AU2017279645B2

AU2017279645B2 - Injectable parasiticidal formulations of levamisole and macrocyclic lactones

Info

Publication number: AU2017279645B2
Application number: AU2017279645A
Authority: AU
Inventors: Atul Awasthi; Jitendra Goswami; Robert Holmes; Alan Johnson; Majid Razzak
Original assignee: Boehringer Ingelheim Animal Health USA Inc
Current assignee: Boehringer Ingelheim Animal Health USA Inc
Priority date: 2010-05-12
Filing date: 2017-12-20
Publication date: 2019-09-19
Anticipated expiration: 2031-05-12
Also published as: AU2019226279A1; AU2017279645A1; AU2016201743A1; AU2016201743B2

Abstract

The present invention provides levamisole-/macrocyclic lactone-containing injectable formulations that are effective against animal pests, including endoparasites, in or on birds and mammals. The invention also provides for an improved method for eradicating, controlling and preventing parasite infestation of birds and mammals.

Description

2017279645 20 Dec 2017

TITLE OF THE INVENTION

INJECTABLE PARASITICIDAL FORMULATIONS OF LEVAMISOLE AND MACROCYCLIC LACTONES

INCORPORATION BY REFERENCE

This Application is a divisional of Australian Patent Application No. 2016201743, which is a divisional of Australian Patent Application No. 2013206630, which is a divisional of Australian Patent Application No. 2011252987, which corresponds to International Patent Application PCT/US2011/036321 in the Australian National Phase, the entire contents of which 0 are incorporated herein by reference.

This application claims priority to U.S. provisional patent application No. 61/333,882, filed May 12, 2010. All documents cited or referenced in the applicant cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, 5 descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

FIELD OF THE INVENTION

The present invention is directed to new injectable parasiticidal formulations comprising levamisole and macrocyclic lactones. The present invention also provides methods for eradicating, controlling, and preventing parasite infestation and/or infection in birds, fish and mammals. The formulations of the invention may be administered to animals, particularly mammals, fish and birds, to prevent or treat parasitic infestation and/or infection.

BACKGROUND OF THE INVENTION

Animals, such as mammals, fish and birds are often susceptible to parasite infestation and/or infection. These parasites may be ectoparasites, such as insects and acarine species, and endoparasites such as filariae and other worms. Endoparasites pose a significant problem to those 30 who raise production animals such as bovines, ovines, and porcines. Thus, there is an ongoing

2017279645 20 Dec 2017 need to develop active pesticidal and parasiticidal compounds to protect animals against attack or infestation/infection by pests.

Many endoparasiticides or anthelmintics exist in the art for treating internal parasites and other animal pests. These endoparasiticides vary in cost as well as their effectiveness to a 5 particular parasite. However, the results of treatment with these pesticides are not always satisfactory because of continued development of resistance by the parasite to the available la

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2017279645 20 Dec 2017 therapeutic agents. Thus, there is a need in the art for more effective parasiticidal agents for treatment and protection of animals, e.g. mammals, fish and birds from infestation by animal parasites.

The avermectin and milbemycin series of compounds are potent anthelmintic and antiparasitic agents against a wide range of internal and external parasites. The compounds which belong to this series are either natural products or are semi-synthetic derivatives thereof. The structure of these two series of compounds are closely related and they both share a complex 16-membered macrocyclic lactone ring; however, the milbemycins do not contain the disaccharide substituent in the 13-position of the lactone ring. The natural product avermectins are disclosed in U.S. Patent No. 4,310,519 to Albers-Schonberg, et al., and the 22, 23-dihydro avermectin compounds are disclosed in Chabala, et al., U.S. Patent No. 4,199,569. For a general discussion of avermectins, which include a discussion of their uses in humans and animals, see Ivermectin and Abamectin, W. C. Campbell, ed., Springer-Verlag, N.Y. (1989). Naturally occurring milbemycins are described in Aoki et al., U.S. Patent No. 3,950,360 as well as in the various references cited in The Merck Index 12^th ed., S. Budavari, Ed., Merck & Co., Inc. Whitehouse Station, N.J. (1996). Semi-synthetic derivatives of these classes of compounds are well known in the art and are described, for example, in U.S. Patent No. 5,077,308, U.S. Patent No. 4,859,657, U.S. Patent No. 4,963,582, U.S. Patent No. 4,855,317, U.S. Patent No. 4,871,719, U.S. Patent No. 4,874,749, U.S. Patent No. 4,427,663, U.S. Patent No. 4,310,519, U.S. Patent No. 4,199,569, U.S. Patent No. 5,055,596, U.S. Patent No. 4,973,711, U.S. Patent No. 4,978,677, and U.S. Patent No. 4,920,148, all incorporated herein by reference in their entirety. A particularly useful macrocyclic lactone is eprinomectin, which is described in US6,174,540, US6,733,767, US5,602,107 to Merck (all incorporated by reference). This compound provides for a very short milk withdrawal period due to it tendency to partition away from the milk of a 25 lactating mammal.

To overcome the problem of resistance, various combinations of anthelmintics have been extensively researched. Unfortunately, combinations of macrocyclic lactone anthelmintics with anthelmintics of other families have proven difficult to formulate at high concentration and with an acceptable viscosity for injection. For example, Closamectin (manufactured by Norbrook) contains ivermectin at 0.5% and closantel at 12.5% and is so viscous that the product is supplied in a special pack that has a warming device. The warming device is activated 15 minutes before

2017279645 04 Jul 2019 any previously described, effective combinations of levamisole and eprinomectin, injectable or otherwise.

It is expressly noted that citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Any 5 foregoing applications, and all documents cited therein or during their prosecution (“application cited documents”) and all documents cited or referenced in the application cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in 0 any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to 5 the present disclosure as it existed before the priority date of each of the appended claims.

Throughout this specification the word comprise, or variations such as comprises or comprising, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

SUMMARY OF THE INVENTION

In one aspect, there is provided a pharmaceutically or veterinarily acceptable formulation adapted to be injected into an animal, which formulation comprises;

a. an effective amount of a levamisole in a concentration of from about 18% to about

25% (w/v);

b. an effective amount of at least one macrocyclic lactone in a concentration of from about 0.1% to about 2% (w/v);

c. a pharmaceutically acceptable system of solvents and surfactants wherein the system provides for acceptable storage stability for both the levamisole and the macrocyclic lactone and wherein the system provides for a viscosity which is suitable for injection;

2017279645 23 Aug 2019 wherein the surfactants comprise at least polyoxyethylenated esters of sorbitan, a polyethylene glycol stearate, or both;

wherein the solvents comprise at least glycerol formal (GF), propylene glycol, dimethyl acetamide (DMA), or combinations thereof, but not n-methyl pyrrolidone (NMP);

wherein the levamisole is levamisole phosphate;

wherein the formulation comprises water for injection; and wherein the water for injection is provided in a quantity sufficient to bring the formulation to a suitable injection volume.

In another aspect, there is provided a method for preventing and/or treating a helminth infestation in an animal, which method comprises administering to an animal an effective amount of a formulation as defined above.

In another aspect, there is provided a method for producing a formulation as defined above comprising the steps of:

a. adding water for injection to a vessel,

b. dissolving the Levamisole in the water,

c. in a separate vessel dissolving Eprinomectin and BHT in DMA,

d. adding the surfactant to the DMA/Eprinomectin blend,

e. transferring the Eprinomectin/DMA blend to the bulk aqueous phase with mixing, and

f. optionally, bringing the batch to volume with added WFI and mix.

In another aspect, there is provided use of a formulation as defined above for the manufacture of a medicament for the prevention and/or treatment of a helminth infestation in an animal.

In another aspect, there is provided use of a formulation as defined above for the prevention and/or treatment of a helminth infestation in an animal.

The invention provides novel parasiticidal formulations comprising levamisole and macrocyclic lactones, particularly avermectins such as ivermectin and eprinomectin, and milbemycins, such as moxidectin, milbemectin, and milbemycin oxime. The formulations are storage-stable and provided in a single solvent system that is miscible in water. Therefore, the formulations according to the present invention may be classified as micellar solutions as opposed to emulsions or biphasic emulsions. The invention also provides methods for the treatment and prevention of parasitic infestation and/or infection of animals.

4a

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The inventive formulations comprising the levamisole and macrocyclic lactones are highly effective for the treatment or prophylaxis of parasites in or on mammals, fish and birds, and in particular, cats, dogs, horses, chickens, pigs, sheep and cattle with the aim of ridding these hosts of all the parasites commonly encountered by mammals, fish and birds. The invention also 5 provides for effective and long-lasting defense against endoparasites, such as helminths, nematodes, filariae, hookworms, whipworms and roundworms of the digestive tract of animals and humans.

Accordingly, the present invention provides methods for preventing and treating parasites in or on animals, comprising administering a parasiticidally effective amount of a storage-stable 0 injectable formulation comprising levamisole and macrocyclic lactone to the animal. The invention also provides a method for combating or controlling animal pests and parasites.

4b

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It is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that the Applicants reserve the right to this invention and hereby disclose a disclaimer of any previously known product, process, or method.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law; e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from, and encompassed by, the following Detailed Description.

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:

FIG. 1 is a flow diagram outlining the preparation of formulations according to the instant invention

FIG. 2 depicts levamisole plasma concentration at time points after administration of formulations according to the instant invention

FIG. 3 depicts eprinomectin plasma concentration at time points after administration of formulations according to the instant invention

DETAILED DESCRIPTION

The present invention provides novel injectable levamisole / macrocyclic lactone formulations with parasiticidal activity, for the treatment or prevention of parasitic infestations and/or infection in an animal. Also provided are methods for the treatment or prevention of parasitic infestations and/or infection in animals, comprising administering an effective amount of the formulation of the invention to the animal.

An important aspect of formulations containing eprinomectin as the macrocyclic lactone component is that said formulations may have shortened withdrawal times before it is

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2017279645 20 Dec 2017 appropriate to obtain milk from lactating mammals. This is because of the macrocyclic lactones, eprinomectin has relatively the lowest milk to plasma partition ratio (-0.2). For a point of reference and definition, a milk to plasma partition ratio of 1 would indicate the active tends to distribute evenly between milk and plasma.

Formulations described herein are particularly effective for controlling endoparasites. Endoparasites include, but are not limited to, nematodes (such as roundworms, hookworms, whipworms and heartworms) and cestodes (tapeworms) and trematodes (flukes). Therefore, the inventive formulations have substantial utility in preventing damage to crops, plants, plant propagation material and wood-containing property, and in controlling and preventing the infestation and/or infection of animals by parasites.

The invention includes at least the following features:

(a) In one embodiment, the invention provides novel injectable formulations comprising levamisole and macrocyclic lactones, which are active against animal pests, including insects and parasites;

(b) methods for treating a parasitic infestation/infection in or on an animal are provided, which methods comprise administering a parasiticidally effective amount of a levamisole / macrocyclic lactone-containing formulation to the animal in need thereof;

(c) methods for the prevention of a parasitic infestation/infection of an animal, which comprise administering a parasiticidally effective amount the levamisole / macrocyclic lactonecontaining formulation to the animal in need thereof;

(d) use of the formulations as a veterinary medicament for controlling pests, including parasites; and (e) 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.

The term acid contemplates all pharmaceutically acceptable inorganic or organic acids.

Inorganic acids include mineral acids such as hydrohalic acids such as hydrobromic acid and hydrochloric acid, sulfuric acid, phosphoric acids and nitric acid. Organic acids include all pharmaceutically acceptable aliphatic, alicyclic and aromatic carboxylic acids, dicarboxylic acids, tricarboxylic acids and fatty acids. In one embodiment of the acids, the acids are straight

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2017279645 20 Dec 2017 chain or branched, saturated or unsaturated C1-C20 aliphatic carboxylic acids, which are optionally substituted by halogen or by hydroxyl groups, or C₆-Ci₂ aromatic carboxylic acids. Examples of such acids are carbonic acid, formic acid, acetic acid, propionic acid, isopropionic acid, valeric acid, α-hydroxy acids such as glycolic acid and lactic acid, chloroacetic acid, benzoic acid, methane sulfonic acid, and salicylic acid. Examples of dicarboxylic acids include oxalic acid, malic acid, succinic acid, tartaric acid, fumaric acid, and maleic acid. An example of a tricarboxylic acid is citric acid. Fatty acids include all pharmaceutically acceptable saturated or unsaturated aliphatic or aromatic carboxylic acids having 4 to 24 carbon atoms. Examples include butyric acid, isobutyric acid, sec-butyric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and phenylsteric acid. Other acids include gluconic acid, glycoheptonic acid and lactobionic acid.

The term “base” contemplates all pharmaceutically acceptable inorganic or organic bases, including hydroxides, carbonates or bicarbonates of alkali metal or alkaline earth metals. Salts formed with such bases include, for example, the alkali metal and alkaline earth metal salts, including, but not limited to, as the lithium, sodium, potassium, magnesium or calcium salts. Salts formed with organic bases include the common hydrocarbon and heterocyclic amine salts, which include, for example, ammonium salts (NH4⁺), alkyl- and dialkylammonium salts, and salts of cyclic amines such as the morpholine and piperidine salts.

In addition, the compounds within the compositions of the invention may exist as hydrates or solvates, in which a certain stoichiometric amount of water or a solvent is associated with the molecule in the crystalline form. The compositions of the invention may include hydrates and solvates of the active agents.

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 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

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2017279645 20 Dec 2017 used herein the “endectoparasiticidal” and variants thereof includes compounds and/or formulations that are active against internal (endo) and external (ecto) parasites.

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 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 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, and are provided as micellar solutions of actives, which are miscible in water for injection, and are particularly effective at controlling / combating parasites, particularly endoparasites. In certain 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 in the formulations of the invention may be at least one avermectin or milbemycin compound. In various embodiments, the at least one macrocyclic lactone compound may be abamectin, avermectin, dimadectin, doramectin, emamectin, eprinomectin, ivermectin, latidectin, lepimectin, selamectin, milbemectin, milbemycin D, milbemycin oxime, moxidectin or nemadectin.

In another embodiment, the at least one macrocyclic lactone active agent may be any one of the avermectins or avermectin monosaccharides modified in the 4' or 4 position described in US 7,704,961, US 7,521,429, US 2006-0105970 Al, US 7,678,740, US 7,632,820, US 7,678,773, US 7,605,134, and US 6,933,260 (each to Merial). It will be appreciated by a skilled person that any of the above-recited avermectins exhibiting a relatively low milk to plasma 30 partition ratio (for example less than about 1, less than about 0.5, and even more favorably, less

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2017279645 20 Dec 2017 than about 0.2) will be a particularly desirable component of formulations according to the instant invention.

In one embodiment the invention provides formulations according to the following:

(a) Levamisole;

(b) At least one macrocyclic lactone;

(c) At least one water miscible organic solvent;

(d) A surfactant;

(e) Water for injection (referred to hereafter as “WFI”).

In another embodiment, the invention provides a formulation comprising:

(a) Levamisole;

(b) At least one of ivermectin, abamectin, or eprinomectin, or a mixture thereof;

(c) At least one water miscible organic solvent;

(d) A surfactant; and (e) WFI.

In still another embodiment, the invention provides a formulation comprising:

(a) Levamisole;

(b) At least one of dimadectin, doramectin emamectin, latidectin, lepimectin or selamectin, or a mixture thereof;

(c) At least one water miscible organic solvent;

(d) A surfactant; and (e) WFI.

In yet another embodiment, the invention provides a formulation comprising:

(a) Levamisole;

(c) At least one water miscible organic solvent;

(d) A surfactant; and (e) WFI.

In another embodiment, the invention provides a formulation comprising:

(a) Levamisole;

(b) At least one of milbemectin, milbemycin D, milbemycin oxime, moxidectin or nemadectin, or a mixture thereof;

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2017279645 20 Dec 2017 (c) At least one water miscible organic solvent;

(d) A surfactant; and (e) WFI.

In a preferred embodiment, the invention provides a formulation comprising:

(a) Levamisole;

(b) At least one of ivermectin or eprinomectin, or a mixture thereof;

(c) At least one water miscible organic solvent;

(d) A surfactant; and (e) WFI.

In another embodiment, the invention provides a formulation comprising:

(a) Levamisole;

(b) At least one of avermectin or avermectin monosaccharide, each modified at the 4’ or 4” position, or a mixture thereof;

(c) At least one water miscible organic solvent;

(d) A surfactant; and (e) WFI.

In an embodiment, the levamisole is levamisole phosphate and is present in an amount sufficient to deliver a dose of at least 4 mg/kg animal bodyweight. In another embodiment, the macrocyclic lactone is either ivermectin or eprinomectin and is present in an amount sufficient to deliver a dose of at least 150 pg/kg animal bodyweight.

In a particular embodiment, the present invention is a stable injectable formulation comprising eprinomectin and levamisole and is suitable for use in cattle. In an embodiment, the formulation is an injectable combination anthelmintic containing about 7 g/L eprinomectin and about 223 g/L levamisole phosphate.

In an emodiment, the formulation requires not more than a 35 day withholding period for either milk or meat.

In an embodiment, the formulation is administered at a dose rate of 1 mL per 35 kg (200pg eprinomectin and 6.4 mg levamisole phosphate per kg, equivalent to 5 mg levamisole HC1 per kg). In another embodiment, the formulation is effective against a broad range of 30 internal parasites including Ostertagla spp., Trichostrongylus axel, Cooperia spp., suckling lice, and certain strains resistant to certain macrocyclic lactones and/or benzamidazoles.

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In an embodiment, the formulations have a shelf life and storage condition of at least 2 years at 2-6 °C. In another embodiment, the formulations are prepared as pack sizes of 100 to 1000 mL. In a particular embodiment, the pack sizes are 500 mL flexible packages, or flexipacks.

In a preferred embodiment, the water miscible solvent will be an organic solvent, including an amide, alcohol, ester or sulfoxide. In another preferred embodiment, the formulations of the invention will comprise a pharmaceutically acceptable amide including, but not limited to, dimethylformamide, dimethylacetamide, 2-pyrrolidone, N-methylpyrrolidone and the like.

Surfactants are well known in the art and may include non-ionic surfactants, cationic surfactants and anionic surfactants. Anionic surfactants include, but are not limited to, alkaline stearates (e.g. sodium, potassium or ammonium stearate); calcium stearate or triethanolamine stearate; sodium abietate; alkyl sulfates, which include but are not limited to sodium lauryl sulfate and sodium cetyl sulfate; sodium dodecylbenzenesulphonate or sodium dioctyl sulphosuccinate; or fatty acids (e.g. coconut oil);

Cationic surfactants include, but are not limited to, any known water-soluble quaternary ammonium salts such as cetyltrimethylammonium bromide and the like; and known amine salts such as octadecylamine hydrochloride and the like.

Non-ionic surfactants include, but are not limited to, optionally polyoxyethylenated esters of sorbitan, e.g. Polysorbate 80, or polyoxyethylenated alkyl ethers; polyethylene glycol stearate, polyoxyethylenated derivatives of castor oil, polyglycerol esters, polyoxyethylenated fatty alcohols, polyoxyethylenated fatty acids or copolymers of ethylene oxide and of propylene oxide. Other surfactants that are suitable for the formulations of the present invention include amphoteric surfactants, such as substituted lauryl compounds of betaine. The surfactant of the 25 inventive formulations may also be a mixture of at least two different surfactants.

In another embodiment, the levamisole is levamisole phosphate and the macrocyclic lactone is either eprinomectin or ivermectin. In some embodiments, the water miscible organic solvent is dimethylacetamide (DMA) and the surfactant is CREMOPHOR EL. Thus in an embodiment, the formulation of the present invention may comprise:

(a) Levamisole phosphate;

(b) Eprinomectin, ivermectin, abamectin, doramectin or moxidectin;

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2017279645 20 Dec 2017 (c) DMA (d) CREMOPHOR EL; and (e) WFI

The inventive formulations may contain other inert ingredients such as antioxidants, 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 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.

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, 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%.

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 25 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 acid/phosphate, glycine/glycimate, tris, glutamic acid/glutamates and sodium carbonate.

Parasites Controlled:

The injectable levamisole / macrocyclic lactone formulations are particularly effective for efficiently controlling endoparasites, such as flukes, hookworms, and helminths such as cestodes, nematodes, and trematodes. Endoparasites further include helminths such as Anaplocephala,

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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, 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, Nematodirus spp., Oesophagostomum spp., Opisthorchis spp., Onchocerca volvulus, Ostertagia spp., Paragonimus spp., Schistosomen spp., Strongyloides fuelleborni, Strongyloides stercoralis, Stronyloides spp., Taenia saginata, Taenia solium, Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella nelsoni, Trichinella pseudopsiralis, Trichostrongulus spp., Trichuris trichuria, Wuchereria bancrofti.

In one embodiment for treatment against ectoparasites, the 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.

In another embodiment for the treatment against ectoparasites, the ectoparasite is from the genera Ctenocephalides, Rhipicephalus, Dermacentor, Ixodes and/or Boophilus. The ectoparasites treated include but are not limited to fleas, ticks, mites, mosquitoes, flies, lice, blowfly and combinations thereof. Specific examples include but are not limited to cat and dog fleas (Ctenocephalides felis, Ctenocephalides sp. and the like), ticks (Rhipicephalus sp., Ixodes 25 sp., Dermacentor sp., Amblyoma sp. and the like), and mites (Demodex sp., Sarcoptes sp., Otodectes sp. and the like), lice (Trichodectes sp., Cheyletiella sp., Lignonathus sp., and the like), mosquitoes (Aedes sp., Culex sp., Anopheles sp., and the like) and flies (Hematobia sp., Musca sp., Stomoxys sp., Dematobia sp., Cochliomyia sp., and the like). In yet another embodiment for the treatment against ectoparasites, the ectoparasite is a flea and/or tick.

Additional examples of ectoparasites include but are not limited to the tick genus Boophilus, especially those of the species microplus (cattle tick), decoloratus and annulatus', myiases such

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2017279645 20 Dec 2017 as Dermatobia hominis (known as Berne in Brazil) and Cochliomyia hominivorax (greenbottle); sheep myiases such as Lucilia sericata, Lucilia cuprina (known as blowfly strike in Australia, New Zealand and South Africa). 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 and humans. These include, for example migrating dipterous larvae.

When an anthelmintic agent is added to the composition of the invention, the composition can also be used to treat against endoparasites such as those helminths selected from the group consisting of Anaplocephala, Ancylostoma, Anecator, Ascaris, Capillaria, Cooperia, Dipylidium, Dirofilaria, Echinococcus, Enterobius, Fasciola, Haemonchus, Oesophagostumum, Ostertagia, Toxocara, Strongyloides, Toxascaris, Trichinella, Trichuris, and Trichostrongylus.

In another embodiment of the invention, the compounds and compositions of the invention are suitable for controlling pests such as insects selected from the group consisting of Blatella germanica, Heliothis virescens, Leptinotarsa decemlineata, Tetramorium caespitum and combinations thereof.

The phytoparasitic nematodes include, for example, Anguina spp., Aphelenchoides spp., Belonoaimus spp., Bursaphelenchus spp., Ditylenchus dipsaci, Globodera spp., Heliocotylenchus spp., Heterodera spp., Longidorus spp., Meloidogyne spp., Pratylenchus spp., Radopholus similis, Rotylenchus spp., Trichodorus spp., Tylenchorhynchus spp., Tylenchulus spp., Tylenchulus semipenetrans, Xiphinema spp.

In addition, with or without the other pesticidal agents added to the composition, the invention can also be used to treat other pests which include but are not limited to pests:

(1) from the order of Isopoda, for example Oniscus asellus, Armadillidium vulgare and

Porcellio scaber;

(2) from the order of Diplopoda, for example Blaniulus guttulatus;

(3) from the order of Chilopoda, for example Geophilus carpophagus and Scutigera spp.;

(4) from the order of Symphyla, for example Scutigerella immaculata;

(5) from the order of Thysanura, for example Lepisma saccharina;

(6) from the order of Collembola, for example Onychiurus armatus;

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2017279645 20 Dec 2017 (7) from the order of Blattaria, for example Blatta orientalis, Periplaneta americana, Leucophaea maderae and Blattella germaniccr, (8) from the order of Hymenoptera, for example Diprion spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis and Vespa spp.;

(9) from the order of Siphonaptera, for example Xenopsylla cheopis and Ceratophyllus spp.;

(10) from the order of Anoplura (Phthiraptera), for example, Damalinia spp., Haematopinus spp., Linognathus spp., Pediculus spp., Trichodectes spp.;

(11) from the class of Arachnida, for example, Acarus siro, Aceria sheldoni, Aculops spp., Aculus spp., Amblyomma spp., Argas spp., Boophilus spp., Brevipalpus spp., Bryobia praetiosa, Chorioptes spp., Dermanyssus gallinae, Eotetranychus spp., Epitrimerus pyri, Eutetranychus spp., Eriophyes spp., Hemitarsonemus spp., Hyalomma spp., Ixodes spp., Latrodectus mactans, Metatetranychus spp., Oligonychus spp., Ornithodoros spp., Panonychus spp., Phyllocoptruta oleivora, Polyphagotarsonemus latus, Psoroptes spp., Rhipicephalus spp., Rhizoglyphus spp., Sarcoptes spp., Scorpio maurus, Stenotarsonemus spp., Tarsonemus spp., Tetranychus spp., Vasates lycopersici.;

(12) from the class of Bivalva, for example, Dreissena spp.;

(13) from the order of Coleoptera, for example, Acanthoscelides obtectus, Adoretus spp., Agelastica alni, Agriotes spp., Amphimallon solstitialis, Anobium punctatum, Anoplophora spp., Anthonomus spp., Anthrenus spp., Apogonia spp., Atomaria spp., Attagenus spp., Bruchidius obtectus, Bruchus spp., Ceuthorhynchus spp., Cleonus mendicus, Conoderus spp., Cosmopolites spp., Costelytra zealandica, Curculio spp., Cryptorhynchus lapathi, Dermestes spp., Diabrotica spp., Epilachna spp., Faustinas cubae, Gibbium psylloides, Heteronychus arator, Hylamorpha elegans, Hylotrupes bajulus, Hypera postica, Hypothenemus spp., Lachnosterna consanguinea, Leptinotarsa decemlineata, Lissorhoptrus oryzophilus, Lixus spp., Lyctus spp., Meligethes aeneus, Melolontha melolontha, Migdolus spp., Monochamus spp., Naupactus xanthographus,

Niptus hololeucus, Oryctes rhinoceros, Oryzaephilus surinamensis, Otiorrhynchus sulcatus, Oxycetonia jucunda, Phaedon cochleariae, Phyllophaga spp., Popillia japonica, Premnotrypes spp., Psylliodes chrysocephala, Ptinus spp., Rhizobius ventralis, Rhizopertha dominica, Sitophilus spp., Sphenophorus spp., Sternechus spp., Symphyletes spp., Tenebrio molitor, 30 Tribolium spp., Trogoderma spp., Tychius spp., Xylotrechus spp., Zabrus spp.;

(14) from the order of Diptera, for example, Aedes spp., Anopheles spp., Bibio hortulanus,

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Calliphora erythrocephala, Ceratitis capitata, Chrysomyia spp., Cochliomyia spp., Cordylobia anthropophaga, Culex spp., Cuterebra spp., Dacus oleae, Dermatobia hominis, Drosophila spp., Fannia spp., Gastrophilus spp., Hylemyia spp., Hyppobosca spp., Hypoderma spp., Liriomyza spp., Lucilia spp., Musca spp., Nezara spp., Oestrus spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Stomoxys spp., Tabanus spp., Tannia spp., Tipulapaludosa, Wohlfahrtia spp.;

(15) from the class of Gastropoda, for example, Arion spp., Biomphalaria spp., Bulinus spp., Deroceras spp., Galba spp., Lymnaea spp., Oncomelania spp., Succinea spp.;

(16) from the class of helminths, for example, Ancylostoma duodenale, Ancylostoma ceylanicum, 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, Nematodirus spp., Oesophagostomum spp., Opisthorchis spp., Onchocerca volvulus, Ostertagia spp., Paragonimus spp., Schistosomen spp., Strongyloides fuelleborni, Strongyloides stercoralis, Stronyloides spp., Taenia saginata, Taenia solium, Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella nelsoni, Trichinella pseudopsiralis, Trichostrongulus spp., Trichuris trichuria, Wuchereria bancrofti.', (17) from the order of Heteroptera, for example, Anasa tristis, Antestiopsis spp., Blissus spp., Calocoris spp., Campylomma livida, Cavelerius spp., Cimex spp., Creontiades dilutus, Dasynus piperis, Dichelops furcatus, Diconocoris hewetti, Dysdercus spp., Euschistus spp., Eurygaster spp., Heliopeltis spp., Horcias nobilellus, Leptocorisa spp., Leptoglossus phyllopus, Lygus spp., Macropes excavatus, Miridae, Nezara spp., Oebalus spp., Pentomidae, Piesma quadrata, Piezodorus spp., Psallus seriatus, Pseudacysta persea, Rhodnius spp., Sahlbergella singularis,

Scotinophora spp., Stephanitis nashi, Tibraca spp., Triatoma spp.;

(18) from the order of Homoptera, for example, Acyrthosipon spp., Aeneolamia spp., Agonoscena spp., Aleurodes spp., Aleurolobus barodensis, Aleurothrixus spp., Amrasca spp., Anuraphis cardui, Aonidiella spp., Aphanostigma piri, Aphis spp., Arboridia apicalis, Aspidiella spp., Aspidiotus spp., Atanus spp., Aulacorthum solani, Bemisia spp., Brachycaudus helichrysii,

Brachycolus spp., Brevicoryne brassicae, Calligypona marginata, Carneocephala fulgida, Ceratovacuna lanigera, Cercopidae, Ceroplastes spp., Chaetosiphon fragaefolii, Chionaspis

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(20) from the order of Lepidoptera, for example, Acronicta major, Aedia leucomelas, Agrotis spp., Alabama argillacea, Anticarsia spp., Barathra brassicae, Bucculatrix thurberiella, Bupalus piniarius, Cacoecia podana, Capua reticulana, Carpocapsa pomonella, Cheimatobia brumata, Chilo spp., Choristoneura fumiferana, Clysia ambiguella, Cnaphalocerus spp., Earias insulana, Ephestia kuehniella, Euproctis chrysorrhoea, Euxoa spp., Feltia spp., Galleria mellonella, Helicoverpa spp., Heliothis spp., Hofinannophila pseudospretella, Homona magnanima,

Hyponomeuta padella, Laphygma spp., Lithocolletis blancardella, Lithophane antennata, Loxagrotis albicosta, Lymantria spp., Malacosoma neustria, Mamestra brassicae, Mods repanda, Mythimna separata, Oria spp., Oulema oryzae, Panolis flammea, Pectinophora gossypiella, Phyllocnistis citrella, Pieris spp., Plutella xylostella, Prodenia spp., Pseudaletia spp., Pseudoplusia includens, Pyrausta nubilalis, Spodoptera spp., Thermesia gemmatalis, Tinea 30 pellionella, Tineola bisselliella, Tortrix viridana, Trichoplusia spp.;

(21) from the order of Orthoptera, for example, Acheta domesticus, Blatta orientalis, Blattella

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(22) from the order of Thysanoptera, for example, Baliothrips biformis, Enneothrips flavens, Frankliniella spp., Heliothrips spp., Hercinothrips femoralis, Kakothrips spp., Rhipiphorothrips cruentatus, Scirtothrips spp., Taeniothrips cardamoni, Thrips spp.;

(23) from the class of Protozoa, for example, Eimeria spp.

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 is particularly, though not exclusively, representative of injectable eprinomectin / levamisole formulations according to the present invention. Example 2 provides evidence that ivermectin-containing formulations according to the present invention are stable and safe in animals. Finally, example 3 provides extensive, though not sole evidence of the significant amount of time and effort that was invested in developing the inventive formulations.

Example 1 - Development of stable injectable levamisole / eprinomectin formulations

Levamisole is water soluble and eprinomectin is nearly water insoluble. Eprinomectin is soluble, for example, in methanol, ethanol, ethyl acetate, and dimethyl acetate (DMA). Applicants are aware of no existing combination injectable formulation comprising eprinomectin and levamisole. The development work fully disclosed and described herein out was carried out to provide a formulation in which the anthelmintics eprinomectin and levamisole (as the phosphate) could be combined in a formulation having physical and chemical properties suitable 25 for injection.

Stress studies have identified at least two formulations that are stable and suitable in terms of irritancy.

Constituents of these formulations are provided in Table 1.

Table 1: Formulation of Eprinomectin levamisole Injection

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Ingredient	Formulation 1	Formulation 2	Function
% w/v Composition	% w/v Composition
Eprinomectin	0.70*	0.70*	Active
Levamisole Phosphate	22.3*	22.3*	Active
BHT	0.1	0.1	Antioxidant
Dimethylacetamide	5.0	5.0	Solvent
Methyl Paraben	0.15	0.15	Preservative
Polysorbate 80	5.0	-	Surfactant
Cremophor EL	-	5.0	Surfactant
Water For Injection	q.s. to 100%		Vehicle

* Corrected for potency

Stability of both formulations has been demonstrated in real-time storage conditions.

Table 2: Stability of Eprinomectin Levamisole Injection Formulation 1

Batch No.		2-8°C	25°C / 60% RH
		Control	1 month	2 month	3 month
Formulation 1.	Description	VLYS	VLYS	VLYS	VLYS
Assay of Eprinomectin	100.9	99.6	98.0	98.6
Assay of Levamisole Phosphate	98.0	97.9	98.3	99.2

VLYS: Very Light Yellow Solution

LYS: Light Yellow Solution

YS: Yellow Solution

Table 3: Stability of Eprinomectin Levamisole Injection Formulation 2

Batch No.

2-8°C

25°C / 60% RH

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		Control	1 month	2 month	3 month
Formulation 2.	Description	VLYS	VLYS	VLYS	VLYS
Assay of Eprinomectin	101.1	100.0	98.0	99.6
Assay of Levamisole Phosphate	98.9	99.3	98.8	100.3

VLYS: Very Light Yellow Solution

LYS: Light Yellow Solution

YS: Yellow Solution

Table 4: Administration of Treatments

Both formulations were also tested in cattle studies to determine the absorption characteristics and injection site acceptability. Twenty-one Hereford Cross 15 month old female cattle were selected for the study from a larger group held at the study site. On Day -1 the study animals were individually identified with uniquely numbered ear tags.

The study animals were weighed and ranked according to body weight. The animal with the median body weight (305 kg) was selected as the control animal. The remaining animals were assigned to two groups of ten by restrictive randomization based on body weight. Treatments were administered as per Table 4.

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Animals in Group 1 were treated with Formulation 1 at a dose rate of lml/35kg body

Animal ID	Group ID	Product/ BN	Weight (kg)	Dose (ml)
2	1	Formulation 1 BN:08	324.0	9.3
3	294.0	8.4
5	334.0	9.5
6	315.0	9.0
9	343.0	9.8
11	297.0	8.5
12	285.0	8.1
13	289.0	8.3
14	302.0	8.6
20	313.0	8.9

1	2	Formulation 2 BN:09	270.0	7.7
4	303.0	8.7
7	288.0	8.2
8	317.0	9.1
10	326.0	9.3
16	331.0	9.5
17	291.0	8.3
18	309.0	8.8
19	296.0	8.5
21	340.0	9.7
15	3	Control	305.0

weight. Animals in Group 2 were treated with Formulation 2 at a dose rate of lml/35kg bodyweight. All calculated doses were rounded up to the nearest 0.2ml. The control animal received no treatment.

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Treatments were administered by subcutaneous injection to the anterior half of the neck, using a new sterile 18 gauge 25mm needle for each injection. The area for injection was clipped and swabbed with 70% isopropyl alcohol prior to injection. The injection sites were inspected and palpated at 24, 48 and 96 hours post treatment and on Days 10, 21 and 35. All observations were recorded.

The degree of injection site swelling was recorded according to the following scale:

= no edema = very slight edema (barely noticed) = slight edema (well defined by definite raising) = moderate edema (raised approximately 1mm) = (raised more than 1 mm and extending beyond the area of injection)

The maximum length, width and height of any visible or palpable site reaction was determined using calipers, and all findings were recorded.

Table 5: Injection Site Reactions Formulation 1

Formulation 1 Batch no:08
Animal ID	Weight (kg)	Dose (mL)	24 hours	48 hours	96 hours	Day 10	Day 21	Day 35
2	324.0	9.3	4	4	4	4	1	0
3	294.0	8.4	4	2	1	0	0	0
5	334.0	9.5	4	1	1	0	0	0
6	315.0	9.0	4	4	4	4	3	1
9	343.0	9.8	0	0	1	0	0	0
11	297.0	8.5	4	3	1	1	0	0
12	285.0	8.1	4	2	1	1	0	0
13	289.0	8.3	4	4	3	2	0	0
14	302.0	8.6	4	4	1	1	0	0
20	313.0	8.9	4	4	1	0	0	0

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Table 6: Injection Site Reactions Formulation 2

Formulation 2 Batch no:09
Animal ID	Weight (kg)	Dose (mL)	24 hours	48 hours	96 hours	Day 10	Day 21	Day 35
1	270.0	7.7	4	4	3	1	0	0
4	303.0	8.7	4	4	1	0	0	0
7	288.0	8.2	4	4	4	0	0	0
8	317.0	9.1	4	4	4	3	0	0
10	326.0	9.3	4	4	0	0	0	0
16	331.0	9.5	4	1	0	0	0	0
17	291.0	8.3	4	4	4	1	0	0
18	309.0	8.8	4	3	1	0	0	0
19	296.0	8.5	1	1	1	0	0	0
21	340.0	9.7	4	4	1	1	0	0

Blood plasma profile

On Day -1, (3 Jan 2010) five animals were randomly selected from each of the two treated groups for blood sampling as illustrated in Table 7:

Table 7: Eprinomectin Levamisole Blood Sampling Schedule

Blood Plasma Group 1	Blood Plasma Group 2
3	1
11	10
12	16
14	18
20	21

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These ten animals plus the untreated control were blood sampled via jugular venipuncture on Day -1 prior to treatment. These same animals were blood sampled at 1, 2, 6, 12, 24, 48 and 96 hours post treatment, and then at Day 10, Day 21 and Day 35 after treatment. Blood samples were collected by caudal venipuncture into green top heparinised Vacutainer’s, and spun in a centrifuge for ten minutes at 3000 RPM. Plasma for each animal was decanted into individually labeled plasma vials and frozen within two hours of collection.

At the conclusion of the animal phase of the study (Day 35) the frozen plasma samples were submitted to an analytical laboratory for Eprinomectin and Levamisole analysis. Samples from 0, 1,2, 6, 12, 24 and 48 hours were analyzed for Levamisole concentration. Samples from 0, 48, 96 hours and 10, 20 and 35 days were analyzed for Eprinomectin concentration.

The mean eprinomectin and levamisole pharmacokinetic profiles in cattle plasma after treatment with the two formulations are presented in the figures below. Results demonstrate that both formulations resulted in almost identical plasma profiles for both levamisole and eprinomectin. FIGs. 2 and 3 summarize the data.

Table 8: Excipients used in the Eprinomectin / Levamisole injectable formulations

Ingredient	CAS No.	Formulation 1	Formulation 2	Function
% w/v Composition	% w/v Composition
BHT	128-37-0	0.1	0.1	Antioxidant
Dimethylacetamide	127-19-5	5.0	5.0	Solvent
Methyl Paraben	99-76-3	0.15	0.15	Preservative
Polysorbate 80	9005-65-6	5.0	-	Surfactant
Cremophor EL	61796-12-6	-	5.0	Surfactant
Water For Injection	7789-20-0	q.s. to 100%	q.s. to 100%	Vehicle

Formulation Development

A number of solvents were evaluated including Glycerol Formal, 2-Pyrol, Propylene Glycol, Miglyol 840 and DMA from 2 - 20% (see Example 2). The preferred batches contained a surfactant such as Cremophor EL, Polysorbate 80 and modified Lecithin in the range 2 - 5%.

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Vehicles trialed included Water, Glycerol Formal and modified vegetable oil. The combination of 5% DMA and 5% CREMAPHOR EL or POLYSORBATE 80 was demonstrated to be quite useful for the levamisole / ivermectin formulations according to the present invention.

Formulations comprising eprinomectin instead of ivermectin were evaluated using CREMAPHOR EL and several other surfactants. The stability data provided above indicated that eprinomectin performed at least as well as ivermectin. A series of batches were prepared comparing the addition of BHT as an antioxidant to batches without antioxidant. Stress studies showed a significant improvement in stability of the actives when BHT was added to the formulation. Finally, Methyl Paraben was selected for a preservative as the marketed product may be provided as a multi-dose pack.

Physicochemical Properties

Assessment of developmental formulation batches was generally performed on the basis of appearance, including color, of the solution and the assay content of both of the active ingredients. The description of appearance for the proposed formulation is that of a clear light yellow to yellow solution. The physicochemical properties investigated during the stress study monitored the assay of the two actives. Density and pH values were also monitored. Density was in the range 1.05 - 1.10 and pH in the range 3.0 - 5.0. Visual assessment shows the product to be flowable with a water-like appearance.

The bulk of the WFI was added to the main manufacturing vessel and methyl paraben, the water soluble preservative, was added first to maintain microbial resistance. Levamisole Phosphate was added next as it water soluble and at a relatively high concentration. In a separate vessel, eprinomectin and BHT were dissolved in DMA and the surfactant added next. This combination was then added to the bulk aqueous phase with mixing. Eprinomectin is not water soluble, so this carried the eprinomectin into and maintained it in solution. Finally the batch was 25 made to volume with WFI and mixed. All mixing was accomplished using simple mechanical agitation. Heat was only required to dissolve the Methyl Paraben, and once all soluble components were dissolved, the WFI was cooled before further processing occurs.

Example 2

Preliminary work focused on stability of ivermectin / levamisole-containing formulations, 30 though subsequent studies (summarized in Example 1) shifted to the related macrocyclic lactone eprinomectin. As mentioned, eprinomectin offers a significant advantage in terms of short

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2-13.

Table 9. Formulation summary: IVM is ivermectin and LEV is levamisole phosphate

Batch No	Batch size	Formulation	Source and batch number of APIs	Purpose, method of manufacture and comments
			% w/v	IVM	LEV
02	100m	Ivermectin	0.70	Hisun	Guilin	Purpose: Preliminarv
	L	Levamisole PO4	22.3	080340	020903	screening trial to determine a
		Glycerol Formal	20.0			suitable base for Ivermectin
		Benzyl Alcohol	1.0			and Levamisole Phosphate
		Polysorbate 80	5.0
		WFI	to 100
03	100m	Ivermectin	0.70	Hisun	Guilin			55°C 5 days
	L	Levamisole PO4	22.3	080340	020903			Assay compared to
		DMA	2.0					initial
		Cremophor EL	2.0					IVM	LEV
		WFI	to 100				02	78.0%	94.4%
04	100m	Ivermectin	0.70	Hisun	Guilin		03	101.2%	94.1%
	L	Levamisole PO4	22.3	080340	020903		04	96.5%	86.3%
		Propylene Glycol	20.0				06	99.6%	99.7%
		Benzyl Alcohol	1.5				07	98.0%	94.5%
		WFI	to 100				08	125.3%	96.1%
06	100m	Ivermectin	0.70	Hisun	Guilin
	L	Levamisole PO₄	22.3	080340	090601	Result: Batch 03 (07) batch 06
		Miglyol 840	20.0			most suitable formulations.
		Phospholipon	1.8			Batch 03, a solution preferred
		Glycerol Formal	2.2			over batch 06 an emulsion.
		WFI	to 100

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07 (Repe at 03)	100m L	Ivermectin Levamisole PO4 DMA Cremophor EL WFI	0.70 22.3 2.0 2.0 to 100	Hisun 070403	Guilin 090601
08	100m L	Ivermectin Levamisole PO4 WFI Montanide	0.70 22.3 30.0 to 100	Hisun 070403	Guilin 090601
09	100m L	Ivermectin Levamisole PO₄DMA Cremophor EL WFI	0.70 22.3 2.0 2.0 to 100	Hisun 080340	Guilin 090601	Purpose: Repeat batch 03 using two different batches of Ivermectin
	55°C 5 days
Percent initial
IVM	LEV
10	100m L	Ivermectin Levamisole PO4 DMA Cremophor EL WFI	0.70 22.3 2.0 2.0 to 100	Hisun 070403	Guilin 090601	09	83.6%	97.9%
10	58.4%	97.6%
Result: Crystallization observed.
11	100m L	Ivermectin Levamisole PO4 DMA Cremophor EL WFI	0.70 22.3 5.0 5.0 to 100	Hisun 080340	Guilin 090601	Purpose: Repeat batch 03 but increase surfactant and solvent levels. Use two different batches of Ivermectin.

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12	100m L	Ivermectin Levamisole PO4 DMA Cremophor EL WFI	0.70 22.3 5.0 5.0 to 100	Hisun 070403	Guilin 090601		55°C 5 days Percent initial
IVM	LEV
11	85.6%	97.2%
12	93.1%	98.3%
Result: Stability improved.
13	300m L	Ivermectin Levamisole PO4 DMA Cremophor EL WFI	0.70 22.3 5.0 5.0 to 100	Hisun 070403	Guilin 090601	Purpose: Repeat batch 12 with stress testing at 40°C and 55°C.
	5 days
Percent initial
IVM	LEV
40° C	98.3%	99.6%
55° C	85.2%	97.3%
Result: Stability improved.

Example 3 - Alternative Formulations:

The following example illustrates the significant time and effort that was invested in developing the stable, injectable formulations according to the present invention. A person of 5 ordinary skill will immediately appreciate the rigorous experimentation that was, and is very often required in order to arrive at the proper blend of actives and excipients to produce an effective, pharmaceutically acceptable formulation. Injectable formulations tend to be particularly challenging to develop, as factors such as excessive viscosity and injection site irritation can preclude the use of otherwise effective excipients (e.g. excipients which provide 10 good stability for the combination of active pharmaceutical ingredients).

Pre-formulation development efforts commenced using the hydrochloride salt of Levamisole. Preliminary solubility data strongly contra-indicated the use of NMP and Glycerin

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Early indications were that CAPMUL MCM (medium chain mono and diglycerides) aqueous based formulations might provide a clinically acceptable base for injection. Batches INJ0003-01 to INJ0003-11 were therefore formulated with Levamisole using either the hydrochloride or the phosphate salts thereof (formulation compositions are summarized in Table 10). CAPMUL MCM and TWEEN 80 with a citrate buffer was the basis of this aqueous formulation. Although considered stable at the planned storage of 2 - 8°C, unacceptable site reactions occurred during clinical investigation.

Table 10: Formulation summary - batches INJ003-01 through INJ003-11

Batch No	Batch size	Formulation	Purpose, method of manufacture and comments
	% w/v
INJ003	400mL	Ivermectin	0.48	Puroose: Preliminary screening trial to determine a
-01		Levamisole HC1	20.0	suitable base for Ivermectin and Levamisole HC1
		Capmul MCM	25.0
		Tween 80	15.0
		Tri Sodium Citrate	1.5			25°C
		Cone. HC1	0.25 mL			Assay compared to initial
		WFI	to 100			Ivermectin	Levamisole HC1
					1 M	99%	98%
					2M	98%	99%
					4M	97%	97%
					6M	95%	98%
					9M	93%	97%
					12M	92%	97% 40°C
						Assay compared to initial
						Ivermectin	Levamisole HC1
					1 M	95%	95%

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INJ003 -02	400mL	Ivermectin Levamisole HC1 Capmul MCM Tween 80 Tri Sodium Citrate Cone. HC1 WFI	0.48 20.0 10.0 10.0 2.0 0.25 mL to 100
INJ003	lOOOmL	Ivermectin	0.48

2M	91%	96%
4M	86%	92%
6M	78%	90%

	55°C
Assay compared to initial
Ivermectin	Levamisole HC1
2 wk	84%	95%
4 wk	70%	93%

Purpose: Preliminary screening trial to determine a suitable base for Ivermectin and Levamisole HC1

	25°C
Assay compared to initial
Ivermectin	Levamisole HC1
1 M	97%	97%
2M	95%	96%
4M	93%	97%
6M	91%	95%
9M	90%	96%
12M	87%	96%

	40°C
Assay compared to initial
Ivermectin	Levamisole HC1
1 M	90%	94%
2M	83%	91%
4M	79%	91%
6M	75%	88%

	55°C
Assay compared to initial
Ivermectin	Levamisole HC1
2 wk	74%	89%
4 wk	69%	83%

Purpose: Batch submit for site reaction study

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-03		Levamisole HC1 Capmul MCM	16.11 25.0
		Tween 80	15.0			25°C
		Tri Sodium Citrate	1.5			Assay compared to initial
		Cone. HC1	0.25 mL			Ivermectin	Levamisole HC1
		WFI	to 100		1 M	98%	99%
					2M	94%	96%
					4M	94%	99%
INJ003	500 mL	Ivermectin	0.48	Puroose: Same formula as batch INJ003-01
-04		Levamisole HC1	20.0
		Capmul MCM	25.0
		Tween 80	15.0			25°C
		Tri Sodium Citrate	1.5			Assay compared to initial
		Cone. HC1	0.25 mL			Ivermectin	Levamisole HC1
		WFI	to 100		1 M	97%	100%
					2M	97%	99%
					4M	96%	98%
INJ003	500 mL	Ivermectin	0.48	Puroose: Same formula as batch INJ003-01
-05		Levamisole HC1	20.0
		Capmul MCM	25.0
		Tween 80	15.0			25°C
		Tri Sodium Citrate	1.5			Assay compared to initial
		Cone. HC1	0.25 mL			Ivermectin	Levamisole HC1
		WFI	to 100		1 M	89%	79%
					2M	92%	81%
					4M	87%	77%
INJ003	500 mL	Ivermectin	0.48	Puroose: Same formula as batch INJ003-01	- with
-06		Levamisole HC1	20.0	parabens
		Capmul MCM	25.0
		Tween 80	15.0
		Tri Sodium Citrate	1.5			25°C
		Methylparaben	0.18			Assay compared to initial
		Propylparaben	0.20			Ivermectin	Levamisole HC1
		Cone. HC1	0.25 mL		1 M	99%	99%
		WFI	to 100

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				2M	100%	98%
4M	96%	99%

INJ003 -07	1500 mL	Ivermectin Levamisole PO₄ Capmul MCM Tween 80 BHT Methylparaben Propylparaben Tri Sodium Citrate Propyl gallate Cone. HC1 WFI	0.57 20.0 25.0 15.0 0.02 0.18 0.20 1.5 0.02 0.25 mL to 100	Puroose: Batch with Levamisole PC)₄ & BHT Results: Sample kept at 55°C were separated into two layers in three weeks
INJ003 -08	1500 mL	Ivermectin Levamisole PO₄Capmul MCM Tween 80 Methylparaben Propylparaben Tri Sodium Citrate Propyl gallate Cone. HC1 WFI	0.57 20.0 25.0 15.0 0.18 0.20 1.5 0.02 0.25 mL to 100	Puroose: Batch with Levamisole PCL & nil BHT Results: Sample kept at 55°C were separated into two layers in three weeks
INJ003 -09	1500 mL	Ivermectin Levamisole PO₄ Capmul MCM Tween 80 BHT Methylparaben Propylparaben Tri Sodium Citrate Propyl gallate Cone. HC1	0.57 20.0 25.0 15.0 0.03 0.18 0.20 1.5 0.02 0.25 mL	Puroose: Batch with Levamisole PCL & BHT with pH 4.5
	2-8°C
Assay compared to initial
Ivermectin	Levamisole PO4
1 M	103	104
2M	105	100
3 M	100	99

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		WFI	to 100
INJ003	1500 mL	Ivermectin	0.57
-10		Levamisole PO4	20.0
		Capmul MCM	25.0
		Tween 80	15.0
		BHT	0.03
		Methylparaben	0.18
		Propylparaben	0.20
		Tri Sodium Citrate	1.5
		Propyl gallate	0.02
		Cone. HC1	0.25 mL
		WFI	to 100

6M	103	101
9M	101	99

	25°C
Assay compared to initial
Ivermectin	Levamisole PO4
1 M	103	103
2M	103	99
3 M	100	95
6M	101	94
9M	93	90

Purpose: Batch with Levamisole PO4 & BHT with pH 4.0

	2-8°C
Assay compared to initial
Ivermectin	Levamisole PO4
1 M	103	103
2M	103	99
3 M	96	102
6M	104	102
9M	100	98

	25°C
Assay compared to initial
Ivermectin	Levamisole PO4
1 M	103	102
2M	103	99
3 M	99	96
6M	99	94
9M	92	89

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INJ003

1500 mL

Ivermectin	0.57
Levamisole PO₄	20.0
Capmul MCM	25.0
Tween 80	15.0
BHT	0.03
Methylparaben	0.18
Propylparaben	0.20
Tri Sodium Citrate	1.5
Propyl gallate	0.02
Cone. HC1	0.25 mL
WFI	to 100

Purpose: Batch with Levamisole PO₄ & BHT with pH 5.0

	2-8°C
Assay compared to initial
Ivermectin	Levamisole PO₄
1 M	101	104
2M	102	102
3 M	101	10
6M	102	100
9M	98	99

	25°C
Assay compared to initial
Ivermectin	Levamisole PO₄
1 M	102	102
2M	102	101
3 M	95	96
6M	98	94
9M	97	86

INJ0003/11 Method of Preparation

1. Ivermectin was dissolved in CAPMUL MCM under stirring.

2. To this Tween 80 was added under stirring.

3. BHT was added to step 1 under stirring.

4. Methyl paraben and Propyl paraben dissolved in 600ml of water under heating.

5. Levamisole phosphate was added to step 3 under stirring at room temperature.

6. Propyl gallate was added to step 4 under stirring.

7. Trisodium citrate and 50% HC1 solution were added to step 5 under stirring.

8. Step 2 was added to step 6 under stirring

9. Adjusted the pH to around 5.00 using 20% HC1 solution, if necessary.

10. The resulting solution was made up to volume with water.

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Table 11: Site reaction test for ivermectin / levamisole injection for cattle - IN J 0003/11

Species:	Bovine
Number:	2 animals
Type:	mixed
Age:	~6 months
Sex:	either
Weight:	Greater than 125kg
Source:	Commercial farm
Health status:	Normal animals in good body condition.
Special requirements:	No anthelmintic treatment in the past 4 weeks
Medication history:	Herd-based treatments as part of normal animal husbandry practice.
Housing and management:	The study animals were maintained as a single mob and rotationally grazed on pasture with continuous access to water throughout the experimental period.

Results'. Two calves were treated. No sting in either animal was noted upon injection. A site reaction occurred in both animals within 1 hour of injection, lasting for the entire 28 days of the trial. The reaction was a slightly raised area at the point of injection getting as large as 150 x 170mm and 110 x 140mm. Such reactions would be unacceptable in a commercial farming situation.

Example 4 - Formulations including other macrocyclic lactones

Tests were conducted to determine whether the invention would be able to be used as a vehicle for delivering a wider range of macrocyclic lactone active ingredients. The test formulations were prepared using POLYSORBATE 80 as the surfactant of choice according to Table 12.

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Table 12. Formulation ingredients

Ingredient	% w/v Composition	Function
Macrocyclic lactone active	0.70*	Active
Levamisole Phosphate	22.3*	Active
BHT	0.1	Antioxidant
Dimethylacetamide	5.0	Solvent
Methyl Paraben	0.15	Preservative
Polysorbate 80	5.0	Surfactant
Water For Injection	q.s. to 100%	Vehicle

* Corrected for potency

The results demonstrated that abamectin, moxidectin and doramectin are suitable macrocyclic lactones for formulations according to the instant invention. When compared to the eprinomectin/levamisole formulation, the general physical parameters are very similar.

Table 13a: Formulations of various macrocyclic lactones levamisole

	Eprinomectin/Levamisole Inj.	Abamectin/Levamisole Inj.
B.No.	SOW 21-08	SOW 21-23
Condition	2-8°C	25°C	40°C	2-8°C	25°C	40°C
Storage Period	5 Days	5 Days	5 Days	5 Days	5 Days	5 Days
Description	*	*	*	*	*	*
Wt/mL	1.0843	1.0843	1.0843	1.0831	1.0831	1.0831
Viscosity	3.5 cP	3.5 cP	3.5 cP	3.3 cP	3.3 cP	3.3 cP
pH	4.22	4.21	4.06	4.17	4.16	4.05

Table 13b: Formulations of various macrocyclic lactones levamisole

Moxidectin/Levamisole

Doramectin/Levamisole

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	Inj.	Inj.
B.No.	SOW 21-24	SOW 21-25
Condition	2-8°C	25°C	40°C	2-8°C	25°C	40°C
Storage	5	5	5	5	5	5
Period	Days	Days	Days	Days	Days	Days
Description	*	*	*	*	*	*
Wt/mL	1.0825	1.0825	1.0825	1.0837	1.0837	1.0837
Viscosity	4.2 cP	4.2 cP	4.2 cP	3.3 cP	3.3 cP	3.3 cP
pH	4.15	4.14	4.02	4.13	4.12	4.01

* Clear very light yellow solution

Example 5 - Other Formulations

Formulations including novel avermectins would be prepared in accordance with Table 12, and could include the compounds and amounts recited in Table 14. The formulations could also include any one of the avermectins or avermectin monosaccharides, modified in the 4' or 4 position, described in US 7,704,961, US 7,521,429, US 2006-0105970 Al, US 7,678,740, US 7,632,820, US 7,678,773, US 7,605,134, and US 6,933,260 (each to Merial).

Table 14. Amounts and identities of novel avermectin compounds

Avermectin	% w/v Composition
4-diallylaminosulfonyloxy-avermectin Bi	0.5-0.9
4-(2-Hydroxyethylaminosulfonyloxy)-avermectin Bi	0.5-0.9
4-dipropargylaminosulfonyloxy-avermectin Bi	0.5-0.9
4-bis(cyanomethyl)aminosulfonyloxy-avermectin B i	0.5-0.9
4-Dicyanomethylaminosulfonyloxy-avermectin Bi	0.5-0.9
4-pyrrolidinosulfonyloxy-avermectin B i	0.5-0.9
4-benzoylaminosulfonyloxy-avermectin B i	0.5-0.9
4-epi-sulfamoyloxy-avermectin Bi	0.5-0.9

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4-methylaminosulfonyloxy-avermectin B i	0.5-0.9
4-ethylaminosulfonyloxy-avermectin B i	0.5-0.9
4-sulfamoyloxy-avermectin Bi	0.5-0.9
4-allylaminosulfonyloxy-avermectin B i	0.5-0.9
Any dimeric avermectin Any hetero-dimeric avermectin/milbemycin	0.5-0.9
Any 4” substituted avermectin	0.5-0.9
Any 4’-4” dually substituted avermectin	0.5-0.9
Any avermectin monosaccharide	0.5-0.9

Example 6 - Determination of the chemical residues and depletion profiles for eprinomectin and levamisole following subcutaneous injection of cattle.

These studies were conducted to determine the residue levels and depletion profile of eprinomectin and levamisole in the tissue of cattle, following the subcutaneous injection of an injectable formulation according to the instant invention, containing 7 g/L eprinomectin and 223 g/L levamisole phosphate. Thirty Short Hom cross cattle were sourced from a commercial farm. On the day of treatment (Day 0) all cattle were weighed, and then, based on live weight, 24 animals were allocated to treatment and tissue collection groups. The treatment groups and sampling times are presented in Table 15.

Table 15. Treatment groups, active ingredients and dose rates

Treatment group	Active ingredient concentration	Dose rate	Number of cattle treated (tissue collection times)
1. Untreated	-	-	2 cattle (2 on Day 21)
2. Test Item	7 g/L eprinomectin + 223 g/L levamisole phosphate	1.25mL/35kg	20 cattle (5 on each of Days 21,28, 35 and 42)
3. Test Item contingency	7 g/L eprinomectin + 223 g/L levamisole phosphate	1.25mL/35kg	2 cattle

Residues. The untreated cattle were removed from the site of treatment before application of the test item. A single injection was made on Day 0 using pre-calibrated plastic graduated 15 disposable syringes and 18 G x 1” hypodermic needles. The calculated dose was administered

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Table 16. Study design

Parameter	Description
Number	24
Species	Bovine
Breed	Short Hom cross (Hereford x Friesian dams, Short Hom sires)
Sex	Females and castrated males
Age	Approximately 8 Months
Body weight Range	200-246 kg
Condition	Excellent condition, healthy

The field study was conducted according to the details outlined in Table 16. In this GLP tissue residue study, twenty (20) treated cattle and two (2) untreated control cattle were sacrificed for tissue collection following the subcutaneous injection of a developmental parasiticide. Two (2) further “contingency” animals were treated and managed with the treated mob. All animals were chosen from a mob of thirty (30) cattle. Upon arrival at the research facility, all cattle were already individually identified with an ear tag with a unique number in their left ear so it was unnecessary to remove that tag and/or replace it with another. On the day of treatment (Day 0) all cattle were weighed and ranked from highest to lowest based on live 20 weight. In order to keep the live weight range as narrow as possible, the heaviest four and lightest two animals from the 30 potential study animals were removed from the study. Labelled discs (20 x treated, 2 x contingency and 2 x untreated) were drawn randomly from a box and assigned to each animal as per ranking, in order of drawing. This meant that the cattle were randomly allocated to treatment groups (treated or untreated). Slaughter days were randomly 25 allocated to treated cattle in blocks of four. Average live weights for the treatment group by collection day are shown in Table 17.

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Table 17. Average live weight of each tissue collection day for treated and untreated cattle

Treatment Group	Collection day
	0	21	28	35	42
Untreated		223
Treated		231	228	227	227
Contingency	215

Cattle that were treated received a white uniquely numbered tag in their right ear while the untreated control cattle received a red uniquely numbered tag in their right ear. Contingency animals received a blue numbered tag in their right ear. Treatment details are presented in Table 18.

Table 18. Active ingredient, dose rates and cattle numbers in treatment groups in accordance with ACVAM guidelines for maximum allowable residues in animal meat, edible offal and fat tissue

Treatment group	Active ingredient concentration	Dose rate	Number of cattle
1. Untreated	-		-	2
2. Test Item	7 g/L eprinomectin and 223 g/L levamisole phosphate	1.25mL/35 kg		20
3. Contingency	7 g/L eprinomectin and	1.25mL/35 kg		2
	223 g/L levamisole phosphate

Table 19. Presents the maximum residue limits for eprinomectin (mg/kg), and levamisole (mg/kg) limit (PQL) for eprinomectin (mg/kg) and levamisole (mg/kg) in animal meat, edible offal and fat tissue

Eprinomectin (mg/kg)	Levamisole (mg/kg)
Fat	0.25	0.01
Liver	1.5	0.1
Kidney	0.3	0.01
Muscle	0.05	0.01

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Table 20. Limit of quantitation (LOQ), limit of detection (LOD) and practical quantitation limit (PQL) for eprinomectin (mg/kg) and levamisole (mg/kg) in animal meat, edible offal and fat tissue

Eprinomectin (mg/kg)	Levamisole (mg/kg)
LOD LOQ PQL	LOD LOQ PQL
Fat tissue	0.001	0.005	0.01	0.001	0.005	0.01
Edible offal & meat	0.001	0.005	0.01	0.002	0.006	0.01

Table 21. Eprinomectin Bia and levamisole concentrations in liver tissue

Tag			Ef5i'3ititHS€idu Bls (las/kg.;	L* vaaa ss &k (iag.^:	Sigi
	(Uike<>rF-?<:ted7	{Cerreffed) (Cas	ayrec ited)
1		L'TC	•-lgs	-' LOD	: LOB -	LOD
;	21	UTC	-••.LOD	-. LOD	··; LOD -	LOD
3	21		5.21	0,3.2	0.011	S.S1
4	21		3.15	0.16	- LGQ -·	LOQ
5	21		024	034	<- LOQ -	LOQ
s	21		¢.823	0.023	- LO-Q -	LOQ
— !	21		022:	S.SLs	< loq <	LOQ
8	2®		013,013	0.013. 0.514	.- LOQ. - LO-Q - LOQ, - LOQ
£	2S		0.04	0.04	: L GO -	LOQ
	2S	E tia.<Levaaaso	0.04$:	0.045	- LOQ -	LOQ
Π	28	^riaamssto^sstrnsele	< LOD	-- LOD	< LOD	LOD
12	2S		OOO	O.t-05	- LOD -	LOQ
1.5	35		<LOQ	< LOQ	< LOQ <	LOQ
14	35		O1S	0.519	··; LOD -	LOD
15	35	Esriri<2mee:hivLev3.aivio3e	0.012	0.-013	: LOG -	LOD
1ΐ	35		<LOD	< LvD	< LOS --	LOD
17	i :		0.005	0.505	< LOD	LOD
1$:	42:	E ^riaomec tm.^;’Levaaas ote	LOD. < LOD	< LSD.. < LOD	-'. LOQ, < LOQ < LOQ, - LOQ
	42:		<LOD	< LOD	< LOQ <	LOQ
20	42		'-.LOD	-. LOD	··; LOQ -	LOQ»
21	42	Esriri<2mee:hivLev3.aivio3e	--LOD	-' LOD	.- LOQ -	LOQ
3?	42		<LOD	- LOD	-. LO D -	LOD

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Table 22. Eprinomectin Bia and levamisole concentrations in kidney tissue

Ear Tag	Interval		ΖριικΐΜίϊΐίΙιιι Bia i atS 'K?/
(Cerrareta!)	(Uac^rrsiEfiii)	(C’<3:iT4£:i«sl)
1	.21	utc	·-. LSD	: LOD	c LSD	··. LCD
2	21	LiTC	cLOD, cLOD	< 10©, -c LOD	-c LOD, < LOD	«. LOD, < LOD
3	21		8.844	8.845	c LSD	c LOD
4	.21		S.82:	8.82	< LSD	< LOD
5	21		C.9M	S.C9S	•c LSD	c LCD
e	21	Εΐ<11ίΙΙ<>Ώ“θ&ΐϊ·Εί:Ν·<Η22>·.:ί?'·“	c LOG	-: LC>Q	c LSD	cLOD
'?	21	E sis	cLOQ. cLOQ	LOQ. LOQ	-c LOD, < LOD	«. LOD, < LOD
s	2S		c LOQ	< 1GQ	•c LOD	c LOD
9	.2$		085	8.886	< LOD	·· LCD
16	2S	E prstts me^iiiVL-e·νjmiii si-e	8.88*	8.887	c LOD	c LCD
11	2S		cLOD	cLOD	c LSD	cLOD
'12	.28	EJ<sfiil£>SSSJ5>l· fctt- L^VaSSl-'i fcifi	c LOD	LCD	< LOD	< LOD
12	35	E’pT.nKtnse^trii.E^T'^amisc^e	cLOD	LCD	•c LSD	c LOD
14	55	E JkiTK»5£SS.?^'?j33.'T.^V3£BJ3 S&f	c LOG	• LOQ	- LSD	•LSD
15	35	E prsjsssjiee vasiiis si-e	c LOQ	<LOQ	c LOD	c LOD
	35	EjKiTil£>Si££ frnv? Axra»yisx*jjj·	cLOD	LOD	•c LOD	LOD
17	35		< LOD. < LOD	< LSD.c LOD	<--L0D. cLQD	LOD. - LOD
IS	42	EvMitKtKiisiiu: /Eey^tiiiss©' i-e	- LSD	cLOD	c LOD	•. LOD
19	42		LSD	LSD	c LSD	cLOD
23	42	E »itq-322 ec&ui/Lev smis-si-e	cLOD	LCD	c LOD	< LOD
21	42	EpTittctaae^tm^va^siiie	c LSD	LCD	•c LSD	.'LOD
?·?	42		< LOD	1GD	< LCD	< LOD

Table 23. Eprinomectin Bia and levamisole concentrations in injection site tissue

Ear Ta.?	laterral	Fr-sdisst	EpPTEriSifietnE: B-iY (iH.g/kg)	Levaiiiiw.te {hs.^-kg?
	n.-ybi*>rre<'tSi^	(C&rueetsti)
1	21	L'TC	c LOD	. LOD	• LCD	LCD
7	21	vTC	c LOD	LOD	LOD	LCD
:3-	21	Ε^<Γ^Ϊ^Π5^ίίίί1·Ζ SVaisilviiS	3 12	3.12	••LOQ	•cLGQ
4	21	.ΕρΏΏ·££Β5££ Τΐττ/Τ.^.-^Γηί-·;··;^	8.815	8.815	•.LCD	••LOD
5	21	E^iu'Ctmeeiin.^evs^isaSe	: LOD	LCD	LCD	•LCD
6	21		<LSQ	• LOQ	LCD	LCD
7	21		c LOD	LOD	LOD	c LOD
8	28	Eprm-omeetLn ^;Ee ani s-s^e	•c LSD, c LOQ	c LOD, c LOQ	cLQB, < LSD	cLOD, LCD
9	2®	Eyirtt^-iNwsia^riFi.N^x'^-yrii^^sis	ii.U	8.11	cLOD	cLOD
59	2S	E:$ΏΏ·££Β5££ ΤΐττΝ.^-.-^τηί-·;··;^	< LOO	cLOQ;	•.LOD	cLOD
11	28	E»riii<?nieehLL^:'Ley3;mii;aifi	·- LSD	LOD	LCD'	•LCD
12	28	Es-rmom^e tuxE evaniisste	cLOQ	LOQ	<LOD	LCD
13	35	E^is;i<meiteLe^?^3ais^e·	<:LOD	LOD	LOD	LCD
14	35		3.035	0.W	cLOD	cLOD
15	35	EyiFtntNWi^'ti-K-fTja.va.yrii·?;!^^	c LOQ	-ICQ	cLOD	•cLOD
1«	35		< LOQ	' LOQ;	•••.LOD	cLOD
17	35^:	E^i*£a<c&£ie£tiE^L.ev3mts-^a	c LSD	eLSD	• LCD	‘.LOD
1®	42	E^im£<£S.€i ώ1Έ$·	c LOD, - ·. l<sd	c. LOD, c LOD	•c LOD . c LCD	cLOD. cLOD
19	42'	E^sEi^jse^feL^^aaiscile	cLOD	c LOD	LOD	c LOD
23	42	^,^'ΏΓ'ι.ΌίίΙ^ίνίίί.'^.^’ν^ΐΟϊϋΰ'ϊδ	cLOD	• LCD	c LOD	cLOD
2:·	42:		< LOD	-.LCD	-.LCD	. LOD
7;>	42	^;Ε«:τ?;ΐηι^ΰ·^	-. LOD	• LOD	cLOB	c. LOD

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Table 24. Eprinomectin Bia and levamisole concentrations in peri-renal tissue of treated and untreated cattle

Est Tag	Iski-erval		Bia {111.2:¾}
	(Ι'ϋΐ'οΐ’Γίϊίΐί'ίΙ) (£s	ίΤί£ ti (Ua<· -srss!? tsd i
J	V		< L©D	LOD	LOD . LOD
7·	2.1	VTC	< LOD	« LCD	LOD --.LOB
5	a ’		:< LOQ	« LOQ <	LOD « LOD
i	•X V		< LOD	« LOD	LOD -LOD
S'	21		< LUS	« LOD '	LOD -«LOD
ί·	21		< LOS	-<· LQD ··	LOD «LOD
	21		«LOD	« LOD <	LOD . LOD
s.	TS*		LOE < LOD	«LOD.. «LOD «LOD, «LOD «LOD, «LOD
y	3S		< LOD	« LOD -	LOD LOD
?· n	2S		« LOD	« LCD <	LOD ··-. LOD
il	2S	Epi’ssismeciiajLs'	< LOD	< LOD -	LOD «LOD
	2S		< LOD	« LOD -	LSD LOD
13	35		<. US	« LOD -	LOD «LOD
54	35	Fxs·: t py3-»nxft?p	< LOD	« LOD	LOD «LCD
55	35	Ejvriaoal· sc: o te	«LOD	« LOD <	LOD < LOD
L5	35		« LUS	« LOD ··	LOD «LOD
5?	3-5		. LOD	«. LOD -	LOD «. L vD
IS	43		··.·. LQS_;. ·< LUD	··.· LOD, « LOD « LOD, « LOD « LOB, « LOB
14	42		« LOE	« LOD	LOE «LOD
28	42	tiiS.· X.S:V3iDilSOt£	«LOD	« LOD <	LOD < LOD
3i	43		« LOD	« LOD	LOD «LOD
7'?	42		«' LOD	< LCD ·	LOD «. LOD

Example 7 - Efficacy of a combination eprinomectin and levamisole injection against sucking lice (Linognathus vituli) in cattle.

Four species of louse have been recorded on cattle in New Zealand. Of these, the biting louse Bovicola bovis and one of the three species of sucking louse, Linognathis vituli are common and dual infections with L. vituli and B. bovis occur. The remaining two species of 10 sucking louse, Haematopinus eurysternus and Solenopotes capillatus are uncommon (Chalmers and Charleston, 1980). The 56 day post-treatment assessment period covered at least two complete lifecycles of the louse.

This study was a randomized efficacy study in naturally infected animals with a negative control group. Prior to the start of the study, a mob of suitably infected animals were identified 15 on a commercial grazing property located in the Waikato region of New Zealand. On Day -13 sixteen animals from the larger mob were purchased and relocated to the study site and identified using uniquely numbered ear tags. Samples of biting and sucking lice were collected from seven study animals and identified by the Study Investigator. On Day 0 all sixteen study animals were

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2017279645 20 Dec 2017 lice counted and allocated to two groups (n=8) based on individual sucking lice counts. All study animals were weighed and treatments were administered subcutaneously. Group 1 was treated with the instant eprinomectin (7g/L) / levamisole phosphate (223g/L) formulation at lmL/35kg, and Group 2 remained untreated as the negative control group for the study. Individual lice counts were performed on each study animal on Days 7, 13 and 48. Group 1 was counted prior to Group 2, to ensure no transmission of lice between untreated animals and animals in the treated group. Injection sites were inspected on Days 7, 13 and 48 post-treatment for lesions or abnormalities.

Table 25. Treatment regime

Group	No. Animals	— Treatment	Dose	Route
1.	io	Eprinomectin/Ievamisole injection	I mU35kg	Subcutaneous
2	10	Dectamax® injectable cndectocide	lmL/50kg	Subcutaneous
3	10	Negative control	NA	NA

Statistical methods. The efficacy of the Investigational Veterinary Product (IVP, i.e. the eprinomectin / levamisole injectable formulation recited above) against both sucking and biting lice at each time point was calculated as the percent reduction in arithmetic and geometric mean lice count on the treated group compared to the untreated control group using Equation 1 and Equation 2, as recommended in WAAVP guidelines.

Equation 1: Abbott's formula n in T after treatment

Efficacy % - (1 -------------------------------) * 100 n in Co after treatment

Equation 2: Henderson-Tilton’s formula n in Co before treatment * n in T after treatment

Efficacy % -(1--_:;------------------—----------------) * 100 n in Co after treatment * n in T before treatment

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Where Efficacy % = efficacy, n = mean number of lice of the relevant species, T = Treatment group and Co = Control group. All statistical analyses were performed using Minitab® for Windows® Release 16.1.0 (Minitab, Inc.). Where data were considered to be normally distributed, as determined by Levene's test of equal variance, means were compared using a Two Sample T -Test. Data that were not normally distributed were compared using an equivalent nonparametric method. Results were considered significant at P<0.05.

Tables 26 and 27 show arithmetic and geometric mean sucking and biting lice counts recorded for Groups 1 and 2 throughout the study. Both biting and sucking lice were present on all animals prior to treatment and at the termination of the study on Day 48. Both tables incorporate the results of the statistical evaluation.

Table 26. Arithmetic and geometric mean suckling lice counts

Group

Arithmetic mean sucking lice__

Day 13 13.6^a

32.1^a

Geometric mean sucking lice

Day 13......

5.8 ^s__

31.0

Day 0 j Day 7 36.3 ^a j 5.1^s

26.8^a i 32.3^b

Day 48

12.3^s

19.3 ^a

Group

Day 0

27.6^

Day 48

10.8 ^s

16.2 ^s

[............Day 7 [___ _3.8^a j 30.6 ^b

Γ 1 [ ^ab means within the same column with the same superscript are not significantly different at p<0.05

24.6

Table 27. Arithmetic and geometric mean biting lice counts

Arithmetic mean biting Lice

Group	Day 0	ΐ Day 7	Day 13 j	Day 48 i
1	67.3^s	Ϊ 12.4^a	21.8^a	‘	17.l^a
2	64.9^s	Ϊ 46.6^b	43.9 ^b		26.0^b	5
		Geometric mean biting Lice			5
Group	DayO	i Day 7	Day 13	........ .........	Day 48
1	60.5^a	10.5^s	20.9^a		15.7^a	§
1	58.2 ^s	45.9¹	41.2^b		25.0 ^b

^ab means within the same column with the same superscript are not significantly different at p<0.05

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The efficacy of the IVP against sucking and biting lice are show in Tables 28 and 29. Each table presents calculations based on Equation 1 and Equation 2. The efficacy of the IVP against sucking lice peaked at 84% and 88%, based on arithmetic mean counts on Day 7 using Equation 1 and Equation 2, respectively. Efficacy had declined over the subsequent two counts to 36% and 53% by Day 48, based on Equation 1 and Equation 2, respectively. The efficacy of the IVP against sucking lice based on geometric mean counts peaked at 88% and 89% on Day 7, but declined to 33% and 41 % by Day 48, using the same two equations.

Table 28. Efficacy against sucking lice based on arithmetic and geometric mean lice counts calculated using Equation 1 and Equation 2.

Arithmetic Efficacy Abbott's Formula sucking lice

Group	\| Day 7 _:	Day 13	Day 48
2	\| 0.84	0.58		0.36
	Arithmetic Efficacy Henderson-Tilton's Formula sucking lice
Group	Day 7	Day 13	Day 48
2	1 0,88	0.69	0.53

Group

Geometric Efficacy Abbott's Formula sucking Hee t

+

Group

.............pay 7 ...... Ρ^νΜ i

I 0-88 [____________0.81..... I _

Georn etric Efficacy Henderson-Tilton'sFormute sucking lice j....... Day 7

0.89

Day 13

0.83 pay 48

0.33

Day48

0.41

Efficacy based on arithmetic mean lice counts against biting lice peaked at a lower level with the IVP achieving a maximum reduction of 73% and 74% in lice numbers, using Equation 1 and Equation 2, respectively and declined to 34% (Equation 1) and 36% (Equation 2) by Day 48.

When efficacy was calculated using the geometric mean lice counts, maximum reductions of77% and 78% were achieved on Day 7 and had declined to 37% and 40%, based on Equation 1 and Equation 2.

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Table 29. Efficacy against biting lice based on arithmetic and geometric mean lice counts calculated using Equation 1 and 2

Arithmetic Efficacy Abbott's Formula biting lice 1
Group	Day 7 \|	Day 23 Ϊ	Day 48 I
2	0.73 ΐ	0.50 i	0.34 ί
Arithmetic Efficacy Henderson-Tilton’s Formula biting lice	_______________i
Group	Day? \|	Day 13	Day 48 \|
2	0.74 \|	0.52 ΐ	0.36 \|
	Geometric Efficacy Abbott's Formula biting lice
Group	Day 7 I	Day 13 \|	Day 48 \|
2	0.77 J	0.49 I	0.37 J

Geometric Efficacy Henderson-Tilton’s Formula biting lice

Group	Day 7 ΐ	Day 13 \|	Day 48
2	0.78 i	0.51		0.40

As expected, the IVP was more effective against sucking lice than biting lice, given that its route of administration was by subcutaneous injection. The highest level of efficacy against sucking lice (89%) was recorded on Day 7 compared to a 78% reduction in biting lice numbers at the same time point. At 13 days post treatment, the highest level of efficacy against sucking lice was 83% compared to 52% against biting lice.

Example 8 - Safety of eprinomectin/levamisole formulations

Thirty male Hereford x Friesian cattle, each weighing between 105-123kgs, were treated according to preceding examples. The animals were healthy and had not been treated with a macrocyclic lactone or levamisole within 3 months prior to Study Day 0. One group received the lx concentration of eprinomectin and levamisole phosphate (see Table 18) administered by 15 subcutaneous injection, while the other group received 3x this concentration. Animals were monitored for pain and swelling, and the results indicated the eprinomectin / levamisole injection is safe to administer the recommended dose, or at least up to 3x the recommended dose (for example, in the event of an accidental overdose).

Example 9. Fecal egg count reduction efficacy of a subcutaneous combination eprinomectin and levamisole injection against a natural infection of gastrointestinal nematodes in cattle.

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This study was a randomized fecal egg count reduction field efficacy study in animals carrying a natural infection, and included positive and negative control groups for comparison. Seven days prior to commencement of the study, ten individual unidentified fecal samples were collected from the paddock and individual fecal egg counts were conducted to confirm infection. Four days prior to commencement of the study, individual fecal samples were collected per rectum from 60 animals otherwise meeting the inclusion criteria. Fecal egg counts (FECs) and a pooled larval culture were performed. Forty-five animals with positive FECs were selected for the study and randomly allocated to three groups each of 15 animals based on FEC. They remained at pasture in a single mob on the source farm for the duration of the study.

On Day 0, the animals were weighed and a fecal sample was collected from each animal per rectum for individual FECs and a pooled larval culture. Group 1 was treated with the IVP, Group 2 was treated with Control Product (CP) and Group 3 remained untreated as the negative control. Treated animals were observed at 30 minutes and 2 hours after treatment. On Days 14, 16 and 28 post-treatment, fecal samples were collected per rectum for individual FECs and pooled group larval cultures. No sample was collected from Animal #60 (Group 3) on Day 14. Injection sites of animals in Group 1 were examined on Days 14 and 28. Further samples were due to be collected on days 42 and 56 but the study was concluded after day 28 due to evidence of nematode resistance to macrocyclic lactone anthelmintics. After the conclusion of the study, the study animals were treated with an oral abamectin, oxfendazole and levamisole anthelmintic (MATRIX C HI-MIN; Merial Ancare).

Randomization Procedure. FECs were determined for 60 animals on Day -4. Fourteen animals were excluded for negative FECs and one animal with a FECs of 1050 eggs per gram was excluded. The remaining 45 animals with positive FECs (range 50-550 eggs per gram) were selected for the study. They were ranked in ascending order of FECs and blocked into blocks of 25 3 animals. A random number was allocated to each animal using Microsoft Excel random number generation. The animals were ranked in ascending order of random number within each block. The animal with the lowest random number in each block was allocated to Group 1, the next to Group 2 and the highest to Group 3. Each of the three groups comprised 15 animals and had a similar average FECs.

Blinding/masking procedure. Laboratory staff performing FECs and group coprocultures were advised which group each animal was in after Day 0, but were not aware of the treatment

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2017279645 20 Dec 2017 allocated to each group. Forty-five Friesian/Jersey cross cattle, aged 10 months, female, and weighing on average 158.6 kg (range 106.5-208.5 kg) on Day 0, were used for the study. All animals were FEC positive, which was determined by the presence in the animals of a mean egg per gram fecal (epg) of 206, with the range being 50-550 epg on Day -4. No anthelmintic had been administered to the cattle within 30 days prior to Study Day 0. Study animals were transferred from the home dairy farm to the study site (a grazing block) approximately 6 weeks prior to Day 0. On Day 0 they were harboring an existing gastrointestinal nematode infection derived from the study site farm and/or the home dairy farm. The IVP (Eprinomectin 7g/L and levamisole phosphate 223 g/L) was administered at 1 mL/35 kg via subcutaneous route. The Control Product (CP) was DECTOMAX® (Pfizer Animal Health), which consists of lOmg/mL doramectin was administered to the animals via subcutaneous route at a dose of lmL/50kg. Meat and milk withholding were each 35 days.

Method of administration/application. The IVP and CP were administered 1 time at Day 0 by subcutaneous injection in the anterior part of the neck. Individual doses were drawn up into a graduated lOmL syringe immediately prior to treatment and were checked by a second person before administration. Doses of IVP and CP were calculated on the basis of the heaviest bodyweight in the relevant group as determined on Day o. Calculated doses were rounded up to the nearest minor increment of the syringe. The IVP was administered at ImL / 35kg. The heaviest animal in Group 1 weighed 208.5 kg and the dose of IVP administered to each animal in Group 1 was 6.0 mL. The CP was administered at lmL/50kg. The heaviest animal in Group 2 weighed 199.0 kg and the dose of CP administered to each animal in Group 2 was 4.0 mL.

All animals were returned to the source herd at the conclusion of the study. All animals were treated with MATRIX C Hi-Min (Merial Ancare) after the conclusion of the study and relevant withholding periods applied. Efficacy of the IVP and CP was determined by fecal egg 25 count reduction for the treated groups compared to the negative control group at each time point.

Efficacy (%E) of the IVP and CP was calculated at each time point, using the formula:

%E= [C-T]/C x 100. Where: C = mean FEC of the control group; T = mean FEC of the treated group. Arithmetic and geometric mean FECs were then calculated. The significance of differences between group FECs was statistically evaluated. Where data were considered to be normally distributed as determined by Levene's test of equal variance, means were compared using One-way Analysis of Variance (ANOV A). Data that were not normally distributed were

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2017279645 20 Dec 2017 compared using an equivalent non-parametric method. Where differences were considered significant, (p<0.05), pair-wise comparisons were used to evaluate differences between groups.

Results. No injection site reactions were observed in Group 1 animals at any time point. FEC and larval culture results are shown in Tables 30-34. Table 30 shows arithmetic mean Day -4 FEC and Day 0 bodyweight. Means in the same column that share a letter (A) are not significantly different (p<0.05). Table 31 shows arithmetic and geometric mean FEC at Days 0 to 42. Based on arithmetic and geometric means, the FECs for Group 1 (treated with the IVP) were significantly less (p<0.05) than both the control group (Group 3) at every timepoint posttreatment and significantly less than Group 2 (treated with DECTOMAX) at Day 14 and 28. Based on arithmetic and geometric means, the FECs for Group 2 (treated with DECTOMAX) were not significantly different from the control group at any timepoint.

Table 30. Group details.

Group	n	Mean (range) FEC Day -4 (epg)	Mean (range) bodyweight Day 0 (kg)
1	15	20^Λ(50-500)	162 (112.5-208.5)
2	15	207^A (50-550)	156 (106.5-199)
3	15	207^A (50-550)	158 (128.5-201)

Table 31. Group FECs.

Group	T reatment	.......................................................ArithmeticmeaiilCIACCepg)............................................
DO	D 14	D 16	D 28
I	IVP	337'	37^Λ	23 ^A	17 λ
2	Dectomax	320 ^A	150’	80	1.83 ^s
3	Control	237 ^A	261^B	183^B	180³
		Geometric mean FEC (epg)
DO	D 14	D 16	D 28
1	IVP	219.3'	4,8³	4.0 ^A	2.0’
2	Dectomax	262.6 ^A	93.5 ^A	27.2 ^{A 8}	44.7'
3	Control	12'1.8^A	136.9-'	87.7’	45.5 ^A

Table 32. Percentage FEC reduction efficacy.

	Treatment	% Efficacy (arithmetic means)	% Efficacy⁷ (geometric means)
\| Oioup	D 14	D 16	D28	D14	D16	D28
1 1	IVP	855	87.3	90.7	96.0	95.4	95.6
i 2	Dectomax	42.5	56.4	-1.9	32.0	69.0	1.8

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Efficacy of the IVP and CP at each timepoint (based on FEC reduction compared to the untreated control group) is shown in Table 32. The percentage of each nematode genus identified in 100 larvae from group coprocultures at each timepoint is shown in Table 33.

Table 33. Larval culture results (%)

Day I....................	Group	........ Treatment	Ostertagia	Trichosirongylus	Cooper ia	Number of larvae spectated
-4	Pooled	NA	50	7	43	100
0	Pooled	NA	46	3	51	100
	1	IVP	100*	0	0	1
14	2	Dectomax	0	4	96	100
	3	NA 1 27	·?	71	100
	1	IVP	wo**	0	0	6
16	2	Dec to max	18	4	78	100
	3	NA ) 30		69	100
	1	IVP	46***	0	54***	35
28	2	Dec to max	1	5	94	100
	3	NA	35	1	64 \| 100

* 1 larva recovered ** 6 larvae recovered ***% of 35 larvae recovered

The larval differential in a coproculture is usually determined from the percentage of each genus identified in the first 100 larvae counted. Small numbers of larvae were obtained from coprocultures from group 1 at days 14, 16 and 28, despite positive FECs. This suggests that egg viability or larval development may have been suppressed in this group. Individual FEC results at each timepoint are shown in Table 34.

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STRONGYLE EEC

Table 34. Individual FEC results

11/	D-4	DO	D14	DI 6	»28
11	100'	100	150	50	0
	15	150	100	0	0	0
	20	100	150	0	0	o 1
21	200	400	100	0	0
22	300	250	0	0	0
35	400	1050	50	0	0
	36	50	200	0	0	100
	38	50	100	0	0	0 ;
	42	500·	400	50	50	0
	46	250	300	150	100	0
	47	150	loo	0	50	0
	57	350	1500	0	50	50
	70	250	100	0	50	50
	72	50	150	50	0	50
86	150	150	0	0	0
	3	200	550	100	200	150
	7	550	600	200	100	450
18	100	100	200	0	50
	23	300	600	250	0	300
	26	100	200	50	50	50
	27	50	100	200	0	0
.........	29	200	300	100	200	150
	30	150	200	0	0	0
	55	so	200	150	50	350
	56	350	600	200	50	550
	64	50	150	50	50	0
	81	300	250	250	100	50
	91	350	600	........ 50	100	400
	96	150	200	400	200	0
97	200	ISO	50	100	250
	13	50	50	50	0	0
	16	250	50	too	150	200
	19	100	100	100	150	200
	28	300	350	750	250	400
	33	550	1000	600	600	150
	40	200	300	250	250	150
	45	150	350	1 50	100	0
	49	50	0	100	50	100
51	200	400	250	200	150
	53	100	100	150	0	0
	60	450	200	No sample	300	650
	85	300	150	100	100	50
	89	150	50	400	200	100
	94	50	100	0	100	0
	102	200 s 350	650	300	550

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Conclusions. Based on arithmetic mean FECs, the levamisole/eprinomectin injection was over 85% effective and this effect was maintained up to 28 days after treatment. DECTOMAX (doramectin) was insufficiently effective. Based on geometric mean FECs, the levamisole/eprinomectin injection was over 95% effective and this effect was maintained up to 28 days after treatment. DECTOMAX was insufficiently effective. Efficacies were evaluated in animals carrying a mixed infection of Ostertagia, Trichostrongylus and Cooperia spp. There was strong evidence of anthelmintic resistance, and in particular, resistance by Cooperia spp. to doramectin.

Example 10 - Efficacy of a combination injectable anthelmintic containing cnrinomcctin and levamisole against common gastrointestinal nematodes in cattle.

Experimental design. This study was a randomized total worm count efficacy and dose confirmation study, with a negative control group. The study determined efficacy against a natural infection of common gastrointestinal nematodes in cattle. Prior to the study, a group of 21 animals meeting the inclusion criteria and suspected of harboring a natural infection of gastrointestinal nematodes were individually identified with uniquely numbered ear tags. The animals were fecal sampled and individual fecal egg counts (FECs) were performed. A bulk fecal sample was cultured and larval differentiation performed to identify the nematode species present. The fourteen study animals with the highest FEC results were selected for the study and introduced to the when study diet while in the paddock on day -5. On Day 0, the study animals were removed from pasture to the indoor facility. The animals were fecal sampled for individual FEC and randomly allocated into two groups of seven. One animal in each group was identified as a spare. Animals were weighed and the NP treatment was administered. Animals in Group 1 25 were treated with the I VP. Animals in Group 2 were left untreated. Treated animals were observed for adverse reactions at approximately 30 minutes and 1, 2 and 4 hours after treatment.

On Day 11, fecal samples were collected for individual fecal egg counts. On Day 13, animals were held off food overnight but continued to have ad libitum access to water. On Day 14, all animals (excluding spares) were humanely euthanized. The gastrointestinal tract of each 30 animal was collected and forwarded directly to the laboratory for processing and subsequent worm counts. The animal phase of the study concluded after the Day 14 procedures had been

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2017279645 20 Dec 2017 performed. Bodyweights were recorded immediately prior to treatment Fecal egg count and total worm count data was entered into an excel spreadsheet for statistical analysis. Individual fecal samples were collected from study animals by rectal grab prior to the study and on Day 0, and Day 11. Samples were delivered to the Laboratory on the day of collection or the day after collection. Individual fecal egg counts were performed on samples collected prior to the study and on Day ° and 11. Bulk larval culture and larval differentiation was performed on samples collected prior to the study. Gastrointestinal tracts (GITs) were collected immediately after euthanasia. The GIT was ligated at each end of the small intestine, large intestine and abomasum. The GIT was allowed to cool at ambient temperature for approximately 2 hours after collection. Each individual GIT was sealed (together with the animal's ear tag) in an individual bag identified by the animal's ear tag number. GITs were delivered to the Analytical Laboratory as soon as possible on the day of collection, accompanied by a sample submission form. Randomization / blinding was carried out as described.

All study animals had positive FECs prior to the study with a mean egg count of 121 epg. Larvae cultured from feces from all animals sampled prior to the study showed the following parasite species, 86% Cooperia spp., and 14% Ostertagia (Teladorsagia) spp. The mean body weight of the treatment group (excluding the reserve animal) on day zero of the study was 98.0kg. The mean body weight of the control group (excluding the reserve animal) on day zero of the study was 96.5kg. IVP was prepared and administered as in Example 9, and comprised eprinomectin (at 7 g/L) and levamisole phosphate (at 223 g/L). The injection site was cleaned and dried and was clipped then swabbed with 70% alcohol before treatment. A new sterile 20G 1.5 inch needle was used for each injection. Administration was performed once during the study, on Day 0, based upon bodyweight at 1 mL/35kg bodyweight. After the conclusion of the study, the spare animals were returned to the source herd. The treated spare was subjected to 25 observation of a 91 day withholding period.

Worm counts were performed according to the VICH Guideline on the efficacy of anthelmintics: general requirements (1998). Counts were made on 2% aliquots of abomasa and small intestine washings that had been sieved through a 38pm sieve. Counts were also made on 10% aliquots of large intestine washings that had been sieved through a 150pm sieve. Total 30 Worm Counts & Fecal Egg Counts: Calculation of IVP percentage efficacy (%E) was performed for each of the worm species (WS) present using the following formula: %E (Mean of (WS) in

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2017279645 20 Dec 2017 controls - mean of (WS) in treatment group) x 100 Mean of(WS) in controls. The same %E calculations were used to analyze the fecal egg count data. Statistics were calculated as described in the preceding Examples.

Results. Fecal egg counts: Mean fecal egg count (FEC) results are shown in Table 35.

Based on FEC results the IVP was> 99.9% effective at reducing FECs at eleven days post treatment.

Table 35. Arithmetic mean FECs and % reduction in FECs of treated animals relative to controls in calves treated with a combination eprinomectin and levamisole injection

1 Study Day	Control Grp FEC (epg)	Treatment Grp FEC (epg)	Efficacy \|
Day 0	1083	91.7	/
1 Day H	250 __________________________________________________________________________________________Ϊ	0*	>99.9% 1

* - Statistically sigeiihcatit dtfleteace fem Ae control group

Table 36. Arithmetic mean worm counts and % reduction in worm numbers of treated animals relative to controls in calves treated with a combination eprinomectin and levamisole injection.

Parasite	Control	Treatment	Efficacy
(ad tilt)............................................ Ostertagia (immature)	1867	0	>99.9% “““>99.9% ““
Trtchostronylus axei (adult)	367	0	>99.9%
I far month its contortus (adult)	42	0	>99.9%
JTaemofichtis contortus (immature)	17	0	>99.9%
Ggupffig (adult)............................................... Cooperia (immature)	18550	0 ..... ______	>99.9%
Trichostrongylus (adult)	33	0	>99.9%
Nematodirus (adult)	17	0	>99.9%
Oesophagostomum (adult)	12	0	>99.9%

Total worm counts. Arithmetic and Geometric mean total worm counts and percentage efficacy of the treatment group relative to the control group are presented in Table 36 and 37. Results demonstrate that the IVP was highly effective in the control of a mixed infection of gastrointestinal parasites in cattle. The IVP was >99.9% effective against the following parasite spp; Ostertagia (adult & immature), Trichostrongylus axei (adult), Haemonchus contortus (adult & immature), Cooperia (adult & immature), Trichostrongylus (adult), Nematodirus (adult),

Oesophagostomum (adult).

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Table 37. Geometric mean worm counts and % reduction in worm numbers of treated animals relative to controls in calves treated with a combination eprinomectin and levamisole injection.

Parasite	Control	Treatment	Efficacy
Ostertagia (adult)	1338		>99.9%
Ostertagia (immature)	8	0	>99.9%
Trlchostronylus axei (adult)	237	0*	>99.9%
Haemonchus contortus (adult)	8	0	>99.9%
Haemonchus contortus (immature)	3	0	>99.9%
Cooperia (adult)	14015		>99.9%
Cooperia (immature)	147	0*	>99.9%
Trichostrongylus (adult)	7	0	>99.9%
Nematodirus (adult)	3	0	>99.9%
Oesophagostomum (adult)	8	0*	>99.9%

* - Statistically significant difference Ssm the control group

Conclusions. This study was designed to determine the efficacy of an eprinomectin / levamisole injection against a mixed infection of common gastrointestinal parasites in cattle. Results demonstrate that the IVP was highly efficacious. Efficacies of >99.9% were recorded against the following parasite spp; Ostertagia (adult and immature), Trichostrongylus axei (adult), Haemonchus contortus (adult and immature), Cooperia (adult and immature), Trichostrongylus (adult), Nematodirus (adult), Oesophagostomum (adult).

Example 11. Efficacy of investigational eprinomectin / levamisole formulation versus commercial product

This study was performed using the methods described in the preceding Examples, except that an additional DECTOMAX treatment group was added.

Results. No injection site reactions were observed in Group 1 animals at either the Day 14 or the Day 28 observations. Table 38 below shows group arithmetic and geometric mean FEC at Days -8 to 50. Means in the same column that do not share a letter (A/B/C) are significantly 20 different (p<0.05). Based on both arithmetic and geometric means, the FECs for Group 1 (treated with the IVP) and Group 2 (treated with DECTOMAX) were significantly less (p<0.05) than the control group (Group 3) at each of the post-treatment timepoints up to and including Day 43. The FECs for Group 1 (treated with the IVP) were also significantly less (p<0.05) than the control group (Group 3) at day 50.

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Table 38. Group mean FECs (epg)

Arithmetic mean FEC

Group	me nt		DO	D 14	D28	D 43	D50 \|
1 1	IVP	146.7* I	96.4^A	6.7*	13.3*	13.3*	23.3* !

Dectomax

Control

43.3^AS i

156.7³ i 100.0^B......]

IVP

Dectomax

Control

140*!...... 833* T 4Ϊ3* | 46.7*'

133.3* f“ 121.4*.......ΐ.......166.7^R 'j 133.3»

Geometric mean EEC

1)0 i 1)14 ! 1)287

12.0*

87.8*

D-8

134.1*

129.1*

1.31.4*

D 14

D43D50

1.3^B s4.0 f—...........

>9.0*°

109-9*.......[52.4*

5³

Efficacy of the IVP and CP at each timepoint (based on FEC reduction compared to the untreated control group) is shown in Table 39.

Table 39. Percentage FEC reduction efficacy

Group	Treatment	% Efficacy (arithmetic means)	% Efficacy (geometric means) I
D 1.4		1)14	ί D2<8	D43	I) 50 ΐ
I D28	D 43 > D 50
1	IVP	96.0	ΐ 90.0	91.5 \| 76.7	99.0	1 99.1	98.8	92.3 I
2	Dec to max	74.0	65.0	70.2 \| 56.7	93.0 ί 86.3	93.2	82.8 i

The percentage of each nematode genus identified in 100 larvae from group coprocultures at each timepoint is shown in Table 40. Only 18 larvae were recovered and identified from the coproculture for Group 1 on Day 14. This suggests that egg viability or larval development may have been suppressed in this Group.

Table 40. Larval culture results (%).

Day	Group	Ί Treatment	Ostertagia	Ti'ichostrongylus	Cooperia	Haenwnchus
0	Pooled	NA	14		85	0
	1	IVP	100*	0	0	0
14-	7	Dectomax	0	0	100	0
	3	.NA	28	13	59 \| 0
	1	IVP	86	0	14	0
28	2	Dectomax	0	0	100	0
	3	NA	19	0	81	0
	1	IVP	41	0	53	0
43	2	Dectomax	6	2	92	0
	3	NA	19	0	81	0
i 1	IVP	14	2	84	0

Dectomax *' 8 larvae recovered.

NA

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Table 41 shows group body weights at Day 0 and Day 50. Means in the same column that do not share a letter (A/B) are significantly different (p<0.05). The mean live weight gain for Group 1 (treated with the I VP) over the 50 day period of the study was significantly greater than for the untreated control group (Group 3).

Table 41. Bodyweights

Group	Treatment	Mean (range) bodyweight Day 0 (k	Mean (range) g) s bodyweight Day 50 (kg'	I Mean (range) weight i gain (kg) ]
1	1VP	177.2(138-221)	I 233.5 (201-266)	56.3^Λ (43-80) \|
2	Dectomax	184.1 (.128-229)	\| 234,5 (153-285)	\| 50.4^(25-71) \|
3	Control	189.6 (163-234)	235.4 (218-291)	\| 45.8° (33-58) \|

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Table 42. Individual FEC results

NS-'-'-No sample

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Conclusions. Based on arithmetic mean FECs, the levamisole/eprinomectin injection had an efficacy of 90% or greater and this effect was maintained up to 43 days after treatment. DECTOMAX (doramectin) was insufficiently effective at all time points. Based on geometric mean FECs, the levamisole/eprinomectin injection was over 98% effective and this effect was maintained up to 43 days after treatment. DECTOMAX was over 86% effective during the same period. Efficacies were evaluated in animals carrying a mixed infection of Ostertagia, Trichostrongylus and Cooperia spp.

Example 12 - Efficacy of investigational eprinomectin / levamisole compared with doramectincontaining product

Methods. This study was performed in accordance with Example 11. The doses of I VP and CP were calculated according to individual body weights. Group 1 was treated with the IVP at the dose rate of ImL/ 35 kg live weight; Group 2 was treated with the Control Product at the recommended dose rate of lmL/50kg; and Group 3 was left untreated as the negative control.

Results. No injection site reactions were observed in Group 1 animals on either Day 17 or Day 28. Table 43 shows group arithmetic mean FEC at Day -6 and mean bodyweights at Day 0 and Day 56. Means in the same column that share a letter (A) are not significantly different (p<0.05).

Table 43. Group details

i Group	i Mean (range) FEC Day-6 i ¹¹ ΐ (epg)	Mean (range) bodyweight Day 0 (kg)	Mean (range) bodyweight ΐ Day 56 (kg) i
	15 i 170(100-300)	159(129-189)	184(155-205) \|
! ²	\| 15 i 180 (100-350)	158 (136-209)	183(152.5-240) \|
i 3 5.........................	i 15 I 187 (100-450)	156(127-197)	178 (149-220) i

Table 44 shows group arithmetic and geometric mean FEC at Days 0-42. Means in the same column that do not share a letter (A/B) are significantly different (p<0.05). Based on geometric means, the FECs for Group 1 (treated with the IVP) were significantly less (p<0.05) than both the control group (Group 3) and the group treated with DECTOMAX (Group 2) on both of Days and 28.

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Table 44. Group FECs

j Group	i Treatment !—	Arithmetic mean (range) EEC (eng) >
------................----...... - -............................... ................;--------------------	D 42
DO s	D 16	D28
1............1........	4.........n?________L	503^A(0-1800) \|	3^s (0-50) \|	3^B(0-50)	60* (0-200) ί
i 2	Ϊ Dectomax i	393^A (0-1050) \| 1	6fiMO-iO50) [	103^A (0-700)	86* (0-200)
Ϊ 3	i Control i	254- (0-550) ΐ 1	37“ (0-600)	125-(0-250)	83* (0-150) ΐ
	1 i		Geometric mean FEC (epg)
j	ϊ i	DO	D16 1	D 28	D42 ?
i 1	IVP i	249.2'	0.3^Λ ΐ	03^A	17.6- i
i 2	s Dectomax i	161.7'· [	28.1^s j	16.8®	35.8^A
j...........3__	s Control [	137-8^A 1	52 7⁸ §	____ 68.0^B_____	30.4*...... j
The efficacy of the IVP and CP at each timepoint (based	on FEC reduction compared to the
untreated	control group is	shown in Table 45.	The percentage of each nematode genus identified

in 100 larvae from group coprocultures at each timepoint is shown in Table 46.

Table 45. Percentage FEC reduction efficacy compared to untreated control

Treatment ί Dectomax % Efficacy (arithmetic means) D 16.........jD 28

97,6 j97.3

-1.7.1 j17,3

1)42

-2.9 % Efficacy (geometric means) j

1)16......| 1)28 [ D42 j

99.0 § 99.6 ΐ 42.2 s

47,0__j 753 j -18.0____

Table 46. Larval culture results (%)

Ϊ Day	Group	Treatment		Cooperia	i Haemonchus
Ostertagia	strongylus
§ 0	Pooled	NA	1 ¹² 1	8	80	⁰
	1	IVP	[ 97 '	0	3	i 0
§ 16	2	Dectomax	i 2 S	0	98	0
	3	NA	1 5 s	0	95	i o
	1	IVP	1 ⁵⁶	0	44	⁰
28	2	Dectomax	1 ¹¹ 1	0	89	1 0
	3	NA	23 1	9	68	j 0
[	1	IVP	; 2 !	0	98	0
⁴²	2	Dectomax	! 3 3	0	97	⁰
	3	NA	⁸	0	92	J 2

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Table 47. Individual FEC results

_n.s — no sample

Conclusions. Based on arithmetic mean FECs, the levamisole/eprinomectin injection was over 97.6% effective and this effect was maintained up to 28 days after treatment. DECTOMAX

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2017279645 20 Dec 2017 was insufficiently effective at all time points. Based on geometric mean FECs, the levamisole / eprinomectin injection was over 99% effective and this effect was maintained up to 28 days after treatment. DECTOMAX was insufficiently effective at all time points. Efficacies were evaluated in animals carrying a mixed infection of Ostertagia, Trichostrongylus and Cooperia spp. There was strong evidence of resistance by Cooperia spp. to doramectin.

* * *

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

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Claims

WHAT IS CLAIMED IS:

1. A pharmaceutically or veterinarily acceptable formulation adapted to be injected into an animal, which formulation comprises;

a. an effective amount of a levamisole in a concentration of from about 18% to about 25% (w/v);

b. an effective amount of at least one macrocyclic lactone in a concentration of from about 0.1% to about 2% (w/v);

c. a pharmaceutically acceptable system of solvents and surfactants wherein the system provides for acceptable storage stability for both the levamisole and the macrocyclic lactone and wherein the system provides for a viscosity which is suitable for injection;

wherein the surfactants comprise at least polyoxyethylenated esters of sorbitan, a polyethylene glycol stearate, or both;

wherein the solvents comprise at least glycerol formal (GF), propylene glycol, dimethyl acetamide (DMA), or combinations thereof, but not n-methyl pyrrolidone (NMP);

wherein the levamisole is levamisole phosphate;

wherein the formulation comprises water for injection; and wherein the water for injection is provided in a quantity sufficient to bring the formulation to a suitable injection volume.
2. The formulation of claim 1, wherein the levamisole phosphate is present in an amount sufficient to deliver a dose of at least 4 mg/kg animal bodyweight.
3. The formulation of claim 1 or claim 2, wherein the macrocyclic lactone is either ivermectin or eprinomectin and is present in an amount sufficient to deliver a dose of at least 150 pg/kg animal bodyweight.
4. The formulation of any one of claims 1 to 3, which further comprises an antioxidant, and a preservative.

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5. The formulation of any one of claims 1 to 4 wherein the surfactant concentration is from about 1% to about 10% (w/v).
6. The formulation of claim 4 or claim 5, wherein the macrocyclic lactone is eprinomectin and its concentration is about 0.5% to about 0.9% (w/v); the surfactant is polyoxyethylene (20) sorbitan monooleate and its concentration is about 3% to about 7% (w/v); the antioxidant is BHT; the solvent is DMA and its concentration is about 1% to about 10% (w/v); and the preservative is methyl paraben.
7. The formulation of any one of claims 1 to 6, wherein the macrocyclic lactone is eprinomectin and wherein the density of the formulation is between 1.05 and 1.10 and the pH is between 3.0 - 5.0.
8. A method for preventing and/or treating a helminth infestation in an animal, which method comprises administering to an animal an effective amount of a formulation according to any one of claims 1 to 7.
9. The method according to claim 8 wherein the formulation is injected.
10. The method of any one of claims 8 or 9 wherein the macrocyclic lactone concentration is from 0.5% to 0.9% (w/v).
11. The method of any one of claims 8 to 10 wherein the helminths are nematodes, cestodes, trematodes.
12. A method for producing a formulation according to any one of claims 1 to 7 comprising the steps of:

a. adding water for injection to a vessel,

b. dissolving the Levamisole in the water,

c. in a separate vessel dissolving Eprinomectin and BHT in DMA,

2017279645 23 Aug 2019

d. adding the surfactant to the DMA/Eprinomectin blend,

e. transferring the Eprinomectin/DMA blend to the bulk aqueous phase with mixing, and

f. optionally, bringing the batch to volume with added WFI and mix.
13. Use of a formulation according to any one of claims 1 to 7, for the manufacture of a medicament for the prevention and/or treatment of a helminth infestation in an animal.
14. Use of a formulation according to any one of claims 1 to 7 for the prevention and/or treatment of a helminth infestation in an animal.

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Mix until clear

Mix until clear

Mix until clear

Mix until clear

Mix until clear

Mix with Silverson

Mix with Silverson

FIG. 1

Aseptic Filling

Solution A

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Levamisole Cone in Plasma of Cattle Treated with Eprin/Lev Inj

1.2 .·................................................................................................................................................................................

B:N :08 'S'· BN :09

10 IS