AU2013205280A1 - Macrocyclic Lactone injectable Formulations - Google Patents

Abstract

The present invention provides a pharmaceutical or veterinary formulation adapted to be injected into an animal, comprising (a) an effective amount of levamisole; (b) an effective amount of at least one macrocyclic lactone selected from abamectin, doramectin, eprinomectin, ivermectin, and moxidectin; (c) at least one surfactant selected from ethoxylated castor oils, polyethyoxylated castor oil, poly oxyl 40 hydrogenated castor oil, or a combination thereof; (d) at least one solvent selected from glycerol formal, propylene glycol, polyethylene glycol, and dimethyl isosorbide; (e) water. The invention also provides a method for preventing or treating internal parasite infestations in animals, comprising administering to an animal an effective amount of the aforementioned formulation.

Description

EDITORIAL NOTE There are two page 2 in the description, the numbering is consecutive 5 MACROCYCLIC LACTONE INJECTABLE FORMULATIONS FIELD OF THE INVENTION The present invention relates to improved injectable anthelmintic formulations containing levamisole and one or more macrocyclic lactone anthelmintic as well as methods for the use 10 of the invention in eradicating, controlling, and preventing parasite infestation in or on livestock. BACKGROUND OF THE INVENTION The control of gastrointestinal parasites is an important aspect of modem livestock farming. 15 Typically these parasites are controlled by specific chemical agents developed to meet certain criteria. These criteria include: - High effectiveness against the target parasites - this could either be via broad spectrum efficacy in which a wide range of parasites are controlled or alternatively via narrow spectrum of activity in which a more limited selection of parasites are controlled. 20 - Wide margin of safety to the host animal - Low potential to cause toxic residues that could cause harm to humans consuming meat or milk products from the animal Over the past 50-60 years there have been 5 major classes of broad spectrum anthelmintic developed. These 5 classes are as follows: 25 Benzimidazoles The first chemical class of modem anthelmintics developed were the benzimidazoles (BZD). The first drug in this class, thiabendazole (TBZ) was introduced in 1961. Other members of the benzimidazole class include fenbendazole, albendazole and oxfendazole. Benzimidazole based anthelmintics interfere with the worm's energy metabolism on a 30 cellular level. The molecule binds to a specific building block called beta tubulin and prevents its incorporation into certain cellular structures called microtubules, which are essential for energy metabolism. Interfering with energy metabolism is a much more basic 206155NZ NZ CS 20130411.docx mode of activity than that which occurs with other classes of anthelmintics. For this reason, benzimidazoles are also able to kill worm eggs. Benzimidazoles have a wide margin of safety and broad spectrum activity. Nicotinic agonists 5 Nicotinic agonists comprise the second class of anthelmintics. They include imidazothiazoles and tetrahydropyrimidines. The imidazothiazole group includes levamisole, while the tetrahydropyrimidine group includes pyrantel pamoate, pyrantel tartrate, and morantel tartrate. The tetrahydropyrimidines mimic the activity of acetylcholine, a naturally occurring 10 neurotransmitter that initiates muscular contraction. Upon exposure to the active ingredient the worm is unable to feed and quickly starves. Tetrahydroyrimidines only affect adult populations of worms. They do not have activity against the larval stages and are ineffective against cestodes (tapeworms) and trematodes (liver flukes). Imidazothiaoles have a similar mode of action causing spastic paralysis of the worms. The 15 group includes the drug levamisole discovered in 1966. Compared to other anthelmintics, levamisole has the narrowest margin of safety, though toxicity is usually the result of excess dosage. Levamisole has a broad spectrum of activity and is effective against many larval stages of parasites; though not arrested larvae. Macrocyclic lactones 20 The next anthelmintic class to be developed was the macrocyclic lactones (MLs) developed from the Streptomyces genus of soil dwelling-organisms. The first drug of the class, ivermectin, was introduced in the early 1980s. Macrocyclic lactones consist of two closely related chemical groups: avermectins and milbemycins. The avermectins include abamectin, ivermectin doramectin and eprinomectin. 25 The milbemycin group is represented by milbemycin oxime and moxidectin, introduced in 1997. All of the macrocyclic lactone anthelmintics have the same mode of action. They interfere with GABA-mediated neurotransmission, causing paralysis and death of the parasite. 206155N7 NZ CS 20130411.docx Macrocyclic lactones are the most potent killer of worms and are more persistent in their effect. The duration of persistent activity varies according to the drug and formulation. Macrocyclic lactones have the unique quality of also killing several types of external parasite such as lice, mites, and ticks. Because of this they are also known as "endectocides" (end 5 ecto-cides), meaning control of internal and external parasites. Generally the macrocyclic lactones have a wide margin of safety in treated livestock and are effective against all stages of worms, including inactive forms. Amino-acetonitrile derivatives In 2009 the first of this new class of anthelmintics was introduced. "Monepantel" acts by 10 paralyzing worms by attacking a previously undiscovered receptor HCO-MPTL-1, present only in nematodes. Spiroindoles In 2010 Derquantel (2- deoxoparaherquamide), the first of yet another new class of anthelmintic was introduced. 15 All of the above anthelmintics have application in the control of a wide variety of "ecto" and "endo" parasites. Of particular concern are those parasites classified under the group of worms known as helminths. Helminthiasis is a prevalent and serious economic problem with domesticated animals such as swine, sheep, horses, cattle, goats, dogs, cats and poultry. Among the Helminths, the group of worms described as nematodes cause widespread, and at 20 times serious infection in various species of animals. Nematodes that are contemplated to be treated by the anthelmintics include, the following genera: Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaridia, Ascaris, 25 Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema, Diphyllobothrium, Diplydium, Dirofilaria, Dracunculus, Enterobius, Filaroides, Haemonchus, Heterakis, Lagochilascaris, Loa, Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus, Oesophagostomum, Opisthorchis, Ostertagia, Oxyuris, Parafilaria, Paragonimus, Parascaris, Physaloptera, Protostrongylus, 206155NZ NZ CS 20130411.docx Setaria, Spirocerca, Spirometra, Stephanofilaria, Strongyloides, Strongylus, Thelazia, Toxascaris, Toxocara, Trichinella, Trichonema, Trichostrongylus, Trichuris, Uncinaria, and Wuchereria. Of the above, the most common genera of nematodes infecting the animals referred to above 5 are Haemonchus, Trichostrongylus, Ostertagia, Nemaodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia, Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostoma, Unicinaria, Toxascaris and Parascaris. Certain of these, such as Nematodirus, Cooperia and Oesophagostomum attack primarily the intestinal tract while others, such as Haemonchus 10 and Ostertagia, are more prevalent in the stomach while others such as Dictyocaulus are found in the lungs. Still other parasites may be located in other tissues such as the heart and blood vessels, subcutaneous and lymphatic tissue and the like. While there have been many anthelmintics developed over the years, of great concern is the development of resistance to anthelmintics belonging to the benzimidazole, nicotinic agonist 15 and macrocyclic lactone classes and the high probability that it will similarly develop to anthelmintics from the more recently developed Amino-acetonitrile and Spiroindole classes.Resistance refers to the process by which exposure of the parasite population to an anthelmintic leads to genetic selection of those parasites able to tolerate it. While resistance can naturally be expected to occur at a certain rate the problem is 20 exacerbated by over-exposure of the parasite population to the anthelmintic or by sub-lethal dosing of the parasite through inaccurate dose administration. Of particular relevance to this application is the development of resistance to those classes of anthelmintic widely used in cattle. Resistance to benzimidazole-based cattle drenches is widespread throughout the world. Cases have been reported that involve resistance in all 25 three major cattle parasites species: Ostertagia, Trichostrongylus and Cooperia. Resistance to the nicotinic agonist derivative class (levamisole/morantel) is well known but is less widespread than benzimidazole resistance. 206155NZ NZ CS 20130411.docx Resistance to the more recent macrocyclic lactone class is also becoming much more common. This is particularly troublesome given that it is by far the most popular form of parasite treatment for farmed cattle. To prevent and manage the problem of anthelmintic resistance farmers have relied on 5 various strategies including: 1. minimizing anthelmintic use by only treating at strategically important times 2. alternating the type of anthelmintic used 3. using combinations of anthelmintics from different classes to reduce the potential of parasites to survive the treatment. 10 From the perspective of ensuring the broadest possible efficacy it is desirable that the formulation contains both a macrocyclic lactone active ingredient and levamisole, as both of these anthelmintics are currently used in injectable form in cattle. Novartis product represents a recent attempt to formulate a combination product containing a macrocyclic lactone anthelmintic and levamisole. It is based on emulsion technology, the 15 formulation described includes levamisole in an aqueous acidic phase and macrocyclic lactone in a lipophilic phase. A third active could potentially be suspended in particulate form in the aqueous phase. The disadvantage of this formulation is the need for the formulation to be shaken or agitated into an emulsion. In addition, the product is chemically complicated including 2 or 3 different phases. 20 The complicated nature of the Novartis formulation is due in part to the different formulation requirements of the actives. Macrocyclic lactone active ingredients are substantially insoluble in water whereas levamisole is highly water soluble. Macrocyclic lactone active ingredients are most stable at a pH of about 6.6 while levamisole is most stable at a pH of less than about 4. 25 As will be appreciated, in addition to the need to be physically stable during storage anthelmintic formulations also need to be safe to administer so that they do not cause excessive skin or tissue irritation. 206155NZ NZ CS 20130411.docx The scarcity approaches is an indication of the degree of difficulty in developing such formulations. Accordingly, there remains a need for new combination macrocyclic lactone/levamisole injectable formulations that are shelf stable, easy to manufacture and non-irritant to the 5 animal. SUMMARY OF THE INVENTION The invention provides new injectable anthelmintic formulations comprising a combination of a levamisole and at least one macrocyclic lactone active ingredient. The advantage of the 10 new invention is that it provides for selection of the most preferred solvent/surfactant combination as well as offering clinical advantages over prior proposals. The formulations are surprisingly non-irritant and capable of high loadings of both active ingredients. They are also stable when stored for prolonged periods under recommended storage conditions. 15 The invention also provides for methods of manufacturing of the preferred combinations. The combination of levamisole and macrocyclic lactones in the inventive formulation is highly effective in the prevention and treatment of parasite infection in animals. Such infections include those due to parasites such as lungworm, gastro-intestinal roundworms, and ectoparasiticides such as biting and sucking lice. 20 In one aspect the invention provides a pharmaceutically or veterinarily acceptable formulation adapted to be injected into an animal, which formulation comprises: (a) an effective amount of a levamisole, selected from levamisole phosphate, levamisole hydrochloride and levamisole base; (b) an effective amount of at least one macrocyclic lactone selected from 25 abamectin, doramectin, eprinomectin, ivermectin and moxidectin; (c) at least one surfactant selected from Ethoxylated castor oils, Polyethoxylated castor oil and Poly Oxyl 40 Hydrogenated Castor Oil, or a combination thereof 206155NZNZ_CS_20130411.docx (d) a solvent system including at least one solvent selected from glycerol formal, propylene glycol, polyethylene glycol and dimethyl isosorbide; and (e) water. Preferably the formulation also includes Vitamin B12. 5 Preferably the Vitamin B 12 is present as hydroxocobalamin or cyanocobalamin (or mixtures thereof). Preferably the surfactant is Cremophor RH40. Preferably the surfactant is Cremophor RH40. 10 Preferably the levamisole is levamisole phosphate and wherein the levamisole is present in an amount sufficient to deliver a dose of at least 4 ptg/kg animal bodyweight. Preferably it includes eprinomectin as well as ivermectin or abamectin. Preferably the concentration of each macrocyclic lactone is from about 0.1% to about 2% 15 (w/v); the levamisole concentration is from about 15% to about 30% (w/v); and the surfactant concentration is from about 5% to about 15% (w/v). Preferably it includes: (a) eprinomectin at a concentration of about 0.5% to about 0.9% (w/v); (b) ivermectin or abamectin at a concentration of about 0.5% to about 0.9% (w/v) 20 (c) levamisole phosphate at a concentration of about 18% to about 25% (w/v); (d) glycerol formal (e) a surfactant selected from Ethoxylated castor oils, Polyethoxylated castor oil and Poly Oxyl 40 Hydrogenated Castor Oil, TWEEN 80 or combinations thereof, at a concentration of at least 5% (w/v); 25 (f) Optionally, one or more further solvents selected from propylene glycol, polyethylene glycol and dimethyl isosorbide; 206155NZ NZ CS 20130411.docx (g) Optionally, one or more materials selected from Buffers, Antioxidants, and preservatives; and (h) water In another aspect the invention provides a pharmaceutically or veterinarily acceptable 5 formulation adapted to be injected into an animal, which formulation comprises: (a) moxidectin at a concentration of about 0.3% to about 0.9% (w/v); (b) levamisole phosphate at a concentration of about 8% to about 25% (w/v); (c) glycerol formal (d) a surfactant selected from Ethoxylated castor oils, Polyethoxylated castor oil 10 and Poly Oxyl 40 Hydrogenated Castor Oil, TWEEN 80 or combinations thereof, at a concentration of at least 5% (w/v); (e) Optionally, one or more further solvents selected from propylene glycol, polyethylene glycol and dimethyl isosorbide. (f) Optionally, one or more materials selected from Buffers, Antioxidants, and 15 preservatives. (g) Water In another aspect the invention provides a pharmaceutically or veterinarily acceptable formulation adapted to be injected into an animal, which formulation comprises: (a) At least one macrocyclic lactone at a concentration of about 0.3% to about 20 0.9% (w/v); (b) levamisole phosphate at a concentration of about 8% to about 25% (w/v); (c) glycerol formal (d) Polyethoxylated castor oil and Poly Oxyl 40 Hydrogenated Castor Oil (e) Propylene glycol 206155NZ NZ CS 20130411.docx (f) Optionally, one or more materials selected from Buffers, Antioxidants, and preservatives. (g) Water A further aspect of the invention is a method for preventing or treating internal parasite 5 infestations in animals, which method comprises administering to an animal an effective amount of a formulation as described above. Preferably the formulation is injected. Preferably the parasites are helminths. Preferably the helminths are nematodes, or cestodes, or trematodes, or any combination 10 thereof. Preferably the method of manufacture comprises the steps of: (a) adding water for injection to a vessel, (b) dissolving the Levamisole in the water, (c) adding the buffers 15 (d) in a separate vessel dissolving at least one macrocyclic lactone in at least one solvent selected from glycerol formal, polyethylene glycol and dimethyl isosorbide, (e) optionally adding at least one antioxidant to the macrocyclic lactone blend (f) adding the surfactant to the macrocyclic lactone blend, (g) transferring the macrocyclic lactone blend to the bulk aqueous phase with 20 mixing, and (h) optionally, bringing the batch to volume with added WFI and mix. Preferably the formulation also contains water. Preferred forms will be described in the examples. 206155NZ NZ CS 20130411.docx IVS BRIEF DESCRIPTION OF DRAWINGS The following Detailed Description, given by way of example, and not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which: 5 DETAILED DESCRIPTION The present invention provides novel injectable combinations of levamisole and at least one macrocyclic lactone active ingredient, for the treatment or prevention of parasitic infestations and/or infection in an animal. Also provided are methods for the manufacture of the novel 10 combinations and methods for the treatment or prevention of parasitic infestations and/or infection in animals, comprising administering an effective amount of the novel injectable combination to the animal. The invention includes at least the following features: (a) In one embodiment, the invention provides novel injectable combination 15 formulations comprising levamisole and at least one macrocyclic lactone active ingredient; (b) methods for the treatment or prevention of a parasitic infestation/infection in or on an animal, which methods comprise administering a parasiticidally effective amount of a levamisole / macrocyclic lactone-containing formulation to the animal in need thereof; (c) use of the formulations as a veterinary medicament for controlling pests, 20 including parasites; and (d) processes for the preparation of the levamisole / macrocyclic lactone-containing formulations. Also contemplated within the scope of the invention are acid or base salts of the compounds in the compositions of the invention, where applicable. 206155NZ NZ CS 20130411.docx Definitions: Terms used herein will have their customary meanings in the art unless specified. As used herein the term "anthelmintic" and variations thereof encompasses one or more nematocidal, trematocidal and cestocidal active compounds. The term "pesticidal" and variations thereof 5 includes any said anthelmintic, any endectoparasiticidal, and/or any ectoparasiticidal compound. Where the context allows the term "ectoparasiticidal" includes compounds effective against any one or more ectoparasites including ticks, lice, fleas, mites and the like. Further, as used herein the "endectoparasiticidal" and variants thereof includes compounds and/or formulations that are active against internal (endo) and external (ecto) parasites. 10 As used herein the term "solubility" refers to the ability of a compound to be dissolved in a specific phase; and "lipophilic" means a greater tendency to an organic, oil or the like phase as opposed to another phase (for example, the aqueous phase). As referred to herein the term "injectable" in the context of fluids or liquids covers viscosities ranging from a free flowing liquid to a thin gel consistency that is capable of 15 being expelled by syringe and suitable for being administered to an animal via injection. As used herein, a suitable or appropriate viscosity for injection may be between 1-50 cp or from 1-10 cp. Routes of injection may be parenteral, for example intramuscular (IM), intraperitoneal (IP), or subcutaneous (SQ). As referred to herein the term "acceptable storage stability" means stable for greater than 3 20 months at room temperature. Formulations of the Invention: The formulations of the invention comprise at least levamisole and at least one macrocyclic lactone active compound which are miscible in water for injection, and are particularly effective at controlling / combating parasites, particularly endoparasites. In certain 25 embodiments, the formulations of the invention are useful in veterinary applications, including for controlling parasites in or on an animal. In one embodiment, the at least one macrocyclic lactone active agent may be selected from abamectin, doramectin, eprinomectin, ivermectin and moxidectin. 206155NZNZCS_20130411.docx The chemical structure of each is as follows: HO,,, U" 101' 0 HOHO BOiO OH HO.., O ,O, . 0 0-0 CH3 O H CH3 00,0HRC H3C 0 OHOH CH O OH Ba:R =H 3 0 H H H B3C RH =CHHOCH

OH

3 HO H Blb: R C3Q 5 Abamectin (AvermeetinBiaBib) 206155NZNZC 20130411.docx 0"' H 3 C.I"H

COH

3 e. 0 3 " CH I O OHH HH 0; HH H 0; 0 0. N. H ~o : H H 'H HjG R~r opfmomni 9, 1 , R. C H OHI H wt 1Qnlil t ~CH', 0 H' 5 CH Eprinomectin 206155NZNZCS_20130411.docx 0 0 0 0 OH O - OH Moxidectin Levamisole is an anthelmintic belonging to a class of synthetic imidazothiazole derivatives. When used in this invention it includes levamisole hydrochloride, levamisole base, 5 levamisole phosphate together with other salts and forms. H N N -S N Levamisole In one embodiment the invention provides formulations according to the following: 10 (a) Levamisole; (b) At least one macrocyclic lactone; (c) Glycerol formal; (d) A surfactant; 206155NZ NZ CS 20130411.docx (e) At least one further organic solvent selected from the group comprising, polyethylene glycol, propylene glycol and dimethyl isosorbide. (f) Water In another embodiment, the invention provides a formulation comprising: 5 (a) Levamisole; (b) At least one of abamectin, doramectin, eprinomectin, ivermectin or moxidectin or a mixture thereof; (c) Glycerol formal; (d) A surfactant, selected from Ethoxylated castor oils, Polyethoxylated castor oil and 10 Poly Oxyl 40 Hydrogenated Castor Oil and Tween; (e) At least one organic solvent selected from the group comprising polyethylene glycol, propylene glycol and dimethyl isosorbide (f) Water In yet another embodiment, the invention provides a formulation comprising: 15 (a) Levamisole; (b) Eprinomectin and Ivermectin; (c) Glycerol formal; (d) A surfactant, selected from Ethoxylated castor oils, Polyethoxylated castor oil and Poly Oxyl 40 Hydrogenated Castor Oil and Tween; 20 (e) At least one organic solvent selected from the group comprising polyethylene glycol, propylene glycol and dimethyl isosorbide (f) Water In yet another embodiment, the invention provides a formulation comprising: 206155NZ NZ CS 20130411.docx (a) Levamisole at between 20-30% w/v; (b) Eprinomectin at between 0.5 to 1.5%, and either Abamectin or Ivermectin at between 0.5 to 1.5% w/v; (c) Glycerol formal at between 5 - 30%; 5 (d) A surfactant, selected from Ethoxylated castor oils, Polyethoxylated castor oil and Poly Oxyl 40 Hydrogenated Castor Oil and Tween at between 5-20%; (e) At least one organic solvent selected from the group comprising polyethylene glycol, propylene glycol and dimethyl isosorbide at between 5-50% (f) Water 10 In the most preferred embodiment, the levamisole is levamisole phosphate and the macrocyclic lactone is eprinomectin with ivermectin. The solvent system is glycerol formal and propylene glycol and the surfactant is CREMOPHOR RH or CREMOPHOR EL. Thus in this embodiment, the formulation of the present invention may comprise: (a) Levamisole phosphate; 15 (b) Eprinomectin and ivermectin; (c) Glycerol formal and Propylene glycol (d) CREMOPHOR; and (e) Water The inventive formulations may also contain other inert ingredients such as antioxidants, 20 preservatives, or pH stabilizers. These compounds are well known in the formulation art. Antioxidant such as an alpha tocopherol, ascorbic acid, ascrobyl palmitate, fumaric acid, malic acid, sodium ascorbate, sodium metabisulfate, n-propyl gallate, BHA (butylated hydroxy anisole), BHT (butylated hydroxy toluene) monothioglycerol and the like, may be added to the present formulation. In certain embodiments, the antioxidants are generally 25 added to the formulation in amounts of from about 0.01 to about 2.0%, based upon total weight of the formulation, with about 0.05 to about 1.0% being especially preferred. 206155NZ NZ CS 20130411.docx . I In some embodiments, preservatives, such as the parabens (methylparaben and/or propylparaben), are suitably used in the formulation in amounts ranging from about 0.01 to about 2.0%, with about 0.05 to about 1.0% being especially preferred. Other preservatives include benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, 5 bronopol, butylparaben, cetrimide, chlorhexidine, chlorobutanol, chlorocresol, cresol, ethylparaben, imidurea, methylparaben, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, potassium sorbate, sodium benzoate, sodium propionate, sorbic acid, thimerosal, and the like. Preferred ranges for these compounds include from about 0.01 to about 5%. 10 Compounds which stabilize the pH of the formulation are also contemplated and may be used in certain embodiments of the inventive formulations. Again, such compounds are well known to a practitioner in the art as well as how to use these compounds. Buffering systems include, for example, systems selected from the group consisting of acetic acid/acetate, malic acid/malate, citric acid/citrate, tataric acid/tartrate, lactic acid/lactate, phosphoric 15 acid/phosphate, glycine/glycimate, tris, glutamic acid/glutamates and sodium carbonate. Parasites Controlled: The injectable levamisole / macrocyclic lactone formulations may additionally contain further anthelmintic or ectoparasitic agents. The formulations contemplated by this invention are particularly effective for efficiently 20 controlling endoparasites, such as Anaplocephala, Ancylostoma, Anecator, Ascaris, Capillaria, Cooperia, Dipylidium, Dirofilaria, Echinococcus, Enterobius, Fasciola, Haemonchus, Oesophagostumum, Ostertagia, Toxocara, Strongyloides, Toxascaris, Trichinella, Trichuris, and Trichostrongylus. Or others from the class of helminths, such as from the class of helminths, for example, Ancylostoma duodenale, Ancylostoma ceylanicum, 25 Acylostoma braziliensis, Ancylostoma spp., Ascaris lubricoides, Ascaris spp., Brugia malayi, Brugia timori, Bunostomum spp., Chabertia spp., Clonorchis spp., Cooperia spp., Dicrocoelium spp, Dictyocaulus filaria, Diphyllobothrium latum, Dracunculus medinensis, Echinococcus granulosus, Echinococcus multilocularis, Enterobius vermicularis, Faciola spp., Haemonchus spp., Heterakis spp., Hymenolepis nana, Hyostrongulus spp., Loa Loa, 30 Nematodirus spp., Oesophagostomum spp., Opisthorchis spp., Onchocerca volvulus, Ostertagia spp., Paragonimus spp., Schistosomen spp., Strongyloides fuelleborni, 206155NZ NZ CS 20130411.docx Strongyloides stercoralis, Stronyloides spp., Taenia saginata, Taenia solium, Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella nelsoni, Trichinella pseudopsiralis, Trichostrongulus spp., Trichuris trichuria, Wuchereria bancrofti. The inventive formulations may also be used for treatment against ectoparasites, the 5 ectoparasite is one or more insect or arachnid including those of the genera Ctenocephalides, Rhipicephalus, Dermacentor, Ixodes, Boophilus, Ambylomma, Haemaphysalis, Hyalomma, Sarcoptes, Psoroptes, Otodectes, Chorioptes, Hypoderma, Damalinia, Linognathus, Haematopinus, Solenoptes, Trichodectes, and Felicola. Additional examples of ectoparasites include but are not limited to the tick genus Boophilus, 10 especially those of the species microplus (cattle tick), decoloratus and annulatus; flies proper, namely those whose adult constitutes the parasite, such as Haematobia irritans (horn fly); lice such as Linognathus vitulorum, etc.; and mites such as Sarcoptes scabici and Psoroptes ovis. The above list is not exhaustive and other ectoparasites are well known in the art to be harmful to animals. 15 EXAMPLES The following examples are provided to illustrate certain embodiments of the invention and are not to be construed in any way as limiting the scope of the invention. Example 1 - Development of stable injectable levamisole / eprinomectin / ivermectin formulations 20 Levamisole is water soluble and eprinomectin and ivermectin are nearly water insoluble. Eprinomectin and ivermectin are soluble, for example, in methanol, ethanol, ethyl acetate, and dimethyl acetate. The development work fully disclosed and described herein out was carried out to provide a formulation in which the anthelmintics eprinomectin, ivermectin and levamisole (as the 25 phosphate) could be combined in a formulation having physical and chemical properties suitable for injection. Stress studies identified three formulations that are stable and suitable in terms of irritancy. 206155NZ NZ CS 20130411.doex 17 Constituents of these formulations are provided in Tables 1-3. Table 1: Formulation of Eprinomectin/Ivermectin levamisole Injection w/v% unction % Ivermectin ctive ingredient 0.7 Eprinomectin ctive ingredient 0.7 Levamisole phosphate ctive ingredient 22.3 Glycerol formal stabilized Solvent 10 Polyethylene glycol (PEG 200) Solvent 10 Cremophor RH40 Surfactant 10 BHT Antioxidant 0.1 Disodium hydrogen phosphate. Buffer 0.95 Citric acid monohydrate Buffer 0.34 Propyl paraben Preservative 0.1 Water Water QS to 100mi Table 2: Formulation of Eprinomectin/Ivermectin levamisole Injection w/v% Function % IvennctinActive 0.7 Ivermectin ingredient Active 0.7 Eprinomectin ingredient Levaisole phosphate Active 22.3% Levamisolephosphate__ingredient Glycerol formal stabilized Solvent 10.0 Propylene glycol Solvent 10.0 Cremophor RH40 Surfactant 10.0 BHT Antioxidant 0.1 Disodium hydrogen phosphate. Buffer 0.95 Citric acid monohydrate Buffer 0.34 Propyl paraben Preservative 0.1 Water for Injection Water QS to 100ml 5 Table 3: Formulation of Eprinomectin/Ivermectin levamisole Injection w/v% Function % Ivermectin Active ingredient 0.7 Eprinomectin Active ingredient 0.7 Levamisole phosphate Active ingredient 22.3 Glycerol formal stabilized Solvent 10.0 206155NZ N7 CS 20130411 docx Dimethyl isosorbide Solvent 10.0 Cremophor RH40 Surfactant 10.0 BHT Antioxidant 0.1% Disodium hydrogen phosphate Buffer 0.95% Citric acid monohydrate Buffer 0.34% Propyl paraben Preservative 0.1% Water for Injection Water QS to 100ml The formulations also proved to be suitable for use with other macrocyclic lactone active ingredients such as moxidectin and abamectin. Method of manufacture of the above formulations in tables 1 to 3. 5 Solution1 1. In a vessel, weigh solvents, surfactant, preservative and antioxidant 2. Mix until dissolved (use a little heat, T=40C). 3. Add Ivermectin and Eprinomectin; mix until clear. Solution2 10 1. In another vessel, add Disodium hydrogen phosphate. 12H20 and Citric acid monohydrate mix until clear - add levamisole phosphate, and mix until clear. Final Blend 1. Add Solution 1 into Solution 2, mix well. 2. Adjust volume to 100ml with buffer pH 4.5 15 3. Check pH and adjust with 10% H 3

PO

4 to 4.5 Example 2: Stability, Efficacy and Safety Stability of all three formulations has been demonstrated in real-time storage conditions. Additionally extensive work was undertaken to demonstrate that the formulation systems were robust enough to use with other levamisole/macrocyclic lactone combinations. 206155NZ NZ CS 20130411.docx Field efficacy studies also demonstrated that the formulations had a low level of irritancy and are highly effective in controlling both internal and external parasites. Example 3 - Inclusion of Vitamin B12 5 The formulations have also proven to be quite suitable for the delivery of Vitamin B 12. Any of the cyanocobalamin analogs and derivatives may be used. In particular hydroxocobalamin or cyanocobalamin (or mixtures thereof) are useful in the formulations of this invention. A satisfactory shelf life for a formulation that includes Vitamin B 12 can be obtained either by 10 refrigeration or by including an overage of the B12 component. Vitamin B12 was also able to be added to test formulations incorporating other macrocyclic lactone active ingredients such as moxidectin and abamectin. Table 4: Formulation of Eprinomectin/Ivermectin levamisole Injection with Vitamin B12 15 w/v% Function % Ivermectin Active ingredient 0.7 Eprinomectin Active ingredient 0.7 Levamisole phosphate Active ingredient 22.3 Vitamin B12 Active ingredient 0.14 Glycerol formal stabilized Solvent 10 Polyethylene glycol (PEG 200) Solvent 10 Cremophor RH40 Surfactant 10 BHT Antioxidant 0.1 Disodium hydrogen phosphate. Buffer 0.95 Citric acid monohydrate Buffer 0.34 Propyl paraben Preservative 0.1 Water Water QS to 100ml 206155NZ NZ CS 20130411.docx Table 5: Formulation of Eprinomectin/Ivermectin levamisole Injection with Vitamin B12 w/v% Function % Ivermectin Active 0.7 ingredient Eprinomectin ingre ent07 Levamisole phosphate ingreent 22.3% Vitamin B12 .Active 0.14 ingredient Glycerol formal stabilized Solvent 10.0 Propylene glycol Solvent 10.0 Cremophor RH40 Surfactant 10.0 BHT Antioxidant 0.1 Disodium hydrogen phosphate. Buffer 0.95 Citric acid monohydrate Buffer 0.34 Propyl paraben Preservative 0.1 Water for Injection Water QS to 1 OOml Table 6: Formulation of Eprinomectin/Ivermectin levamisole Injection with Vitamin B 12 w/v% Function % Ivermectin Active ingredient 0.7 Eprinomectin Active ingredient 0.7 Levamisole phosphate Active ingredient 22.3 Vitamin B 12 Active ingredient 0.14 Glycerol formal stabilized Solvent 10.0 Dimethyl isosorbide Solvent 10.0 Cremophor RH40 Surfactant 10.0 BHT Antioxidant 0.1% Disodium hydrogen phosphate Buffer 0.95% Citric acid monohydrate Buffer 0.34% Propyl paraben Preservative 0.1% Water for Injection Water QS to 100ml 5 206155NZNZ_CS_20130411.docx Method of manufacture of the above formulations from Tables 4 to 6 incorporating Vitamin B12 Solution 5 4. In a vessel, weigh solvents, surfactant, preservative and antioxidant 5. Mix until dissolved (use a little heat, T=40C). 6. Add Ivermectin and Eprinomectin; mix until clear. Solution2 10 2. In another vessel, add Disodium hydrogen phosphate. 12H 2 0 and Citric acid monohydrate mix until clear - add levamisole phosphate, vitamin B12 (such as cyanocobalamin or hydroxocobalamin) and mix until clear. Final Blend 15 4. Add Solution 1 into Solution 2, mix well. 5. Adjust volume to 100ml with buffer pH 4.5 6. Check pH and adjust with 10% H 3

PO

4 to 4.5 ADVANTAGES 20 The formulations contemplated by this invention are effective for efficiently controlling endoparasites and ectoparasites. The formulations are also suitable for the inclusion of Vitamin B12. The examples disclose levamisole / macrocyclic lactone-containing injectable formulations in glycerol formal that are effective in preventing and controlling internal and external 25 parasites of animals. 206155NZNZ_CS_20130411.docx VARIATIONS Various alterations or modifications can be made to the specific examples without departing from the spirit or scope of the invention as claimed. 206155NZNZ_CS_20130411.docx

Claims (18)

1. A pharmaceutically or veterinarily acceptable formulation adapted to be injected into an animal, which formulation comprises: (a) an effective amount of a levamisole, selected from levamisole phosphate, 5 levamisole hydrochloride and levamisole base; (b) an effective amount of at least one macrocyclic lactone selected from abamectin, doramectin, eprinomectin, ivermectin and moxidectin; (c) at least one surfactant selected from Ethoxylated castor oils, Polyethoxylated castor oil and Poly Oxyl 40 Hydrogenated Castor Oil, or a combination thereof 10 (d) a solvent system including at least one solvent selected from glycerol formal, propylene glycol, polyethylene glycol and dimethyl isosorbide (e) water.

2. A formulation as claimed in claim 1, also including Vitamin B12.

3. A formulation as claimed in claim 2, wherein the Vitamin B12 is present as 15 hydroxocobalamin or cyanocobalamin (or mixtures thereof).

4. A formulation as claimed in claim 3, wherein the surfactant is Cremophor RH40.

5. A formulation as claimed in claim 1, wherein the surfactant is Cremophor RH40. 20

6. A formulation as claimed in claim 5, wherein the levamisole is levamisole phosphate and wherein the levamisole is present in an amount sufficient to deliver a dose of at least 4 pig/kg animal bodyweight.

7. A formulation as claimed in any one of claims 1 to 6, which includes eprinomectin as well as ivermectin or abamectin. 25

8. A formulation as claimed in any one of claims 1 to 7 wherein the concentration of each macrocyclic lactone is from about 0.1% to about 2% (w/v); the levamisole concentration is from about 15% to about 30% (w/v); and the surfactant concentration is from about 5% to about 15% (w/v). 206155N7 NZ C9 20130411 clnex

9. A formulation as claimed in claim 4 including: (a) eprinomectin at a concentration of about 0.5% to about 0.9% (w/v); (b) ivermectin or abamectin at a concentration of about 0.5% to about 0.9% (w/v) (c) levamisole phosphate at a concentration of about 18% to about 25% (w/v); 5 (d) glycerol formal (e) a surfactant selected from Ethoxylated castor oils, Polyethoxylated castor oil and Poly Oxyl 40 Hydrogenated Castor Oil, TWEEN 80 or combinations thereof, at a concentration of at least 5% (w/v); (f) Optionally, one or more further solvents selected from propylene glycol, 10 polyethylene glycol and dimethyl isosorbide. (g) Optionally, one or more materials selected from Buffers, Antioxidants, and preservatives. (h) water.

10. A pharmaceutically or veterinarily acceptable formulation adapted to be injected into 15 an animal, which formulation comprises: (a) moxidectin at a concentration of about 0.3% to about 0.9% (w/v); (b) levamisole phosphate at a concentration of about 8% to about 25% (w/v); (c) glycerol formal (d) a surfactant selected from Ethoxylated castor oils, Polyethoxylated castor oil 20 and Poly Oxyl 40 Hydrogenated Castor Oil, TWEEN 80 or combinations thereof, at a concentration of at least 5% (w/v); (e) Optionally, one or more further solvents selected from propylene glycol, polyethylene glycol and dimethyl isosorbide. 206155NZ NZ CS 20130411.docx -* I (f) Optionally , one or more materials selected from Buffers, Antioxidants, and preservatives. (g) Water.

11. A pharmaceutically or veterinarily acceptable formulation adapted to be injected into 5 an animal, which formulation comprises: (a) At least one macrocyclic lactone at a concentration of about 0.3% to about 0.9% (w/v); (b) levamisole phosphate at a concentration of about 8% to about 25% (w/v); (c) glycerol formal 10 (d) Polyethoxylated castor oil and Poly Oxyl 40 Hydrogenated Castor Oil (e) Propylene glycol (f) Optionally, one or more materials selected from Buffers, Antioxidants, and preservatives. (g) Water. 15

12. A method for preventing or treating internal parasite infestations in animals, which method comprises administering to an animal an effective amount of a formulation according to any one of claims 1 to 11.

13. A method as claimed in claim 12, wherein the formulation is injected.

14. A method as claimed in any one of claims 12 to 13, wherein the parasites are 20 helminths.

15. A method as claimed in claim 14, wherein the helminths are nematodes, or cestodes, or trematodes, or any combination thereof.

16. A method for producing the formulation as claimed in any one of claims 1 to 11, comprising the steps of: 206155NZNZ_CS_20130411.docx (a) adding water for injection to a vessel, (b) dissolving the Levamisole in the water, (c) adding the buffers (d) in a separate vessel dissolving at least one macrocyclic lactone in at least one 5 solvent selected from glycerol formal, polyethylene glycol and dimethyl isosorbide, (e) optionally adding at least one antioxidant to the macrocyclic lactone blend (f) adding the surfactant to the macrocyclic lactone blend, (g) transferring the macrocyclic lactone blend to the bulk aqueous phase with mixing, and 10 (h) optionally, bringing the batch to volume with added WFI and mix.

17. A formulation as claimed in claim 1, substantially as herein described with reference to any one of the examples.

18. A method for producing the formulation as claimed in any one of claims 1 to 11, substantially as herein described. 15 PIPERS Attorneys for the Applicant ALLEVA ANIMAL HEALTH LIMITED 206155NZNZ_CS20130411.docx