in aqueous conditions, particularly the acidic aqueous conditions required for stability of nicotinic receptor agonists. The ability to prepare such a combination composition is a particularly surprising result and avoids the need for preparing complex bi-phasic compositions. This is a particularly important aspect of the invention as it provides options for the user that were previously not thought to be available.

In another particularly preferred form of the invention, the composition further includes a benzimidazole, either in addition to an anthelmintic nicotinic receptor agonist or in place of an anthelmintic nicotinic receptor agonist. Benzimidazoles for use in the invention are well known to a person skilled in the art. Benzimidazoles include, for example, but not limited to albendazole, oxfendazole, fenbdendazole, mebendazole, rycobendazole, parrbendazole, triclabendazole, febantel, netobimin, thiabendazole, cambendazole. In compositions of the invention containing one or more benzimidazoles, the benzimidazole is dispersed as suspended particles in the aqueous solution. The same suspension aids are optionally be used 25 to suspend the particles of benzimidazole as the particles of macrocyclic lactone. Alternatively, other suspension aids can optionally be used. Where the benzimidazole is suspended in the composition, micronized particles of benzimidazole are beneficially used. Such micronized forms of benzimidazole are commercially available, or can be milled just prior to use, or as part of the method of manufacturing the anthelmintic composition. Most conveniently the 30 benzimidazole is commercially obtained in micronized form, for example with a particles size of 90% of particles being less than 10 microns. If the benzimidazole is milled to the required particles size as part of the method of manufacture, the benzimidazole is preferably milled while in contact with water and/or at least one suspension aid. Where benzimidazole is present in the composition it is preferably at a concentration between about 80-20 g/L, more preferably at 35 about 60-30 g/L, more preferably at about 50-40, most preferably at about 45 g/L. WO 2013/165264 PCT/NZ2013/000078 14

Accordingly, in a particularly preferred aspect the invention provides an anthelmintic composition including a micronized macrocyclic lactone, a benzimidazole, levamisole, and water. Preferably, the composition further includes at least one suspension aid capable of suspending the micronized particles of macrocyclic lactone and/or particles of benzimidazole. Preferably the benzimidazole is also micronized.

Other anthelmintic agents which have complimentary activity with the macrocyclic lactone may optionally be added to the composition. These may be, for example, but not limited to, praziquantel, amino acetonitrile derivatives (AAD), and/or closantel. It would be within the knowledge of a person skilled in the art to incorporate such additional actives, with known formulation requirements. However, for example, such additional actives may be suspended in particle form in the aqueous solution or dissolved in the aqueous solution.

The invention further provides a method of manufacturing an anthelmintic composition including the step of suspending micronized macrocyclic lactone particles in water.

In a preferred embodiment the macrocyclic lactone is milled to give micronized particles following addition to the water. However, alternatively, the micronized particles of macrocyclic lactone are formed using suitable techniques (for example milling) prior to addition to the water.

Reference to “milling” or “milled” should be taken to include forms of grinding, bashing and crushing which will provide the required particles size of macrocyclic lactone. It is not limited to a milling with a rotating tool. Such matters would be known to the skilled person.

In a particularly preferred embodiment, the wetting agent and/or dispersant are added to the water along with the macrocyclic lactone. The mixture is then milled using a suitable mill, for example a Horizontal Bead Mill, to give micronized particles of macrocyclic lactone. The inventors have found addition of the wetting agent and/or dispersant to the water prior to milling provides a particularly stable suspension of the micronized macrocyclic lactone in the water.

Where the method includes the addition of a benzimidazole to the composition, the benzimidazole is preferably also in the form of micronized particles. The micronized benzimidazole is preferably dispersed in a further portion of water, prior to addition to the composition of micronized macrocyclic lactone in water. The micronized benzimidazole is PCT/NZ2013/000078 WO 2013/165264 15 preferably dispersed in the further portion of water with a suspension aid. The suspension aid can be the same as that used in the micronized macrocyclic lactone/water suspension, or can alternatively be a different suspension aid. Suitable suspension aids are discussed in the above with reference to the composition. 5

Where the method includes the addition of an anthelmintic nicotinic receptor agonist, it is preferably dissolved in water, either prior to addition to the composition or on addition to the composition. Preferably, the anthelmintic nicotinic receptor agonist is dissolved in a further portion of water prior to addition to the macrocyclic lactone/water composition. 10

EXAMPLES

Example 1 - Triple active 15 Table 1

Formulation 1 Function of components Amount Micronized Abamectin Active 2-2.1 g/L Levamisole HCL Active 80 g/L Micronized Oxfendazole Active 45.3 g/L Cobalt EDTA Mineral 39.6 g/L Sodium selenate Mineral 2.4 g/L Citric Acid Anhydrous Buffer 15 g/L Sodium Citrate Buffer 8 g/L Polyethylene glycol 40 stearate (PEG 40 stearate) Wetting agent 20 g/L Polyethylene glycol 6000 (PEG 6000) Dispersant 25 g/L Xantham Gum (a polysaccharide) Thickening agent 2.2 g/L Aerosil 200 (colloidal silicon dioxide) Suspension stabiliser 10 g/L Formalin/ Formaldehyde Solution 35%w/w Preservative 2.0 g/L Potassium sorbate Preservative 1.8 g/L Water purified Vehicle to 1L

This example shows a preferred composition of the invention including a macrocyclic lactone (abamectin), a benzimidazole (oxfendazole), an anthelmintic nicotinic receptor agonist (levamisole) and minerals (cobalt EDTA and sodium selenate). WO 2013/165264 PCT/NZ2013/000078 16

The composition also includes suspension aids to suspend the micronized particles of abamectin and oxfendazole in the water (aqueous solution) to provide a substantially homogeneous composition (see Example 5 for stability of suspension).

Example 2 - Dual active

Table 2

Formulation 2 Function of components Amount Micronized Abamectin 2 q/L Active 2.1 g/L Levamisole HCL Active 80 g/L Cobalt EDTA Mineral 39.6 q/L Sodium selenate Mineral 2.4 g/L Citric Acid Anhydrous Buffer 15 g/L Sodium Citrate Buffer 8 g/L Polyethylene glycol 40 stearate (PEG 40 stearate) Wetting agent 20 g/L Polyethylene glycol 6000 (PEG 6000) Dispersant 25 g/L Xantham Gum (a polysaccharide) Thickening agent 2.2 g/L Aerosil 200 (colloidal silicon dioxide) Suspension stabiliser 10 g/L Formalin/ Formaldehyde Solution 35%w/w Preservative 2.0 g/L Potassium sorbate Preservative 1.8 g/L Water purified Vehicle to 1L

This example shows a preferred composition of the invention including two actives - a macrocyclic lactone (abamectin) and an anthelmintic nicotinic receptor agonist (levamisole). WO 2013/165264 PCT/NZ2013/000078 17

Example 3 - single active

Table 3

Formulation 3 Function of components Amount Micronized Abamectin Active 2g/L Cobalt EDTA Mineral 39.6 g/L Sodium selenate Mineral 2.4 g/L Polyethylene glycol 40 stearate (PEG 40 stearate) Wetting agent 20 g/L Polyethylene glycol 6000 (PEG 6000) Dispersant 25 g/L Xantham Gum (a polysaccharide) Thickening agent 2.2 g/L Aerosil 200 (colloidal silicon dioxide) Suspension stabiliser 10 g/L Formalin/ Formaldehyde Solution 35%w/w Preservative 2.0 g/L Potassium sorbate Preservative 1.8 g/L Water purified Vehicle to 1L

This example shows a preferred composition of the invention including abamectin as the macrocyclic lactone.

Example 4 - quadruple active

Table 4

Formulation 4 Function of components Amount Micronized Abamectin Active 1.05 g/L Levamisole HCL Active 40.0 g/L Micronized Oxfendazole Active 22.7 g/L Praziquantel Active 18.8 g/L Cobalt EDTA Mineral 12.6 g/L Sodium selenate Mineral 1.2 g/L Citric Acid Anhydrous Buffer 7.5 g/L Sodium Citrate Buffer 4.0 q/L Polyethylene glycol 40 stearate (PEG 40 stearate) Wetting agent 20 g/L Polyethylene glycol 6000 (PEG 6000) Dispersant 25 g/L Xantham Gum (a polysaccharide) Thickening agent 2.2 g/L PCT/NZ2013/000078 18

Aerosil 200 (colloidal silicon dioxide) Suspension stabiliser 10 g/L Formalin/ Formaldehyde Solution 35%w/w Preservative 2.0 g/L Potassium sorbate Preservative 1.8 g/L Water purified Vehicle to 1L WO 2013/165264

This example shows a preferred composition of the invention including a macrocyclic lactone (abamectin), a benzimidazole (oxfendazole), an anthelmintic nicotinic receptor agonist (levamisole) and a further anthelmintic agent (praziquantel) with minerals (cobalt EDTA and sodium selenate).

Example 5 - quadruple active

Table 5

Formulation 5 Function of components Amount Micronized Ivermectin Active 1.05 g/L Levamisole HCL Active 40.0 g/L Micronized Oxfendazole Active 22.7 g/L Praziquantel Active 18.8 g/L Cobalt EDTA Mineral 12.6 q/L Sodium selenate Mineral 1.2 g/L Citric Acid Anhydrous Buffer 7.5 g/L Sodium Citrate Buffer 4.0 g/L Polyethylene glycol 40 stearate (PEG 40 stearate) Wetting agent 20 g/L Polyethylene glycol 6000 (PEG 6000) Dispersant 25 g/L Xantham Gum (a polysaccharide) Thickening agent 2.2 g/L Aerosil 200 (colloidal silicon dioxide) Suspension stabiliser 10 g/L Formalin/ Formaldehyde Solution 35%w/w Preservative 2.0 g/L Potassium sorbate Preservative 1.8 g/L Water purified Vehicle to 1L

This example shows a preferred composition of the invention including a macrocyclic lactone (ivermectin), a benzimidazole (oxfendazole), an anthelmintic nicotinic receptor agonist (levamisole) and a further anthelmintic agent (praziquantel) with minerals (cobalt EDTA and sodium selenate). WO 2013/165264 PCT/NZ2013/000078 19

Example 6 - Manufacturing process

The manufacturing for the Triple active formulation shown in Example 1 is as follows.

Part A

Add the following to a pre-mix tank. • Dissolve 0.5kg of Polyethylene glycol 40 stearate and 0.7kg of Polyethylene glycol 6000 in 14kg of water. • Add 2.0kg of Abamectin API and mix to keep in solution.

Mill via the Horizontal Bead Mill so 99% of particles are less than 100pm diameter. Less than 2pm diameter was targeted.

Rinse the mill with water so the final weight of the solution is 20kg.

Part B

Add the following to a batch tank. • 300kg of Water, • 80kg of Levamisole HCL, • 39.6kg of Cobalt EDTA, • 2.4kg of Sodium selenate, • 15kg of Citric acid, • 8kg of Sodium citrate, • 2.0kg of Formalin/Formaldehyde Solution, • 1.8kg of Potassium sorbate.

Mix until all ingredients are dissolved.

PartC

Add the following to a tank • Dissolve 19.5kg of Polyethylene glycol 40 stearate and 24.3kg of Polyethylene glycol 6000 in 300kg of water. • Add 45.3kg of Oxfendazole and mix until dispersed.

Add Part A into Part B and mix until dispersed.

Add Part C into Part A+B and mix until dispersed. WO 2013/165264 PCT/NZ2013/000078 20 .Add 10kg of Aerosil 200.

Add 2.2kg of Xantham gum.

Mix until evenly dispersed.

Make up to 1000L with water and mix until evenly dispersed.

The oxfendazole used in the method was obtained in micronized form (air milled) with 90% of particles less than 10 microns in diameter.

To obtain the double active formulation of Example 2 or the single active formulation of Example 3, the respective active ingredients can be left out of the manufacturing method. To obtain the quadruple active formulation of Example 4 the further active (Praziquantel) is added after Part C.

Example 7

Stability testing of a preferred composition of the invention including abamectin, oxfendazole, levamisole (Formulation 1) is shown in Table 6.

Table 6

Real Time 40°C Active TimeO 3 Month 6 Month 9 Month 12 Month 3 Month 6 Month 9 Month 12 Month Oxfendazole 100.0% 99.6% 99.3% 98.5% 99.1% 99.8% 99.8% 99.6% 99.1% Levamisole HCL 100.0% 99.4% 99.5% 99.7% 99.7% 100.4% 100.0% 99.8% 99.5% Abamectin 100.0% 99.7% 99.0% 98.8% 97.9% 100.0% 99.5% 99.0% 97.1%

No change in appearance, specific gravity or pH was noticeable after 12 months of real time and accelerated stability.

No distortion of the packaging was noticeable after 12 months of real time and accelerated stability.

The decomposition of the Oxfendazole content was a maximum of 0.9% in one batch at room temperature. The decomposition of the Levamisole HCL content was a maximum of 0.5% in one batch at room temperature. The Abamectin content was 2.1% at RT, 2.9% at 40°C after 12 months of real time and accelerated stability. All results are within expiry specifications for WO 2013/165264 PCT/NZ2013/000078 21 products of this type.

The results demonstrate the surprising stability and homogeneity of micronized macrocyclic lactone particles in the composition. No, or very little, deterioration in the levels of abamectin is observed in the trials. In addition it should be noted there was no change in the appearance of any of the batches, either at room temperature (real time) or 40 °0, i.e. the compositions have not split or separated. This demonstrates that the micronized particles of abamectin are suspended in the compositions to form a substantially homogeneous composition which is stable over the time period and conditions of the testing.

Example 8

Stability testing of a preferred composition of the invention including ivermectin, oxfendazole, levamisole, and praziquantel (Formulation 5) is shown in Table 7.

Table 7

Real time Active Time 0 6 Month Oxfendazole 100.0% 99.2% Levamisole HCL 100.0% 99.8% Praziquantel 100.0% 99.4% Ivermectin 100.0% 98.9%

The results demonstrate the surprising stability of micronized macrocyclic lactone particles of ivermectin in the composition. Very little, deterioration in the levels of ivermectin is observed in the trials.

Example 9

Two comparative trials in sheep, one in hoggets (approximately 1 year old, Trial 1) and lambs (approximately 4 months, Trial 2) were conducted on the same farm at different times of the year to demonstrate the effectiveness of compositions of the invention.

The farm was selected as it was known to included worm strains with some degree of resistant to not only to the imidazothiazoles (levamisole), macrocyclic lactones (including ivermectin and abamectin) and benzimidazoles (including oxfendazole) when used individually, but also when PCT/NZ2013/000078 WO 2013/165264 22 these were used together including two (dual resistant) or three (triple resistance) active combinations. It is believed this resistance pattern allows a better assessment of the relative effectiveness of each individual anthelmintic active, including in combination to determine a formulation has similar, greater or poorer activity and bioavailability relative to standard registered formulations. Both trials were conducted in sheep naturally infected with a mixed roundworm infection and there was a heavy tapeworm present and confirmed in the trial conducted in lambs. The selection of animals in each trial was performed in a manner to ensure each treatment group had similar worm burdens (by allocating using pre-treatment egg counts, with egg count and worm burden correlated strongly in young sheep), and the animals were weighed and the dose calculated to individual bodyweight (estimated to the nearest 0.1ml), which was then administered by plastic syringe over the back of the tongue by an experienced operator so that each animal received the same dose of active ingredient (mg active/kg of bodyweight), and the worm and tapeworm counts, faecal egg counts and tapeworm eggs were performed blinded to group to prevent bias.

Trial 1

In Trial 1 the compositions and methods of the invention were compared to registered formulations currently on the market in New Zealand; Registered Product 1 and Registered Product 2. The levels of active ingredients of Registered Product 1 and Registered Product 2 are shown in Tables 8 and 9. The levels of actives are according to the labels and registration details for the respective products. Registered Product 1 has equivalent levels of active ingredient to the composition of the invention shown in Example 1 (Formulation 1). Registered Product 2 has equivalent levels of active ingredients to the composition of the invention shown in Example 2 (Formulation 2).

Table 8 - Registered Product 1 (comparative)

Abamectin 2 g/L Levamisole HCI 80 g/L Oxfendazole 45.4 g/L Selenium (present as sodium selenate) 1 g/L Cobalt 4.4 g/L WO 2013/165264 PCT/NZ2013/000078 23

Table 9 - Registered Product 2 (comparative)

Abamectin 2 g/L Levamisole HCI 80 g/L Selenium (present as sodium selenate) 1 g/L Cobalt (as disodium cobalt EDTA) 5 g/L

Trial 1 was conducted on 10 month old Romney and Romney Suffolk cross sheep of mixed sex on the same farm as the roundworm and tapeworm study (Trial 2 below). In order to have sufficient trial animals 30 animals from the trial farm (White tags) and 32 animals of similar breed and weight were purchased from another farm (Green tags) in mid-July and all were dosed with a registered triple combination product (Ivermectin+Levamisole+Oxfendazole) at 1.5 times the standard label dose as a clean out drench then allowed to naturally re-infect with worm larvae from pasture on the trial farm .

The 62 trial animals were faecal sampled and weighed Day 3 (3 days prior to treatment day) and 40 animals with the highest egg counts were selected for the trial. These animals were individually tagged and weighed at Day -3 and had a mean strongyle faecal egg count of 909 epg (300-2500epg, with all animals having positive counts) and had a mean weight of 38.1kg (27.9-48.9kg). Animals were allocated based on egg count to give 5 groups of 8 animals with similar mean and distribution of egg count. The treatment dose was based on individual live-weight at Day 0 and dosed using a 10 ml syringe with the dose estimated to the nearest 0.1ml. The animals were dosed over the base of the tongue by an experienced operator. All formulations were dosed as 1ml per 10kg.

At Day 5 after treatment there was a complete reduction in faecal egg count with no eggs detected in any treated group while all animals in the untreated controls had a positive egg count with a mean count of 500epg (100-31 OOepg). At Day 7, six animals with the highest egg counts in each group at Day -3 were sacrificed to perform worm counts which are presented in the following tables (Tables 10-12). WO 2013/165264 PCT/NZ2013/000078 24

Table 10 - Reduction in worm count in Abomasum following treatment Mean worm count and percentage reduction -Haemonchus adult Mean worm count and percentage reduction — Ostertagia adult Mean worm count and percentage reduction — Trichostrogylus adult Control 141.7 875 533.3 Registered Product 1 0.0(100%) 0.0 (100%) 0.0 (100%) Formulation 1 (Triple) 0.0(100%) 25 (97.1%) 0.0 (100%) Registered Product 2 0.0 (100%) 41.7 (95.2%) 0.0 (100%) Formulation 2 (Dual) 0.0 (100%) 25 (97.1%) 0.0 (100%)

Table 11 - Reduction in worm count in small intestine following treatment Mean worm count and percentage reduction -Trichostrogylus adult Mean worm count and percentage reduction - Cooperia adult Control 3508.3 3891.7 Registered Product 1 0.0(100%) 0.0 (100%) Formulation 1 (Triple) 0.0(100%) 0.0 (100%) Registered Product 2 16.7 (99.5%) 0.0(100%) Formulation 2 (Dual) 0.0(100%) 0.0(100%) WO 2013/165264 PCT/NZ2013/000078 25

Table 12 - Reduction in worm count in large intestine following treatment

Mean worm count and percentage reduction -Oesophagostomum adult Mean worm count and percentage reduction — Chabertia adult Mean worm count and percentage reduction — Trichuris adult Control 48.3 8.3 28.3 Registered Product 1 0.0(100%) 0.0(100%) 0.0(100%) Formulation 1 (Triple) 0.0(100%) 0.0 (100%) 0.0(100%) Registered Product 2 0.0(100%) 0.0(100%) 0.0(100%) Formulation 2 (Dual) 0.0(100%) 0.0(100%) 0.0(100%)

The data shows despite all formulations (including Registered Product 2) apparently giving complete control, very small numbers of Ostertagia survived. The Ostertagia in the trial are triple resistant which was known and documented as occurring where the animals were sourced.

Registered Product 2 had slightly more Ostertagia survive than Formulation 2 (97.1% reduction versus 95.2% for Registered Product 2).

Both Formulation 2 (dual) and Formulation 1 (triple) gave similar control of this resistant Ostertagia at 97.1% and fully controlled all other worms present. Registered Product 2 however did not fully control small intestinal Trichstrongylus (99.5%) while Formulation 2 (dual) and both Registered Product 1 and Formulation 1 (triple) did. This occurred in 2 of 6 animals treated with Registered Product 2.

As shown in the roundworm and tapeworm study (Trial 2 below) both the abomasal and small intestinal Trichostrongylus appeared fully sensitive to ivermectin (Registered Ivermectin Liquid Product - control) and abamectin (that is more potent and present in Registered Product 2) would be expected to control this worm genera even more effectively, especially in combination with levamisole. Trichostrongylus is however the dose limiting worm for the macrocyclic lactones. A possible explanation for the difference in activity between Registered Product 2 and Formulation 2 (dual) is that the level of abamectin in Registered Product 2 is slightly lower or less available than in Formulation 2 (dual) so that some levamisole resistant Trichostrongylus PCT/NZ2013/000078 WO 2013/165264 26 are able to survive the dose of abamectin. This could also be the explanation for slightly lower

Ostertagia control.

While Registered Product 2 is still highly effective the slightly lower control of Ostertagia and Trichstrongylus is not desirable. Formulation 2 (dual) appears to deliver the actives more effectively than Registered Product 2 based on these trial results.

Although Registered Product 1 appeared the most effective in this trial it was considered this was most likely the result of incomplete removal of macrocyclic lactone and triple resistant Ostertagia in the animals originating from the farm (white tags) treated with registered triple combination product. As can be seen in Trial 2 (below) the ivermectin quadruple drenches did not fully remove these worms, so the clean out drench (registered triple combination product) containing the same actives is likely also to have left surviving worms resulting in higher numbers of these in the white tagged sheep originating from the property compared with the Green tagged animals. The highest number of white tagged sheep were in the Groups that received the compositions of the invention (Formulation 1 and 2) with 4 white tagged sheep in the 6 sheep (4/6) chosen for sacrifice and worm count. In the group that was treated with Registered Product 2 there were 3 white tagged sheep out of the 6 sacrificed (3/6), and only 2/6 in the group that were treated with Registered Product 1. Thus it is considered this was a harsher test for both the Formulation examples and the better estimate of efficacy is in Trial 2, where this effect was not present.

The compositions and methods of the invention appear equally or possibly slightly more available and biologically active as available registered formulations.

Trial 2

In Trial 2 quadruple Formulations 4 and 5 of the invention were trialled and compared against registered formulations containing the same actives but manufactured/formulated by using other methods.

The compositions of the invention contained four actives, a macrocyclic lactone (ivermectin or abamectin), levamisole, oxfendazole and praziquantel. These were compared with registered formulations that contain these same four actives at the same concentration and deliver the same dose rate of active (mg/kg) to the animal. The effectiveness of the quadruple formulations PCT/NZ2013/000078 WO 2013/165264 27 was compared against both untreated controls and also a single macrocyclic lactone drench, a micellar formulation of ivermectin (Registered Ivermectin Liquid Product - control Group 1b). Ivermectin was included to demonstrate the degree of macrocyclic lactone (ivermectin) resistance of the worm population and also to demonstrate its relative lack of effect on tapeworm.

Trial 2 was conducted in newly weaned 4 month old lambs with natural roundworm (strongyle) and tapeworm infection. In this trial there were six treatment groups all 8 lambs each (Total 48). These consisted of:

Group 1. untreated control,

Group 2. treated with Formulation 5 of the invention (quad with ivermectin),

Group 3. treated with Formulation 4 of the invention (quad with abamectin),

Group 4. treated with Registered Product 3 (comparative quad with ivermectin formulation currently in the market)

Group 5. treated with Registered Product 4 (comparative quad with abamectin formulation currently in the market)

Group 1b. treated with a macrocyclic lactone (Ivermectin) alone with no praziquantel or recognised tapeworm active.

The 48 trial lambs were selected to ensure similar round worm burdens in each group (6 groups) and similar tapeworm burden (5 groups) prior to treatment. The trial lambs had a mean live weight of 28kg (24.0-31.7kg) with 41 male and 7 female lambs that were mainly Romney or Romney Suffolk crosses.

Dosing of the animals occurred 2 days after selection with the untreated controls remaining undosed, but all other groups dosed by in 10 ml syringes to the nearest 0.1ml.

Each group was faecal sampled at two days pre-treatment (Day -2), then at 6 days (Day 6) and 8 days (Day 8) after treatment. At 8 days after treatment 6 lambs from each group were selected for slaughter and the entire gastrointestinal tract (abomasum, small intestine and large intestine isolated, collected and process for a strongyle (roundworm) and tapeworm count.

Strongyle egg counts were counted at all faecal sampling points but tapeworms eggs (not segments) were assessed at Day 6 post-treatment (all animals, 6 groups of 8=48). PCT/NZ2013/000078 WO 2013/165264 28

Nematodirus eggs were detected at Day 8 at slaughter (6 groups of 6= 42 animals) and were counted at this time in all animals.

Efficacy for the data was based on reduction of counts was based on the formula:

Efficacy or/

Percentage reduction (%) = Mean count of control group - Mean count of treated group x 100

Mean of control group

Efficacy was calculated as a reduction relative to untreated controls using arithmetic mean (AM), expressed as a percentage (%). The mean count was calculated using group arithmetic means (AM) as this provides a conservative measure of efficacy and resistance.

The efficacy figures were defined as per the recommended by the World Association for the Advancement of Parasitology (WAAVP) guidelines. Summary tables are presented of efficacy using this classification to simplify the data.

As per WAAVP guidelines and terminology highly effective (HE) was greater (>) than 98%, effective (E) 90-98%, moderately effective (ME) 80-89% and ineffective or inactive (IA) less than 80% (<80%) is shown in Tables 13-16. Where any of the combinations was less than fully effective (100%), the actual efficacy for all treatments is shown.

Table 13 - Summary Table of Faecal Egg Count Reductions (Efficacy) and Larval culture

Treatment Group Strongyle Eggs Day 6 Strongyle Eggs Day 8 Nematodirus Eggs Day 8 Tapeworm Eggs Day 6 Larval numbers /50g Day 6 Larval numbers/5 og Day 8 2 HE HE HE HE 3 3 3 HE HE HE HE 2 0 4 HE HE HE HE 2 15 5 HE HE HE HE 0 21 1b ME( 84%) IA (53%) IA (79%) IA (48%) 7,230 4,400 1 1,200epg 1,700epg 233epg 2.9 25,000 60,000

WAAVP guidelines highly effective (H E) >98%, effective (E) 90-98%, moderately effective (ME; 80-89% and ineffective or inactive (IA) less than 80% (<80%) WO 2013/165264 PCT/NZ2013/000078 29

Table 14 - Summary Table of Abomasal Worm Count Reductions (Efficacy) Treatment Group Haemonchus contortus Teladorsagia/Ostertagia species Trichostrongylus species Adult LL4 Adult L4 Adult L4 2 HE 1 HE HE (98.4%) HE (100%) HE NA 3 HE HE HE (99.8%) HE (100%) HE NA 4 HE HE HE (99.2%) E (94.3%) HE NA 5 HE HE HE (99.6%) HE (100%) HE NA 1b IA ( 71.3%) HE ME(84.4%) IA (68.6%) HE NA 1 666.7 58.3 4050 292 292 0 NA= Not app icable with no worm count s for this stage in controls.

Table 15 - Summary Table of Small Intestinal Worm Count and Tapeworm Volume Reductions Treatment Group Trichostrongylus spp Cooperia spp Nematodirus spp Tapeworm Volume Adult Adult L4 Adult L4 2 HE HE HE HE HE HE (98%) (1/6 positive) 3 HE HE HE HE HE HE (100%) (0/6 positive) 4 HE HE HE HE HE HE (99%) (1/6 positive) 5 HE HE HE HE HE E (94%) (5/6 positive) 1b HE E (95%) HE E (94%) E (98%) IA (60.1%) (4/6 positive) 1 1242 1933 17 1975 417 65ml (5/6 positive) WO 2013/165264 PCT/NZ2013/000078 30

Table 16 - Summary Table of Large Intestinal Worm Reductions (Efficacy)

Treatment Group Oesophagostomum spp Chabertia spp Trichuris spp Adult Adult Adult 2 HE HE HE 3 HE HE HE 4 HE HE HE 5 HE HE HE 1b HE HE HE 1 152 32 15

In the tables Groups 2 and 3 were treated using the compositions of the invention. Groups 4 and 5 were treated using formulations available on the market (for comparison). Groups 1b is to show the level of macrocyclic lactone resistance (ivermectin resistant), and 1 are untreated controls.

Groups 2 and 4 were treated with combination formulations containing ivermectin. Groups 3 and 5 were treated with combination formulations containing abamectin.

As shown in Table 13 (egg counts and larval cultures) all combination formulations were highly effective (HE) at reducing egg counts at Days 6 and 8 with no strongyle eggs or Nematodirus eggs at these times, and no Tapeworm eggs were detected at Day 6. It was noted that the larval numbers rose in both the comparative groups (groups 4 and 5) at Day 8 (expected as the result of decreased volume of faecal matter) but did not do so in the compositions of the invention (groups 2 and 3), with no larvae recovered from group 3 (formulation 3 of the invention) at Day 8. Low egg and larval numbers post treatment are desirable as it suggests both high efficacy and worm kill and also reduced pasture contamination from parasites particularly resistant worm strains.

The worm count data (Tables 14,15 and 16) generally reflected observations in the egg counts and larval cultures. Ivermectin alone (Group 1b) fully controlled adult Trichostrongylus species in both the abomasum (true stomach) and small intestine, but was not full effective against adult and larval (LL4) stages of Haemonchus or Teladorsagia (Ostertagia) in the abomasum or the adult stages of Cooperia and Nematodirus in the small intestine, but was still fully effective against all the large intestinal worms present. It was not effective against tapeworm with some reduction in tapeworm volume (60%) but 4/6 animals still had detectable surviving tapeworms PCT/NZ2013/000078 WO 2013/165264 31 present. This pattern was consistent with ivermectin (ML) resistant Haemonchus and Teladorsagia (Ostertagia) strains, with emerging ivermectin resistance in Cooperia and Nematodirus, and ivermectin sensitive Trichostrongylus species. The tapeworm results confirmed findings from other studies that ivermectin has little activity against tapeworm infection in sheep.

In contrast all 4 quadruple active formulations (groups 2, 3, 4 and 5) all gave highly effective control (greater than 98%) against all roundworm species and worm stages present with the exception of the comparative Ivermectin quadruple formulation (Group 4) which was only effective (94%) against immature L4 larvae Teledorsagia (Ostertagia). In contrast the ivermectin version of the invention (Group 2) gave full control of this stage, as did both the abamectin version of the invention (Group 3) and the comparative abamectin formulation (Group 5).

The only roundworm stage not fully controlled (100%) by the quadruple formulation was adult stages of Teledorsagia (Ostertagia). The abamectin formulations (Groups 3 and 5) gave the greatest control relative to the ivermectin formulations (Groups 2 and 4) which is consistent with abamectin having greater potency against roundworms than ivermectin. The abamectin compositions of the invention (Group 3) gave the highest control (99.8%) against this highly macrocyclic lactone resistant and triple resistant parasite, followed by the comparative abamectin quadruple formulation (99.6%) (Group 5), followed by the registered ivermectin quadruple formulation (99.2%, but it was less effective against L4 stages)(Group 4) and then the ivermectin quadruple formulation of the invention ( 98.4%)(Group 2). All however still classed as highly effective against this stage.

These results confirm that the macrocyclic lactone component (abamectin or ivermectin) of the invention is equally available and possibly in some cases slightly more active than current registered formulations. The activity of the macrocyclic lactone component could be demonstrated as the worm strains particularly the Ostertagia and Trichostrongylus species were also known from other studies to be benzimidazole, levamisole and benzimidazole+levamisole resistant in addition to having resistance to the macrocyclic lactone family.

Against Tapeworm the abamectin quad version of the invention (Group 3) also demonstrated the greatest action with complete reduction in tapeworm volume with no tapeworm segments PCT/N Z2013/000078 WO 2013/165264 32 found in any of the animals. Both the ivermectin quads, both the comparative product (Group 4) and that of the invention (Group 2), gave high and very similar reductions of tapeworm volume (99 and 98% respectively) with very small amounts of tapeworm detected in 1/6 animals. In contrast the abamectin version of the comparative quadruple formulation (Group 5) was less effective not only reducing tapeworm by 94% but tapeworm segments and material was detected in 5 of the 6 animals in the group treated with this product. Based on this data the most effective formulation based on both worm count and tapeworm volume and animal infected was the abamectin quadruple version of the invention (Group 3). The comparative abamectin quadruple formulation gave similar efficacy to roundworms (marginally lower against resistant adult stages of Teladorsagia (Ostertagia), with lower activity against tapeworm. The ivermectin versions of the quadruple formulations (Groups 2 and 4) were very similar in roundworm and tapeworm control except for slightly less activity detected in Teladorsagia (Ostertagia) L4 larvae in the comparative ivermectin quadruple formulation product (Group 4). Based on the ivermectin data (Group 1 b) these LL4 larval stages appeared more resistant than the adult stages, making their survival more likely if ivermectin was less available in a formulation.

Both findings in both the ivermectin and abamectin version of the composition invention suggest the macrocyclic lactone component is either equally or possibly more available and active than current registered formulations available on the market.

Example 10

The size of the particles in Formulation 2 (Dual Active) was tested using a Mastersizer 2000. The results are shown in Table 17.

The results show the particles in the formulation have a mean size of 15.6 - 31.0pm. These results are the measurement of the particles when suspended in the aqueous composition. Without wishing to be bound by theory, the particle size measured may be agglomerates including the micronized macrocyclic lactone particles. Alternatively it may be that, while less than 2 pm particle size was targeted when preparing the composition (e.g. as shown in Example 6) the accuracy of the particle size measurement may not have been sufficient to identify the actual milled particle size used. WO 2013/165264 PCT/NZ2013/000078 33

Table 17 - particle size distribution

Size (pm) Volume In % 0.05 - 0.06 0 0.06-0.12 0 0.12-0.24 0 0.24- 0.49 0.13 0.49 - 0.7 0.39 0.7- 0.98 0.46 0.98 - 2 1.59 2-3.9 2.11 3.9 -7.8 5.54 7.8-15.6 20.73 15.6-31.0 43.93 31 -37 12.35 37-44 10.86 44-53 1.91 53-63 0 63-74 0 74 - 88 0 88-105 0

General

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of “including, but not limited to”.

The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference. PCT/NZ2013/000078 WO 2013/165264 34

Reference to any prior art in this specification is not, and should not be taken as, an 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 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 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 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. An anthelmintic composition including: micronized macrocyclic lactone particles; polyethylene glycol with average molecular weight of 6000-8000 as a suspension aid; and water.

2. The composition of claim 1, wherein the particles in the composition are less than 100μηη.

3. The composition of claim 1 or 2, wherein the polyethylene glycol is polyethylene glycol 6000.

4. The composition of any one of claims 1 to 3, wherein the composition includes at least one additional suspension aid selected from one or more of: a wetting agent, a thickening agent, a suspension stabiliser.

5. The composition of claim 4, wherein the wetting agent is a polyethylene glycol stearate.

6. The composition of claim 4 or 5, wherein the wetting agent is polyethylene glycol 40 stearate.

7. The composition of any one of claims 4 to 6, wherein the thickening agent is xantham gum.

8. The composition of any one of claims 1 to 7, wherein the composition includes levamisole.

9. The composition of any one of claims 1 to 8, wherein the pH of the composition is between about 2 and about 5.

10. The composition of any one of claims 1 to 9, wherein the composition includes at least one benzimidazole.

11. The composition of any one of claims 1 to 10, wherein the composition includes a further anthelmintic agent.

12. A method of manufacturing an anthelmintic composition including the step of suspending micronized macrocyclic lactone particles in water using polyethylene glycol with average molecular weight of 6000-8000 as a suspension aid.

13. The method of claim 12, wherein the macrocyclic lactone is micronized by milling following addition of the macrocyclic lactone to the water.

14. The method of claim 12, wherein the macrocyclic lactone is in micronized form prior to addition to the water.

15. The method of any one of claims 12 to 14, wherein the micronized macrocyclic lactone particles are less than 100μηι diameter in the composition.

16. The method of any one of claims 12 to 15, wherein the method further includes addition of at least one additional suspension aid to the composition selected from one or more of: a wetting agent, a thickening agent, a suspension stabiliser.

17. The method of claim 16 when dependent on claim 13, wherein the macrocyclic lactone is micronized by milling following addition of the macrocyclic lactone to a mixture of the water and the polyethylene glycol with average molecular weight of 6000-8000 and/or a wetting agent.

18. The method of any one of claims 12 to 17, wherein the method includes addition of least one anthelmintic nicotinic receptor agonist to the composition.

19. The method of claim 18, wherein the anthelmintic nicotinic receptor agonist is dissolved in water, either prior to addition to the composition or on addition to the composition.

20. The method of any one of claims 12 to 19, wherein the method includes addition of least one benzimidazole to the composition.

21. The method of claim 20, wherein the benzimidazole is in the form of micronized particles.

22. The method of claim 20 or 21, wherein the benzimidazole is dispersed in water with at least one suspension aid prior to addition to the composition.

23. The method of any one of claims 12 to 22, wherein the method includes addition of at least one buffer system that buffers the composition at a pH of about 2 and about 5.

24. An anthelmintic composition manufactured by the method of any one of claims 12 to 23.