WO 2014/098623 Al 26 June 2014 (26.06.2014) W P O P C T

Home , Eprinomectin

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2014/098623 Al 26 June 2014 (26.06.2014) W P O P C T

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 31/365 (2006.01) A61P 33/10 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/355 (2006.01) A61P 33/14 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, A61K 9/00 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/NZ20 13/000240 KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (22) International Filing Date: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 19 December 2013 (19. 12.2013) OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, (25) Filing Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (26) Publication Language: English ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 604990 20 December 2012 (20. 12.2012) NZ kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, (71) Applicant: ALLEVA ANIMAL HEALTH LIMITED UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, [NZ/NZ]; 15 Caiman Place, Birkenhead, Auckland, 0626 TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (NZ). EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, (72) Inventor: HOLMES, Robert William Lachlan; 15 TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Caiman Place, Birkenhead, Auckland, 0626 (NZ). KM, ML, MR, NE, SN, TD, TG). (74) Agents: PIPER, James William et al; 5A Pacific Rise, Mt Wellington, Auckland, 1060 (NZ).

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(54) Title: INJECTABLE EPRINOMECTIN FORMULATION AND ANTHELMINTIC USE THEREOF (57) Abstract: A method of treating a herd of cattle in a milk ing shed by injecting an animal at an injection site at or near the rear of the animal with an anthelmintic injectable formula tion comprising eprinomectin dissolved in an organic solvent. The injections can be applied at positions X of a rotary milk ing shed whilst the cows are in their bails on the rotary plat form. Injection positions for a herringbone milking shed are also described and illustrated. The injectable formulation has a nil milk withholding time so that it can be used on lactating cows. w o 2014/098623 Al III III II II III I IIII II ll lll III I III III II I II

Declarations under Rule 4.17: Published: — as to applicant's entitlement to apply for and be granted — with international search report (Art. 21(3)) a patent (Rule 4.1 7(H)) — before the expiration of the time limit for amending the — as to the applicant's entitlement to claim the priority of claims and to be republished in the event of receipt of the earlier application (Rule 4.17(Hi)) amendments (Rule 48.2(h)) INJECTABLE EPRINOMECTIN FORMULATION AND ANTHELMINTIC USE THEREOF

FIELD O F THE INVENTION

This invention relates t o the treatment of cattle especially in large herds.

BACKGROUND OF THE INVENTION

Treatment of cattle t o prevent and control parasite infection is an important aspect of modern farming, especially the treatment of roundworm.

Typical roundworm parasite known t o infect cattle include parasite belonging t o the

following genera: Haemonchus, Trichostrongylus, Ostertagia, Nemaodirus, Cooperia, Ascaris,

Bunostomum, Oesophagostomum, Chabertia, Trichuris, Strongylus, Trichonema,

Dictyocaulus, Capillaria, Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostoma, Unicinaria,

Toxascaris and Parascaris. Certain of these, such as Nematodirus, Cooperia and

Oesophagostomum attack primarily the intestinal tract while others, such as Haemonchus

and Ostertagia, are more prevalent in the stomach while others such as Dictyocaulus are

found in the lungs. Still other parasites may be located in other tissues such as the heart and

blood vessels, subcutaneous and lymphatic tissue and the like.

The effect on cattle health and well-being can be significant, and can range from mild ill-

health through t o death. In adult cattle even sub-clinical parasitism can result in productivity

loss through reduced body condition, reduced weight gain and reduced milk production.

Typically these parasites are controlled by specific chemical agents developed t o meet

certain criteria. These criteria include:

- High effectiveness against the target parasites - this could either be via broad-

spectrum efficacy in which a wide range of parasites are controlled or alternatively

via narrow spectrum of activity in which a more limited selection of parasites are

controlled. - Wide margin of safety to the host animal

Low potential t o cause toxic residues that could cause harm t o humans consuming

meat or milk products from the animal

Over the past 50-60 years there have been 5 major classes of broad-spectrum anthelmintic developed that can be used t o provide treatment of livestock for parasitism. These 5 classes

are as follows:

Benzimidazoles.

The first chemical class of modern anthelmintics developed was the benzimidazoles (BZD).

The first drug in this class, thiabendazole (TBZ) was introduced in 1961. Other members of the benzimidazole class includes fenbendazole, albendazole and oxfendazole.

Benzimidazole based anthelmintics interfere with the worm's energy metabolism on a

cellular level. The molecule binds t o a specific building block called beta tubulin and

prevents its incorporation into certain cellular structures called microtubules, which are

essential for energy metabolism. Interfering with energy metabolism is a much more basic

mode of activity than that which occurs with other classes of anthelmintics. For this reason,

benzimidazoles are also able t o kill worm eggs. Benzimidazoles have a wide margin of safety

and broad spectrum activity.

Nicotinic agonists

Nicotinic agonists comprise the secod class of anthelmintics. They include imidazothiazoles

and tetrahydropyrimidines . The imidazothiazole group includes levamisole, while the

tetrahydropyrimidine group includes pyrantel pamoate, pyrantel tartrate, and morantel

tartrate.

The tetrahydropyrimidines mimic the activity of acetylcholine, a naturally occuring

neurotransmitter that initiates muscular contraction. Upon exposure t o the active

ingredient, the worm is unable to feed and quickly starves. Tetrahydroyrimidines only affect

adult populations of worms. They do not have activity against the larval stages and are

ineffective against cestodes (tapeworms) and trematodes (liver flukes). Imidazothiaoles have a similar mode of action causing spastic paralysis of the worms. The group includes the drug levamisoie discovered in 1966.

Compared t o other anthelmintics, levamisoie has the narrowest margin of safety, though toxicity is usually the result of excess dosage. Levamisoie has a broad spectrum of activity and is effective against many larval stages of parasites; though not arrested larvae.

Macrocyclic lactones

The next anthelmintic class t o be developed was the macrocyclic lactones (MLs) developed from the Streptomyces genus of soil dwelling-organisms. The first drug of the class, ivermectin, was introduced in the early 1980s.

Macrocyclic lactones consist of two closely related chemical groups: avermectins and milbemycins. The avermectins include abamectin, ivermectin doramectin and eprinomectin.

The milbemycin group is represented by milbemycin oxime and moxidectin, introduced in

1997.

All of the macrocyclic lactone anthelmintics have the same mode of action. They interfere with GABA-mediated neurotransmission, causing paralysis and death of the parasite.

Macrocyclic lactones are the most potent killer of worms and are more persistent in their effect. The duration of persistent activity varies according t o the drug and formulation.

Macrocyclic lactones have the unique quality of also killing several types of external parasite such as lice, mites, and ticks. Because of this they are also known as "endectocides" (end- ecto-cides), meaning control of internal and external parasites. Generally, the macrocyclic lactones have a wide margin of safety in treated livestock and are effective against all stages of worms, including inactive forms. Amino-acetonitrile derivatives

In 2009, the first of this new class of anthelmintics was introduced. "Monepantel" acts by paralyzing worms by attacking a previously undiscovered receptor HCO-MPTL-1, present only in nematodes.

Spiroindoles

In 2010 Derquantel (2- deoxoparaherquamide), the first of yet another new class of anthelmintic was introduced.

Some of these anthelmintic types have been used t o treat adult dairy cattle. Over the years, there have been anthelmintic formulations from at least the benzimidazole, nicotinic

agonist and macrocyclic lactone classes that have been used t o fulfil this requirement.

Formulation types have included slow-release bolus formulations, oral drenches and in-feed

materials and, more recently, topical or "pour-on" formulations of the macrocyclic lactone

class of chemicals. It is this latter class that has now become the de-facto norm for

treatment of lactating dairy cows. "Nil-milk" or zero-milk withholding period pour-on

formulations have been developed with all of the commercially available macrocyclic

lactone active ingredients including; abamectin, ivermectin, doramectin, eprinomectin and

moxidectin.

The chemical structure of each of these active ingredients is as follows:

Abamectin (AvermectinBlaBlb) Ivermectin B a &Ivermectin l b Eprinomectin

In each case, the pour-on formulation consists of a quantity of the macrocydic lactone active dissolved in one or more organic solvents. This is then applied topically t o the back of the animal from where it is absorbed through the skin. The dose rate typically applied is

500mcg/kg at a dose volume of around lmL/lOkg or lmL/20kg. In New Zealand, such formulations have been the only nil-milk treatments available for at least the past 20 years.

This has been due t o their supposed convenience and the absence of alternative treatment

forms and methods of treatment. In large dairy herds, it can be difficult t o treat cows with an anthelmintic due t o the design of modern dairy sheds and the lack of effective formulations designed for use in such situations.

The need for speed in milking has led t o advances in the design of milking installations making it difficult t o treat cows using conventional pour on, drenching or injection techniques. Each cow typically weighs in excess of 500kg making it impractical t o manipulate them, particularly when the farmer may be treating 500 or more cows at a time. Most milking installations use either herringbone configuration sheds, or rotary milking platforms.

Treatment with an injectable formulation is desirable as when compared to the topical delivery route. Injectable formulations are capable of delivering much higher blood levels of the drug. Injectable anthelmintic formulations (designed for injection to the anterior neck portion of a cow) containing a macrocyclic lactone anthelmintic are usually formulated to deliver 200mcg/kg of the drug. Typically, this is administered from a 1% formulation via a lmL/50kg dose rate. This size of injectable anthelmintic dose is very impractical for such large animals. If a typical injectable were administered a 500kg cow would generally be given a dose of around lOmls. This would mean a standard 500mL pack would only be capable of treating 50 cows. On many large farms, a rotary platform holds 70-80 cows. In addition, lOmL is a relatively large dose that would take a reasonable period of time to inject. There is also a tendency for doses of this size t o leak from the injection site, particularly when the injection is administered subcutaneously. The design of the rotary platform makes it difficult for the farmer t o access the anterior portion of the neck where such treatments are typically administered. Attempting to reach between or across cows to reach the anterior neck portion could also be highly dangerous and subject the farmer to possible injury. The time taken to do this would also make it impracticable.

There is at present no nil-milk withholding injectable anthelmintic formulations currently available. This means that it is only possible t o treat cows at a time of year when lactation is not occurring.

In New Zealand, the rotary cow shed has become the most popular form of milking parlour. This is due t o the high speed at which large groups of cows can be milked. Unlike older walk through and herringbone designs, the flow of cows through a rotary cow shed is continuous. As shown in figure 1, the un-milked cows walk into stalls o n t o a large platform, which moves in a gradual circular movement around a central point. It is important t o note that the cows are facing toward the centre with their posterior end toward the outside of the platform. Such a positioning means that it is easy for the milker standing at position X t o place the milking machine "cups" on the teats of the cow.

As the platform gradually rotates, the cow is milked. At the end of the rotation the cups are removed either automatically, o r by a second milker standing at position Y. After cup removal the cow then backs off the platform.

Due t o speed and convenience, this style of shed is very popular with farmers milking large herds of 500 cows o r more. Farmers prefer t o treat their cows with an anthelmintic while they are located or moving on a rotary platform. Treatment may for example take place while the cows are being milked or when the cows are specifically brought on t o the rotary platform for some other purpose such as administration of intramammary antibiotics, vaccines etc. If they are t o be treated whilst lactating then the treatment has t o have an approved "nil milk withholding time" (MWT).

In some other cases, the farmer may alternatively treat the cows while they are moving down a race or while they are stationery within the race. Even in such situations the large numbers of animals means that is desirable that the time taken t o treat each animal is relatively fast.

Because of various factors, including the use of rotary sheds, very large animal numbers, and safety t o the farmer, pour-on type formulations have become the preferred form of anthelmintic delivery as they:

Do not require the farmer t o access the head or neck parts of the cow, which can be very difficult or in some cases very dangerous to access. Enables the cow t o be easily treated while it is on a milking platform.

To keep up with the speed of the milking platform each cow should be able t o be treated in a similar period of time t o what it would take the farmer t o put the milking machine cups on the teats of the cow. If treatment were t o take longer than this time the farmer would not keep up with the speed at which the platform is moving.

Pour- On formulations permit this but they do have limitations. These limitations include:

Relatively low levels of drug delivered into the blood when compared t o other formulation types Susceptibility t o poor delivery due t o effect of rain, UV and run-off - Tendency t o run-off the back of the cow; this problem is particularly so with dairy breed animals as they tend t o have very smooth coats which do not act as a very effective barrier t o the movement of the anthelmintic solution.

Such factors would make it desirable that farmers have an alternative means of treatment specifically designed t o enable them t o effectively treat large herds, and in particular lactating dairy cows at a speed sufficient t o keep up with the movement of a rotary milking shed platform. OBJECT O F THE INVENTION

It is an object of the invention t o provide a method o r a formulation which can be used t o treat cattle effectively and quickly, typically while located or moving on a rotary platform o r at least provide the public with a useful choice.

STATEMENT OF INVENTION

In one aspect, the invention provides a method of treating cattle by injecting an animal at an injection site at or near the rear of the animal with an anthelmintic injectable formulation comprising eprinomectin in a suitable liquid carrier.

More preferably, the invention provides a method of treating cattle by injecting an animal at an injection site at or near the rear of the animal with a nil milk withholding time approved anthelmintic injectable formulation comprising eprinomectin in a suitable liquid carrier, wherein the concentration of eprinomectin is greater than 1% w/v.

Preferably, the injection site is chosen from the group comprising: the base of the tail, the tail fold, and the depressions on either side of the tail head of the cow.

Preferably, the liquid carrier is an organic solvent and anthelmintic injectable formulation comprises eprinomectin dissolved in the organic solvent.

Preferably, each animal is provided with an injection delivering at least 200mcg/kg of eprinomectin: per kg of body weight of the animal.

Preferably, each animal is injected whilst being held head first in bail of a milking installation such as a rotary milking shed, or a herringbone-milking shed.

Preferably, anthelmintic injectable formulation is a nil milk withholding injection containing both eprinomectin and vitamin E.

In another aspect, the invention can be said to be the use of an anthelmintic injectable formulation containing eprinomectin as an injection applied at an injection site at or near the rear of the animal.

In another aspect, the invention involves the use of eprinomectin in the manufacture of an anthelmintic injectable formulation to be applied at an injection site at or near the rear of the animal.

In another aspect, the invention provides an injectable anthelmintic formulation suitable for treatment of cattle, the formulation comprising:

More than 1% w/v Eprinomectin;

benzyl alcohol;

sorbitan monooleate; a Vegetable oil or derivative; and one or more co-solvents.

This allows a lower dose/higher concentration of eprinomectin thereby reducing the injection time per animal.

Preferably, the formulation contains at least 1.5% eprinomectin w/v.

Preferably the co-solvent(s) is selected from glycerol formal, dimethyl acetamide and dimethyl isosorbide.

Preferably the formulation further includes Vitamin Eacetate.

Preferably the formulation further includes water and a water-soluble vitamin such as Vitamin B12.

In another aspect the invention provides a method of treating a herd of cattle with an anthelmintic injectable formulation containing eprinomectin dissolved in an organic solvent and in which the dosage recommendations provide for a dose rate of no less than

200mcg/kg and the injection site t o be at or near the rear of each animal.

Preferably the injection site is at the base of the tail, the tail fold or the depression on either side of the tail head of the cow.

Preferably the formulation contains greater than 1.5% w/v eprinomectin.

Preferably the method involves treating cattle with a nil milk withholding injection containing both eprinomectin and vitamin E.

In another aspect the invention provides an injectable anthelmintic formulation suitable for treatment of cattle containing:

- At least 1.5% Eprinomectin Vitamin Eacetate or other oil soluble trace element Benzyl alcohol - Sorbitan monooleate - A Vegetable oil or derivative (e.g. Miglyol) One or more co-solvents Optional antioxidant Preferably the co-solvent is selected from glycerol formal, dimethyl acetamide and dimethyl isosorbide.

Preferably the injectable anthelmintic formulation also contains a water-soluble vitamin such as Vitamin B12 as well as water and other optional elements, including surfactants, water, antioxidants, buffers and preservatives.

In the present invention two factors have been addressed to develop the improved method of treatment:

Development of a nil-milk residue injectable formulation that also allows for the co¬ administration of other beneficial agents - Development of a method of injectable treatment that can reduce the time taken to administer an injectable treatment to cattle without reducing the quality of the treatment.

The method of treatment subject t o this invention is such that it enables the farmer t o treat animals by injection in a period of time substantially similar to that needed to administer pour-on treatments.

These and other aspects of this invention which should be considered in all its novel aspects will be apparent from the following description which is given by way of example only with reference t o the accompanying drawings and examples.

DRAWINGS:

Figure 1 is a schematic drawing of a conventional rotary cowshed.

Figure 2 is a graph showing the mean concentration of Eprinomectin (over time) in bovine plasma after treatment with Eprinomectin injection or Pour-on.

Figure 3 is a front on view of the posterior end of a cow showing the preferred application site.

Figure 4 is a top view of a conventional herringbone milking shed.

Figure 5 is a further schematic drawing of a conventional rotary cowshed. DETAILED DESCRIPTION OF THE INVENTION

Figure 3 shows the preferred injection site (20) on the cattle. This site has the advantage of being easily accessiable, contains sufficiently "loose" skin t o enable the area to be 'tented' t o permit subcutaneous administration.

In a traditional herringbone milking configuration as shown in figure 4, cattle enter (32) the shed from the yard (36), wherein the milker/farmer (30) is located in a central race located below the level of the cattle, which is accessed by the steps (31). Typically the milker/farmers eye level is in line with the prosterior end of the cattle. The suction cups are then placed on the cattle by the farmer in preparation for for milking. Once milking is completed, the suction cups are removed and the the exit gate (33) is opened allowing the cattle t o exit out to pasture (34).

As it can been seen in figure 4, cows are presented in an inline position on either side of a central race. In such a situation it is very difficult for a farmer/milker (30) t o access the back of the cow to permit treatment with a topical/pour-on formulation. The inventive formulation permits treatment at a larger number of admnistration points (marked with an X). The advantages of a nil-milk injectable of low volume that can be adminstered in the rear of the cow are quite obvious in this situation.

In the rotary milking parlour situation, as shown in figures 1 and 5, the cows are on a raised central platform while the Milker ( 43) is on a lower level. The cattle enter the rotary platfrom from the yard (40) by means of a backing gate (41), forcing the cattle towards the entry point (42). Once on the central rotary platform, suction cups are then placed on the cattle by machine or manually by the milker. Milking continues for one rotation (44). Once completed, the suction cups are then removed and the cow exits out the exit gate(45).

When treating with a typical nil-milk topical formulation the only practical position in which the cow can be treated is at the entry (42)or exit (45) points. The inventive formulation permits treatment at a larger number of admnistration points (such as those marked with an X) even while the platform is turning.

EXAMPLE 1:

Formulation

Various injectable formulations of eprinomectin were trialled in which eprinomectin and Vitamin E oil were dissolved in a blend of Benzyl alcohol, sorbitan monooleate and caprylic capric triglyceride (Miglyol). While the active ingredients remained within their proposed shelf-life specifications there was an undesirable tendency for the formulation t o crystallise upon refrigeration.

w/ v AL16-37 Eprinomectin 2 Vit E acetate 5 Benzyl Alcohol 10 Sorbitan monooleate (Span 80) 4 Caprylic capric triglyceride (Miglyol 840) Qs

Various means were tried t o eliminate this tendency. Included in these trials was the testing of a range of co-solvents.

Alternative manufacturing methods were also attempted. Results demonstrated that the addition of a small quantity of a co-solvent such as glycerol formal is a preferred means of eliminating this problem without significantly changing the base formulation:-

Method 1:

1. In a mixing vessel, add Caprylic capric triglyceride (Miglyol 840). 2. Add and dissolve eprinomectin. 3. Add Benzyl alcohol, Glycerol formal and Sorbitan monooleate (Span 80).

4. Add and dissolve vitamin E. 5. Make up volume with Caprylic capric triglyceride (Miglyol 840).

Method 2:

1. In a mixing vessel, add Caprylic capric triglyceride (Miglyol 840). 2. Add BA, G F and Sorbitan monooleate (Span 80), and mix. 3. Add and dissolve eprinomectin.

4. Add and dissolve vitamin E. 5. Make up volume with Caprylic capric triglyceride (Miglyol 840). w/v% AL16-40 AL16-41 AL16-42 Eprinomectin 2 2 2 Vit E acetate 5 5 5 Benzyl Alcohol 10 10 10 Sorbitan monooleate (Span 80) 4 4 4 Co-solvent 3% GF 4% G F 5% GF Caprylic capric triglyceride (Miglyol 840) qs qs qs Method 2 2 2

The results of these studies demonstrated that a level of around 3% or more would required to prevent crystallisation of the 5% Vitamin Acetate present in the formulation.

Observations:

Tests were then conducted to determine the suitability of the formulation in the treatment of cattle.

The first study conducted was t o determine blood levels of eprinomectin obtained by administration of the injectable product when compared t o a topical formulation of eprinomectin. Results are presented in Table 1:

Table 1: Geometric mean AUC and Cmax values for animals treated with an Eprinomectin injection or an Eprinomectin pour-on

a,b = means within a column with different superscript are significantly different from each other. Figure 2 is a graph that shows the mean concentration of Eprinomectin in bovine plasma after treatment with Eprinomectin injection or Pour-on.

Such blood levels would mean that the endoparasite protection delivered by the tested eprinomectin vitamin E injection would be likely be superior t o that of the marketed eprinomectin pour-on formulation.

Injection Efficiency

Injection efficiency is a function of the following factors:

- Time taken t o access and locate the injection needle at the injection site on the animal; Time taken t o inject the dose; Effectiveness of the dose (absence of injection site leakage, absence of injection site abscesses, clinical outcome).

To treat large animals in a production herd situation it is simply not possible t o access the neck/shoulder of the animal in a milking shed or large herd situation.

In New Zealand the average herd size is approx. 350 cows. In some cases 1,000 or more cows are present. Even if it were t o take one minute for the farmer t o move t o a position in which the neck/shoulder area could be accessed this would be almost 6 hours of time taken t o treat an average herd size. In actual fact Dairy NZ statistics indicate that cow throughput at peak lactation ranges from an average of 149 cows/hour for small 40 bail rotary sheds, t o 447 cows/hour for 80 bail rotaries. That is one cow milked for every 8-24 seconds. This is an indication of the speed at which a dairy rotary platform can move.

A second aspect t o treatment of large animals by injection is that the practice can be inherently dangerous as the cow may react violently t o attempts t o move its head or t o lift its neck into a position where the injection can be given. There are also practical difficulties involved with "tenting" the skin so that the injection can be given subcutaneously. The time taken t o inject the anthelmintic solution also means that the head/neck must be held relatively stationery for a period of time.

Treatment of a cow by injection at other sites at or near the rear of the animal is preferred. However there are a limited number of sites on the body of the animal which allow for easy access, easy "tenting" of the skin and are not likely t o cause tissue damage/residue concerns at the injection site if the cow is slaughtered. Of the potential sites the base of the tail, the tail fold, or the depression on either side of the tail head would offer the best combination of these factors. When coupled with a formulation that reduces the dose size the administration of an anthelmintic in the desired timeframe would become easy t o accomplish.

In reality the times when cows are likely t o be treated are:

In a race, with the cows stationary; - In a race, with cows moving through; On a dairy platform while being milked; On a dairy platform while not being milked;

The inventive concept of a high concentration eprinomectin-based injection combined with a low administration dose, convenient injection site and nil milk-withholding periods offers an effective and desirable alternative t o pour-on formulations in all of these situations.

Where the treatment occurs while the cow is being milked on a rotary platform the method ensures that the milker was not unnecessarily inconvenienced by the need t o administer the treatment.

Where the animals are treated in a race situation the method of treatment also ensures greater safety t o the farmer and less stress t o the animal.

The general instructions given t o users on packs of the formulation would be:

This product is t o be given by subcutaneous injection only. The recommended dose is ImL per 100kg bodyweight (200mcg eprinomectin per kg). Inject under loose skin in one of the two sites indicated in the illustration:

A. Anterior portion of the neck B. In the depression on either side of the tail head (this injection site is recommended for treatment of cattle in dairy shed situations or in situations where unintended animal movement could pose a risk t o the user).

Milk Residue Study

A study was undertaken in which a group of lactating dairy cows were treated with the test product at study hour 0 at a dose volume of over lmL/100 kg (slightly in excess of 200mcg/kg eprinomectin). Cows were milked twice daily and milk specimens were collected from study hour 0 t o study hour 96.

Milk yields and milk fat percentage were not significantly affected by the treatment. Statistical analysis of milk eprinomectin concentrations indicated that even at the highest observed level of eprinomectin (24 hours post treatment) the UCL (upper confidence level) remained below the MRL of 0.02 mg/kg. This demonstrated that the formulation achieved the objective of providing for a high sustained peak of eprinomectin in the blood without leading t o residues which would restrict its use in lactating dairy cows.

Injection Site Tolerance

Comparative injection site tolerance studies were undertaken to determine the suitability of the tail depression as a site for injection with the invention. The study demonstrated that the invention was well tolerated and caused no adverse reactions.

Stability

Longer term stability testing has also demonstrated that the formulation provides for very good stability of the active ingredients over at least 12 months at room temperature.

ADVANTAGES OF THE PREFERRED EMBODIMENT

Treatment with an injectable formulation is better than a topical delivery (pour on) as injectable formulations are capable of delivering much higher blood levels of drug.

The method allows rapid treatment by injection the preferred formulation at an injection site on the rear of the animal typically while it is trapped in the bail of a (rotary) milking shed.

The injectable formulation provides a nil-milk withholding injectable anthelmintic formulation. This means that it is possible to treat cows with this injectable formation all the year round whether they are lactating or not.

The method provides a specific method of injectable anthelmintic treatment suitable for large herds and particularly lactating dairy cows, which can be administered in a dairy shed situation.

The preferred formulation also provides for the supplemental delivery of other trace elements important t o the health of cattle, in particular Vitamin Eand/or Vitamin B12

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

1. A method of treating cattle by injecting an animal at an injection site at or near the rear of the animal with a nil milk withholding time approved anthelmintic injectable formulation comprising eprinomectin in a suitable liquid carrier, wherein the concentration of eprinomectin is greater than 1% w/v.

2. A method of treating a herd of cattle with each animal being treated as claimed in claim 1 wherein the injection site is chosen from the group comprising: the base of the tail, the tail fold, and the depressions on either side of the tail head of the cow.

3. A method of treating a herd of cattle as claimed in claim 2, wherein the liquid carrier is an organic solvent and anthelmintic injectable formulation comprises eprinomectin dissolved in the organic solvent.

4. A method of treating a herd of cattle as claimed in claim 3, wherein each animal is provided with an injection delivering at least 200mcg/kg of eprinomectin : per kg of body weight of the animal.

5. A method of treating a herd of cattle as claimed in claim 4, wherein each animal is injected whilst being held head first in bail of a milking installation such as a rotary milking shed, or a herringbone-milking shed.

6. A method of treating a herd of cattle as claimed in claim 5, wherein anthelmintic injectable formulation is a nil milk withholding injection containing both

eprinomectin and vitamin E.

7. Use of an anthelmintic injectable formulation containing eprinomectin as an injection applied at an injection site at or near the rear of the animal.

8. Use of eprinomectin in the manufacture of an anthelmintic injectable formulation to be applied at an injection site at or nearthe rear of the animal.

9. An injectable anthelmintic formulation suitable for treatment of cattle, the formulation comprising: at least 1.5% w/w Eprinomectin; benzyl alcohol; sorbitan monooleate; a Vegetable oil or derivative; and one or more co-solvents. 10. An injectable anthelmintic formulation as claimed in claim 9, wherein the co- solvent(s) is selected from glycerol formal, dimethyl acetamide and dimethyl isosorbide.

11. An injectable anthelmintic formulation as claimed in claim 9, wherein the formulation further includes Vitamin Eacetate.

12. An injectable anthelmintic formulation as claimed in claim 10, further including water, and a water-soluble vitamin such as Vitamin B12.

INTERNATIONAL SEARCH REPORT International application No. PCT/ Z2 13/000240

A. CLASSIFICATION OF SUBJECT MATTER A61K 31/365 (2006.01) A61K 31/355 (2006.01) A61K 9/00 (2006.01) A61P 33/10 (2006.01) A61P 33/14 (2006.01)

According to International Patent Classification (IPC) or to both national classification and IPC B . FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols)

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) EPOQUE (epodoc, WPI, Medline): Keywords; inject, parenteral, intra-dermal, infra-muscular subcutaneous, hypodermic, macrocyclic lactone, macrolide, avermectin, eprinomectin, ivermectin, selamectin, doramectin, abamectin, moxidectin, milbemycin, rear, tail, rump, hind, hindquarter, haunch, hip, glycerol formal, glyceryl formal, dimethyl acetamide, dimethyl isosorbide, vitamin E, tocopherol, tocotrienol, cow, cattle, bovine, herd, benzyl alcohol, sorbitan, polysorbate, tween & like terms

STN (CAPlus, Agricola): Keywords: avermectin, eprinomectin, 123997-26-2/RN, ivermectin, selamectin, doramectin, abamectin, moxidectin, milbemycin, inject, parenteral, intra-dermal. intra-muscular subcutaneous, hypodemiic, rear, tail, rump, hind, hindquarter, haunch, hip, benzyl alcohol, sorbitan, polysorbate, tween & like terms

PatentScope; EspaceNet; Google: Name applicant; Inventor; Keywords: inject, parenteral, eprinomectin, cow, cattle, rear, tail, rump, hind

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

Documents are listed in the continuation of Box C

Further documents are listed in the continuation of Box C X See patent family annex

ψ Special categories of cited documents: "A" document defining the general state of the art which is not later document published after the international filing date or priority date and not in considered to be of particular relevance conflict with the application but cited to understand the principle or theory underlying the invention "E" earlier application or patent but published on or after the document of particular relevance; the claimed invention ca not be considered novel international filing date or cannot be considered to involve an inventive step when the document is taken alone "L" document which may throw doubts on priority claim(s) or document of particular relevance; the claimed invention cannot be considered to which is cited to establish the publication date of another involve an inventive step when the document is combined with one or more other citation or other special reason (as specified) such documents, such combination being obvious to a person skilled in the art "O" document referring to a n oral disclosure, use, exhibition document member of the same patent family or other means P document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search Date of mailing of the international search report 30 May 2014 30 May 2014 Name and mailing address of the ISA AU Authorised officer

AUSTRALIAN PATENT OFFICE Ross Heisey PO BOX 200, WODEN ACT 2606, AUSTRALIA AUSTRALIAN PATENT OFFICE Email address: [email protected] (ISO 9001 Quality Certified Service) Telephone No. 0262833185

Form PCT/ISA/210 (fifth sheet) ( uly 2009) INTERNATIONAL SEARCH REPORT International application No.

C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT PCT/NZ2013/000240

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

BR PI1001224-9 A2 (CLARION BIOCIENCIAS LTDA.) 15 March 201 1 Y Abstract; page 2/7, line 22 - page 3/7, line 1 ; page 3/7, line 22 - page 5/7, line 14; 1-12 claims 1-3

BR PI1001593-0 A2 (CLARION BIOCIENCIAS LTDA.) 03 January 2012 Y Abstract; page 2/7, line 22 - page 3/7, line 17; page 4/7, line 27 - page 6/7, line 4; claim 1-12 1

US 2010/0266628 A l (RAZZAK et al.) 2 1 October 2010 Y Abstract; para. 0050, 0064, 0067-0069, 0072, 0077, 0089, 0103; Tables 2, 4, 7, 10-1 1, 1-12 15; Fig 1; claims 8, 10-1 1

US 4389397 A (LO et al.) 2 1 June 1983 Y Abstract; col. 4, lines 22-40; Examples 1-3 9-12

WO 201 1/143479 Al (MERIAL LIMITED) 17 November 2011 Y Abstract; page 10, lines 27-3 ; Tables 1, 8-9 1-12

WO 201 0/1 16267 Al (PFIZER INC.) 14 October 201 0 Y Abstract; page 2, lines 17-19 and 25-27; page 3, lines 8-31; page 4; lines 14-32; page 1-12 0, lines 20-34; Examples 1, 4-6; claims 5, 10

WO 201 2/001083 A2 (CEVA SANTE ANIMALE SA) 05 January 2012 Y Abstract; para. 0020, 003 1-0034, 0052; Example 1; claims 3, 7-8, 14 1-12

ROEBER, D.L., et al., "Frequencies of injection-site lesions in muscles from rounds of dairy and beef cow carcasses", J . Dairy Sci., 2002, vol. 85, pp. 532-536 Y Abstract; Figure 1; Discussion at page 534, RH col.; Conclusions 1-8

BAOLIANG, P., et al., "Pharmacokinetics of Eprinomectin in Plasma and Milk following Subcutaneous Administration to Lactating Dairy Cattle", Veterinary Research Communications, 2006, Vol. 30, pp. 263-270 Y Abstract; page 269, lines 10-22 1-8

JIANG, H., et al., "Residue depletion of eprinomectin in bovine tissues after subcutaneous administration", J. Agric. Food Chem., 2005, vol. 53, pp. 9288-9292 Y Abstract; Results and Discussion at page 9290, RH col.; page 9291 , RH col. last para - 1-8 page 9292, LH col.

Form PCT/ISA/210 (fifth sheet) (July 2009) INTERNATIONALSEARCH REPORT International application No. Information on patent family members PCT/NZ2 1 0 024 This Annex lists known patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.

Patent Document/s Cited in Search Report Patent Family Member/s

Publication Number Publication Date Publication Number Publication Date

BR PI 1001224-9 A2 15 March 201 1 None

BR PI1001 593-0 A2 03 January 2 12 None

US 2010/0266628 A l 2 1 October 2010 US 83 13752 B2 20 Nov 2012 AU 2010237070 A 1 03 Nov 201 CA 2758276 A 1 2 1 Oct 2010 CO 6450627 A2 3 1 May 2012 EP 241913 1 A l 22 Feb 2012 MX 201 1010710 A 25 Jan 2012 WO 2010120195 A l 2 1 Oct 2010

US 4389397 A 2 1 June 1983 AR 229354 A 1 29 Jul 1983 AU 551280 B2 24 Apr 1986 AU 7361281 A 11 Feb 1 82 CA 1162857 A l 28 Feb 1984 D 345081 A 05 Feb 1982 D 163637 B 23 Mar 92

EP 0045655 A2 10 Feb 1 82 EP 0045655 B 20 Ma 1985

ES 8607721 Al 16 Nov 1986 GR 74604 Al 29 Jun 1984

TE 51460 B l 24 Dec 1986 JP S5765330 A 20 Apr 1982 J H057371 B2 28 Jan 1993 KR 860001 503 B l 30 Sep 1986 NZ 197796 A 09 Nov 1984 OA 6863 A 28 Feb 1983 PT 73471 A 0 1 Sep 198 1 PT 73471 B 06 Jul 1983

YU 183 18 1 A 29 Feb 1984

ZA 81053 17 A 30 Mar 1983

WO 201 1/143479 A l 17 November 201 1 AR 081 187 A l 04 Jul 2012

AU 201 1252987 A l 13 Dec 2012 AU 201 1252987 B2 02 May 2013 EP 2568980 A l 20 Mar 2013

Due to data integration issues this family listing may not include 0 digit Australian applications filed since May 2001 . Form PCT/ISA/21 (Family Annex)(July 2009) INTERNATIONAL SEARCH REPORT International application No. Information on patent family members PCT/NZ2 13 0 024 This Annex lists known patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.

Patent Document/s Cited in Search Report Patent Family Member/s

Publication Number Publication Date Publication Number Publication Date

NZ 603699 A 30 Aug 2013

US 201 1281920 A l 17 Nov 201 1

WO 2010/1 16267 A 14 October 2010 A 075428 A l 30 Mar 201 1 AU 2010233409 A l 11 Aug 201 1

AU 2010233409 B2 17 May 2012

CA 2750626 A l 14 Oct 2 10 CN 1023 16876 A 11 Jan 2012

CN 1023 16876 B 17 Jul 2013 CO 6410307 A2 30 Mar 2012

EP 2396006 A l 2 1 Dec 201 1

EP 2396006 B l 19 Dec 2012 H 1161087 A l 11 Apr 2014 JP 20 125 17994 A 09 Aug 2012

KR 201 101 1623 1 A 25 Oct 20 11 MX 201 1008614 A 09 Sep 201 1 NZ 594389 A 28 Sep 2012

US 201 1294751 A l 0 1 Dec 201 1

US 8551962 B2 08 Oct 2 13

WO 2012/001083 A2 05 January 2012 None

End of Annex

Due to data integration issues this family listing may not include 0 digit Australian applications filed since May 2001 . Form PCT/ISA/21 (Family Annex)(July 2009)