WO2006022562A1 - Improved chemical delivery formulations - Google Patents

Abstract

Formulations are described to improve the delivery of poorly soluble drug agents such as anthelmintic compounds on injection to animals. In one particular example of a complexed ricobendazole anthelmintic formulation given to a sheep, drug absorption and by association bioavailability, was increased significally compared to existing formulations. In addition the invention formulations were well tolerated by test animals with minimal side effects or tissue reactions. One advantage of the formulations of the present invention is that dosages may be able to be decreased owing to the superior absorption and resulting comparable efficacy with other methods.

Description

IMPROVED CHEMICAL DELIVERY FORMULATIONS

TECHNICAL FIELD

The invention relates to improved chemical delivery formulations. In particular, the invention relates to formulations that more readily solubilise and/or disperse poorly soluble chemical compounds and related methods for improving solubility and/or dispersion. More specifically, the invention relates to formulations containing poorly soluble anthelmintic chemical compounds that are formulated so as to increase the solubility and/or dispersion characteristics of the anthelmintic compound. Related methods are also described pertaining to the formulation properties and uses.

BACKGROUND ART

Drug delivery formulations are well known in the art and include vehicles, excipients, fillers, solvents, and a wide variety of other substances to enable compounds such as pharmaceutical drugs to be administered to patients (human and animal).

A preferred administration pathway for many drugs is by injection. Injections are favourable as:

• They are typically fast acting as the drug is delivered direct to the site or directly into the blood stream;

• They often require less active agent (and therefore less cost and/or side effects) as there are minimal losses associated with other pathways such as digestion; and,

• Food effects on drug bioavailability are avoided

• "First-pass" metabolism is avoided

Anthelmintic chemical compounds are widely known agents that are destructive to worms and used for removing internal parasitic worms in animals including humans.

There are many different types of anthelmintic compound, each with varying degrees of parasitic activity and chemical properties. One characteristic of many anthelmintic compounds is that they are poorly soluble in aqueous environments such as within animal tissue. A current method of addressing this problem is to use a variety of formulation compounds to assist in dispersion. Such compounds can markedly increase the cost of a drug and/or may introduce other undesirable effects such as side effects on administration and more complicated administration procedures.

Benzimidazole compounds are one family of anthelmintic compounds having a general structure of:

Benzimidazole compounds in particular are sparingly soluble in aqueous environments. Benzimidazole compounds are a ring system including a benzene ring fused with an imidazole ring. Imidazole is a five membered heterocyclic compound present in various biologically important compounds. Benzimidazole compounds exist in nature as part of the vitamin B₁₂ molecule.

A derivative of benzimidazole is albendazole with the structure:

Albendazole is a known broad spectrum anthelmintic effective against many parasites.

Ricobendazole is a benzimidazole anthelmintic, which is an active metabolite generated by oxidation of albendazole, with the structure:

Ricobendazole is one of the most modern of the benzimidazoles and has confirmed activity against all major internal parasites and their eggs in at least sheep.

Other benzimidazole anthelmintics related to ricobendazole include fenbendazole with a structure of:

oxfenbendazole with a structure of:

and parbendazole with a structure of:

A problem found in the prior art with existing anthelmintic compounds including benzimidazole anthelmintics, is the need to deliver the compound as an oral suspension due to the particularly poor solubility of this type of compound in aqueous environments. As discussed above, a preferred method of administration is by injection however, as the solubility of the anthelmintic is often poor, injections are not readily absorbed and often may cause pain to the animal or human patient due to inadequate absorption or precipitate formation.

Reference will now be made to ricobendazole however, this should not be seen as limiting as it should be appreciated by those skilled in the art that the properties exhibited by ricobendazole also relate to other anthelmintic compounds.

Ricobendazole is characteristic of the poor solubility properties of at least some anthelmintic compounds. Ricobendazole is only soluble in water at extremes of pH level i.e. less than pH 2 or greater than pH 11. As a consequence of the low solubility, existing formulations are normally administered orally to the animal or human.

Two injectable ricobendazole formulations known to the inventors exist that utilise a pH of less than 2 (Bayverm PI™ and Sintyotal-R™) to achieve a desired solubility. In the inventors experience however, it is estimated from pharmacokinetics that the bioavailability of these formulations is low at 37-40%. It is understood that this is because the majority of the active agent precipitates out of solution on entry into the subject's body as the acidic pH is quickly neutralised once the injection mixes with extracellular tissue fluid. The resulting precipitated active must then redissolve to be effective which either does not occur or, occurs too slowly to be effective.

A further problem noted with low pH formulations is pain, swelling and inflammation at the injection site. These reactions may also be associated with the anthelmintic compound such as a reaction from the subject against precipitated drug.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country. It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION

The invention broadly relates to various formulations containing anthelmintic compounds poorly soluble in aqueous environments to minimise the impact of irritant compounds within the composition; improve the pharmacokinetics of the irritant compounds; and regulate the concentration of irritant compounds at the injection site.

The term 'anthelmintic compound' refers to chemical agents that are destructive to worms and typically used for removing internal parasitic worms in animals including humans.

The term 'complexed' or 'complexing' refers to anthelmintic compound being physically or chemically bound to another compound which results in an alteration in the solubility of the resulting combination.

The term 'emulsion' refers to a preparation of one liquid distributed in small globules throughout the body of a second liquid.

The term 'micro-emulsion' refers to an isotropic, thermodynamically stable mixture of oil, water and surfactants.

The term 'aqueous environment' refers to substantially liquid water environments including, but not limited to, extracellular tissue fluid. According to one aspect of the present invention there is provided an injectable formulation including at least one anthelmintic compound which is complexed with at least one complexing compound wherein the complexing compound is characterised by its ability to alter the solubility and/or dispersion properties of the anthelmintic compound.

Preferably, the complexing compound is a β-cyclodextrin compound of the formula:

or,

or an analogue or derivative thereof. More preferably, the complexing compound is a cyclodextrin compound selected from:

(a) hydroxypropyl β-cyclodextrin compounds of the formula:

where R is CH₂CHOHCH₃ or H; or,

(b) sulphabutyl ether β-cyclodextrin compounds with the formula:

where R is (CH₂)₄SO₃Na or H; or,

(c) α-cyclodextrin with six sugar units in the cyclodextrin ring; or,

(d) γ-cyclodextrin with eight sugar units in the cyclodextrin ring.

Most preferably, the complexing compound is hydroxypropyl-β-cyclodextrin with the formula:

where R is CH₂CHOHCH₃ or H.

In preferred embodiments, the injectable formulation includes approximately 20%w/v hydroxypropyl-β-cyclodextrin.

In one embodiment, the injectable formulation also includes benzyl alcohol.

It is the inventors' experience that the injectable formulation described above releases the anthelmintic compound in a rapid manner once administered to an animal or when placed in an aqueous environment.

According to a further aspect of the present invention, there is provided an injectable formulation including at least one anthelmintic compound which is in the form of a micro- emulsion wherein the anthelmintic compound is characterised by having poor solubility and/or dispersion characteristics in an aqueous environment.

Preferably, the micro-emulsion formulation includes at least one medium chain triglyceride compound and at least one emulsifier. Most preferably, the micro-emulsion formulation includes 75% wt of at least one medium chain triglyceride compound and at least one emulsifier.

According to a further aspect of the present invention there is provided an injectable formulation including at least one anthelmintic compound which is in the form of a water in oil emulsion wherein the anthelmintic compound is characterised by having poor solubility and/or dispersion characteristics in an aqueous environment.

Preferably, the water in oil emulsion formulation includes ethyloleate and at least one emulsifier. Most preferably, the water in oil emulsion formulation includes 42% wt ethyloleate and 3% wt emulsifiers.

The above embodiments should not be seen as limiting, as it should be appreciated by those skilled in the art, that ranges of constituents outside of the above ranges may also be used without departing from the scope of the invention. Further, use of medium chain triglyceride, ethyloleate and emulsifiers should also not be seen as limiting, as it should be appreciated by those skilled in the art, that other pharmaceutically and physiologically acceptable compositions may also be used without departing from the scope of the invention. It is the inventors' experience that the water in oil emulsion described above releases the anthelmintic compound in a slow manner.

In preferred embodiments, the above formulations are characterised by their ability to retain anthelmintic compounds in solution within internal aqueous droplets of the systems and as a result, alter the solubility and/or dispersion properties of the anthelmintic compound. More preferably, the injectable formulations above are particularly advantageous where the anthelmintic compound has a solubility in an aqueous environment of less than 10mg of anthelmintic per ml of aqueous solution.

Expressed another way, the injectable formulations above are particularly advantageous where the anthelmintic compound is characterised by having a bioavailability of less than approximately 50% absorption of the anthelmintic compound in the blood stream.

Preferably, the injectable formulation the aqueous environment that the formulation is administered to is extra-cellular fluid.

Preferably, the anthelmintic compound is a benzimidazole compound of the formula:

In one embodiment, the benzimidazole compound is albendazole:

and combinations, analogues and derivatives thereof.

In another embodiment, the benzimidazole compound is ricobendazole:

and combinations, analogues and derivatives thereof.

In another embodiment, the benzimidazole compound is fenbendazole:

and combinations, analogues and derivatives thereof. In another embodiment, the benzimidazole compound is oxfenbendazole:

and combinations, analogues and derivatives thereof.

In another embodiment, the benzimidazole compound is parbendazole:

and combinations, analogues and derivatives thereof.

Preferably, the formulation is administered by means selected from: intravenous, subcutaneously, intramuscular.

Preferably, the anthelmintic compound is acidified with acid to a pH of 2 or less. Preferably, the acid is hydrochloric acid. According to a further aspect of the present invention there is provided a method of treating a parasite infection in an animal by administration of an injectable formulation substantially as described above.

According to a further aspect of the present invention there is provided a method of increasing the solubility and/or dispersion of an anthelmintic compound in an aqueous environment by complexing the anthelmintic compound with at least one complexing compound. Preferably, the anthelmintic compound is a benzimidazole compound and the complexing agent is a cyclodextrin compound. According to a further aspect of the present invention there is provided a method of increasing the solubility and/or dispersion characteristics of an anthelmintic compound in an aqueous environment by mixing the anthelmintic compound into a micro-emulsion. Preferably, the micro-emulsion includes at least one medium chain triglyceride compound and at least one emulsifier.

According to a further aspect of the present invention there is provided a method of increasing the solubility and/or dispersion characteristics of an anthelmintic compound in an aqueous environment by mixing the anthelmintic compound into a water in oil emulsion. Preferably, the water in oil emulsion includes ethyloleate and at least one emulsifier. According to a further aspect of the present invention there is provided a method of increasing the bioavailability of an anthelmintic compound wherein the anthelmintic compound is characterised by having poor solubility and/or dispersion characteristics in an aqueous environment, via any one of the steps of:

(a) complexing the anthelmintic compound with at least one cyclodextrin compound;

(b) mixing the anthelmintic compound into a water in oil emulsion; or,

(c) mixing the anthelmintic compound into a micro-emulsion. Preferably, the anthelmintic compound above is a benzimidazole compound.

According to a further aspect of the present invention there is provided the use of a formulation substantially as described above in the manufacture of a medicament for the treatment of a parasite infection in an animal.

According to a further aspect of the present invention there is provided the use of a formulation substantially as described above in the manufacture of a medicament to increase the solubility and/or dispersion of an anthelmintic compound wherein the anthelmintic compound is characterised by having poor solubility and/or dispersion characteristics in an aqueous environment.

It should be appreciated by those skilled in the art that in preferred embodiments the formulations, methods and uses described above may substantially address the solubility and/or dispersion and bioavailability problems of prior art formulations. They may also minimise the impact of irritant compounds within the composition; improve the pharmacokinetics of the irritant compounds; and regulate the concentration of irritant compounds at the injection site. A further advantage is that, as the bioavailability is increased, dosages of agent may be decreased. BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which:

Fiαure 1 shows a graph of normal ricobendazole solubility as a function of pH level;

Fiqure 2 shows a graph of ricobendazole solubility as a function of hydroxypropyl-β- cyclodextrin (ΗP-β-CD¹) concentration;

Fiqure 3 shows a graph comparing plasma creatine kinase levels in test sheep A and B tested over time;

Fiαure 4 shows a graph comparing plasma creatine kinase levels in test sheep C, D and E tested over time;

Fiαure 5 shows a skin section of normal tissue;

Fiαure 6 shows a skin section from positive control sheep C;

Fiαure 7 shows the variation in time of ricobendazole concentration in blood plasma of tested sheep;

Fiαure 8 shows the variation in time of albendazole sulfone concentration in blood plasma of tested sheep;

Fiαure g shows a schematic of a sheep back showing the location of injections sites;

Fiαure 10 shows microscopic observations of tissue collected at the injection site. A: perivascular dermatitis; B: steatitis; C: muscle damage; D: dermal necrosis;

Fiαure 11 shows graph illustrating the average skin temperature at the injection sites after subcutaneous injection of two ricobendazole formulations (labelled F for formulations 1 and 3) and vehicles (labelled V for formulations 2 and 4) in sheep. A: Skin temperature at the injection site and reference sites after injection of formulations 1 and 2 (n=6); B: formulations 3 and 4 (n=5); C: changes at injection sites relative to the reference sites. Data presented as mean values with standard error bars;

Figure 12 shows a graph comparing changes in plasma creatine kinase after subcutaneous administration of two ricobendazole formulations (formulations 1 and 3) at 0.1 ml/kg along with the respective vehicle (formulations 2 and 4) at the opposite side of sheep back at the same dose. Data represented as the mean ± standard error; Figure 13 shows graphs comparing the plasma concentration-time profiles of ricobendazole (solid symbols) and albendazole sulfone ABZSO₂ (open symbols) in sheep A, B, D₁ E, H₁ and I after subcutaneous administration of formulations 1 and 3; Figure 14 shows graphs comparing plasma concentration-time profiles of ricobendazole (solid symbols) and albendazole sulfone ABZSO₂ (open symbols) in sheep C, F, G, J₁ and K after subcutaneous administration of formulations 1 and 3; and,

Figure 15 shows a graph illustrating mean plasma concentration-time profiles of ricobendazole (solid symbols) and albendazole sulfone ABZSO₂ (open symbols) after subcutaneous administration in sheep of two ricobendazole containing formulations (outline triangles for formulation 3, n=5; and solid triangles for formulation 1 , n=6). Data represented as the mean ± standard error. BEST MODES FOR CARRYING OUT THE INVENTION

The formulation of the present invention will now be described with reference to a series of three experiments.

Experimental Background

Ricobendazole is a poorly soluble anthelmintic and is used for the purposes of the experiment. This should not be seen as limiting as it should be appreciated by those skilled in the art that the formulation methods may also be applied to other poorly soluble anthelmintic compounds.

Low pH has been the preferred approach to achieve the desired solubility and bioavailability at the target dose. This is because ricobendazole has higher solubility levels in aqueous environments at extremes of pH level (see Figure 1 ).

Existing market products Bayverm PI™ and Sintyotal-R™ use low pH (pH = 1) solution and co-solvents to improve solubility but have been reported as causing site irritation and poor bioavailability (37% to 40%). It is understood by the inventors that this may be caused by local drug precipitation at the injection site. To address the above problems, the inventors have developed formulations using ricobendazole compound to minimise the immediate irritation reactions resulting from existing formulations.

There are various methods to evaluate the irritation of parenteral formulations in the literature. The methods used in this experiment included: • Observation of animal response to the injection;

• Visual observation of the injection sites;

• Palpation of the injection site;

• Skin temperature readings at injection sites;

• Plasma creatine kinase levels (levels can alter due to damage of superficial muscular tissue); and

• Histology examinations of the injection site of the skin.

Experiments Undertaken

Experimentation was split into three key experiments. The first experiment was an in vitro characterisation trial to determine the degree of precipitation exhibited by the proposed formulations.

The second experiment was an in vivo trial using sheep to determine the effectiveness of the proposed formulations.

The third experiment was an additional in vivo trial using sheep to further determine the effectiveness of a complexed anthelmintic formulation.

[AI Experiment 1 - In Vitro Characterisation

Formulation Preparation

The formulations used include:

1. A water in oil emulsion; 2. A micro-emulsion formulation; and,

3. A complexed formulation using hydroxylpropyl-β-cyclodextrin as the complexing agent.

With respect to the complexed formulation, the inventors have found that cyclodextrin improves the solubility of anthelmintics that are poorly soluble in aqueous environments. An example of the degree of solubility change observed is shown in Figure 2.

To characterise the proposed formulations, three formulations were prepared, all of which contained approximately 5%w/v ricobendazole, as follows:

1. Water-in-oil emulsion

Formula for 100 ml 5%w/v ricobendazole water-in-oil emulsion injection Ingredients

Ricobendazole 5 g

2 M hydrochloric acid 15 ml

Water for injection 29.8 ml

Ethyl oleate 42 g

Benzyl alcohol 1 g

Butylated hydroxytoluene (BHT) 0.02 g

Sorbitan monooleate (Span 80) 1.5 g

Polyglycerol polyricinoleate (PGPR) 1.5 g

Method of preparation a. Preparation of aqueous phase

Step 1. Dissolve ricobendazole in 2M HCI by heating up to 50-60 ⁰C;

Step 2. Add water for injection at 60 ⁰C slowly with stirring;

Step 3. Cool down to room temperature and pass the solution through a 0.2 micron filter and fill into vials. b. Preparation of oil phase

Dissolve BHT, PGPR and Span 80 in ethyl oleate by stirring; pass the solution through a 0.2 μm filter and fill into vials (solution may be heated so to pass filter easily). Alternatively ethyl oleate can be sterilized by heating at 150 ⁰C for 1 hour.) c. Emulsion preparation

Prior to use add aqueous phase into the oil phase, emulsifying by hand-shaking till milky emulsion formed (2 minutes). Micro emulsion

Formula for 100 ml 5%w/v ricobendazole micro emulsion injection

Ingredients

Ricobendazole 5 g

2M hydrochloric acid (ml) 15 ml

Water for injection 9.5 ml

Medium chain triglyceride (MCT) 15 g Sorbitan monooleate (Span 80) 15 g

Solutol HS 15 2O g

Labrasol® 20 g Benzyl alcohol 1 g

Method of preparation a. Preparation of aqueous phase

Step 1. Dissolve ricobendazole in 2M HC I by heating up to 50-60 °C;

Step 2. Add water for injection at 50-60 ⁰C slowly with stirring;

Step 3. Cool down to room temperature and pass the solution through a 0.2 micron filter and fill into vials. b. Preparation of oil phase

Mix MCT, benzyl alcohol, Span 80, Solutol and Labrasol by stirring, pass the solution through a 0.2 μ filter and fill into vials (solution may be heated so to pass filter easily). c. Micro emulsion preparation

Prior to use add aqueous phase into the oil phase, emulsifying by hand-shaking till clear solution formed. 3. Cyclodextrin solution

Formula for 100 ml 5%w/v ricobendazole injection

Ingredients

Ricobendazole 5 g

2M hydrochloric acid 15 ml

Hydroxylpropyl-β-cyclodextrin 20 g

Benzyl alcohol 1 ml

Water for injection 100 ml Method of preparation

Step 1. Dissolve ricobendazole in 2M HCI by heating up to 60 °C;

Step 2. Dissolve hydroxylpropyl-β-cyclodextrin in about 50 ml water for injection; Step 3. Add solution from step 2 into solution from step 1 while stirring; Step 4. Add benzyl alcohol, make up volume with water for injection; Step 5. Pass the final solution though a 0.2 μ filter and fill into vials. Control Formulation A control formulation was also prepared being a ricobendazole hydrochloride (HCI) solution (pH<2). As shown in Figure 1 , ricobendazole exhibits increased solubility at pH extremes.

Methodology

The droplet size of the emulsion was determined by laser diffraction.

Drug release from the emulsion and micro-emulsion was conducted in modified Franz diffusion cells and released ricobendazole measured by UV spectroscopy.

Light microscopy was used to examine the interface between the formulations and a diluting phosphate buffer (SPB: pH 7.4, 0.08M). pH changes to SPB buffer on titration with the formulations were measured and drug precipitation from the cyclodextrin formulation was compared with the control solution by light scattering at 550nm.

Characterisation Results

The water in oil emulsion formulation was found to have a mean droplet size of 2.5μm.

Cumulative release of ricobendazole from the emulsion and micro-emulsion was found to be proportional to the square root time (R²>0.99). Light microscopy on dilution with SPB buffer showed formation of areas of water/oil/water emulsion at the interface between the water in oil emulsion and buffer and no evidence of droplet rupture.

For the micro-emulsion an emulsion layer appeared at the interface.

No drug crystallization was seen for the water in oil emulsion or micro-emulsion but was observed with the cyclodextrin formulation.

On addition to SPB buffer (6%v/v), the pH of the buffer decreased by:

• 4 units for the control solution;

• 3.5 units for the micro-emulsion formulation;

• 1.4 units for the cyclodextrin formulation; and, • <0.1 units for the water in oil emulsion formulation.

Light scattering experiments showed that drug precipitation from the cyclodextrin solution was reduced compared with a control solution.

Summary of Characterisation Findings

The characterisation experiment showed that for in vitro experiments, the proposed formulations do not readily precipitate out when compared to a control low pH formulation used in the prior art. As a result they show promise for in vivo applications. fBI Experiment 2 - In Vivo Characterisation

Formulation Preparation The formulations used include:

1. A complexed formulation using hydroxylpropyl-β-cyclodextrin as the complexing agent;

2. A micro-emulsion formulation; and,

3. A water in oil emulsion. To investigate the tissue tolerance and pharmacokinetics of the proposed formulations, three formulations were prepared, all of which contained approximately 5%w/v ricobendazole, as follows:

• A water in oil emulsion was prepared by shaking 55% of an acidic ricobendazole solution with 42% ethyloleate and 3% emulsifiers.

• A micro-emulsion was prepared by mixing 75%v/v medium chain triglyceride with emulsifiers and 25% of an acidic solution of ricobendazole.

• A cyclodextrin formulation was prepared by mixing an acidic solution of ricobendazole with 20%w/v hydroxypropyl-β-cyclodextrin.

These formulations were equivalent to that used in Experiment 1 for the in vitro trial. Control Samples

The irritation and bioavailability of the three ricobendazole formulations were measured and compared against a positive control (a low pH solution (<2) and a negative control (a saline solution). The positive control formulations contained approximately 5%w/v ricobendazole. Vehicle control solutions were: a 0.3M HCI solution (positive control); the micro emulsion, water in oil emulsion; and cyclodextrin solution prepared with normal saline as the aqueous phase (negative control). Animal Selection

Five female sheep weighing 69.1+ 3.4 kg in good health and with no signs of disease were used in the trials.

No study animal had pre-existing visible lesions on the injection site, nor had they received any injections to either side of the back during the previous six weeks.

Animals were housed within a test facility during the study.

The sheep received a tranquiliser by intravenous (IV) injection of Acezine (acepromazine, 0.2 mg/ kg). While under anesthetic, a jugular IV catheter (14 GA, 2 in, or 2.1 mm, 5.1 cm) was inserted and fixed for sampling of blood during the course of the experiment. Formulation Administration

Formulations were administered subcutaneously to each sheep by a veterinarian on one side of the back, each formulation including a ricobendazole dose of approximately 0.1 ml/kg.

Respective vehicle formulations (excluding ricobendazole) of approximately the same volumes were administered at the contralateral side of each sheep.

All injection sites were shaved before administration to allow easy observation of the injection site.

The formulations administered are summarised in Table 1 below. Table 1 - Formulations administered in the Experiment

Tissue Irritation

Visual observation

During the study visual observations were made regarding the reactions of the sheep to each injection along with reaction of the tissue at the injection sites. Visual observations were done specifically to determine any signs of immediate post injection discomfort, any injection site swelling or redness and the reaction of the sheep on palpation of the injection site.

Skin Temperature An infrared non-touching thermometer was used to determine the skin temperature at the injection sites.

To verify tissue inflammatory reactions, measurements were taken of the:

• Maximum skin temperature;

• The time at which the maximum temperature was reached (peak time).

• The duration during which elevated skin temperatures were noted.

• The calculated total area under temperature-time curve above 34°C which was taken as a baseline skin temperature

Creatine kinase (CK) level

To determine if damage had occurred to superficial muscle tissue, blood samples were taken at time intervals of 0, 1 , 2, 4, 6, 12, 18, 24, 30, 36, 48 hours, then taken daily in the first week, and then by jugular vein puncture in the second week of the trial period.

To ensure blood samples remained stable, samples were immediately centrifuged at 3000rpm for 5 minutes as each blood sample was taken and the plasma transferred into Eppendorf tubes and kept at -20⁰C until analysis was completed. Injection Site Histology

After two weeks the injection sites were removed for histological examination by biopsy technique after sheep were given IV injection of Acezine (acepromazine, 0.2 mg/ kg) followed by local injection of 2-3ml 2% lignocaine.

Biopsy samples taken were 12mm in diameter including the epidermis, dermis and subcutis layers. Each biopsy sample specimen was placed in 10% formalin solution until histological examination was completed.

Pharmacokinetics

The pharmacokinetics of ricobendazole and its main metabolite albendazole sulphone were measured using solid phase extraction (SPE) and analysis by high performance liquid chromatography. Solid phase extraction procedure

1. Solid phase extraction (SPE) cartridges (Strata^® C 18-E, 200 mg, 3 ml reservoir, Phenomenex, New Zealand) were conditioned by washing with 3 ml of methanol followed by 3 ml of water. 2. 1 ml aliquots of sheep plasma were spiked with 10μl internal standard (oxfendazole 50 μg/ml) and vortexed for 1 minute.

3. Samples were diluted 1 :1 with water, then loaded onto the conditioned SPE cartridges and washed with 0.5ml 40% methanol followed by 9ml water then dried by vacuum (2OkPa) at ambient temperature for 5 minutes. 4. Compounds to be analysed were eluted with 2 ml methanol.

5. Methanol was evaporated with a stream of nitrogen gas and the dried material was re- dissolved in 200 μl mobile phase.

6. Reconstituted samples were centrifuged and the supernatant was transferred into the glass autosampler vials for HPLC assay. HPLC analysis of ricobendazole and albendazole sulphone

The HPLC system (Shimadzu, Kyoto, Japan) comprised a LC 10-AT quaternary pump equipped with a SiI-IOAD auto-sampler, a SPD-10A variable wavelength detector and CTO- 10ASVP column oven (30⁰C) all controlled by a computer using Class-VP 6.1 software.

Separation was carried out on a 4.6mm x 250mm column connected with a 4.6mm x 30mm pre-column (both C18 Prodigy, 5μ, Phenomenex^®, NZ).

The calibration curve was linear over a concentration range of 10 to 1000ng/ml for ricobendazole (R² > 0.999) and the main metabolite of ricobendazole, albendazole sulfone (R² > 0.999).

Intra-day and inter-day precision and accuracy at the low, medium and high concentration for ricobendazole and albendazole sulphone, are listed in Table 2 below.

The extraction recoveries at these concentrations for the ricobendazole metabolite, albendazole sulfone were all over 96 % and the internal standard was 97.2 ± 4.2%. Table 2. Intra-day and inter-day precision and accuracy of ricobendazole and albendazole sulfone

Nominal cone. Cone, found Precision Accuracy

(mean) (ng/ml) _bdllleoeaono c _izeue_rr (ng/ml) (% CV) _Sf A_b (% of nominal)

Intra-day (n=5)

20 19.7 ± 1.1 5.5 98.3 ± 5.4

400 403.8 ± 9.5 2.4 101.0 ± 2.4

° CO 800 808.6 ± 35.7 4.4 100.5 ± 4.5

Inter-day (n=5) im_¬ 20 20.2 ± 1.2 5.9 101.0 ± 6.0

400 409.4 ± 10.1 2.5 102.4 ± 2.5

800 806.3 ± 17.1 2.1 100.8 ± 2.1

Intra-day (n=5)

20 20.4+ 1.5 7.4 102.0 ± 4.1

400 398.7 + 20.0 5.0 99.7 ± 4.6 800 798.8 ± 46.7 5.9 99.8 ± 4.1 Inter-day (n=5)

20 20.6 + 1.3 6.4 103.2 ± 6.6 400 406.4 ± 9.4 2.3 101.6 ± 2.3

800 801.9 + 22.0 2.7 100.2 ± 2.7

Pharmacokinetic Analysis Drug and metabolite information gathered from the experiments were analysed by software (GraphPad Prism, USA) based on the statistical moment theory.

The linear trapezoidal rule was used to calculate the area under the curve (AUC) and the area under the first moment curve (AUMC). The AUC and AUMC extrapolated beyond the last observed point C _n was obtained from C J λ, and C_n Ik (t_n+1/ λ) respectively, λ is the slope obtained from the regression of natural logarithm of concentration versus time in the terminal phase.

The mean residence time (MRT) was calculated as the ratio of AUMC to AUC.

The maximum concentration C_maX and the time when it occurred T_max were observed data. Relative bioavailability of different formulations compared to the positive control low pH solution was estimated by comparison of AUC for each formulation.

Results

Irritation study

Sign of pain upon injection

Table 3 below records the animal response upon subcutaneous injection.

Table 3. Observed pain of the animals on injection

Sheep C (the positive control) showed obvious signs of pain. A transient pain response was observed for sheep A when the complexed drug was administered.

No other pain responses were observed when remaining formulations and vehicles were administered to sheep B, D and E.

After administration, no animal was observed to exhibit signs of pain or discomfort during the two weeks subsequent to administration. Swelling and Palpation Response

Swelling at the injection site was only observed in sheep C (positive control) for 2 to 6 hours post drug administration. Swelling was most intensive after approximately 2 hours, with maximal edema dimension 6 X 6.3cm. Edema observed on the formulation side was slightly flatter (less inflamed) than the vehicle side. Pain response to palpation of the injection site (see Table 4 below) was observed in sheep C (positive control) at both sites for between 1 Vi hours to 6 hours after injection. Pain response to palpation of the injection site was also observed for Sheep E (micro-emulsion) for approximately 2 hours post injection of the formulation side. No animal exhibited signs of stress in the two weeks following administration of the formulations. Further, no signs of discomfort were observed in the general behavior of the test sheep.

Table 4 - Response of the sheep to Palpitation of the Injection Site

Changes of skin temperature at the injection sites

Results from the skin temperature analysis are shown below in Table 5. Table 5 - Temperature Information Results

Formulation Used Water in oil Low pH solution Micro-emulsion Saline Complexed emulsion (Positive Control) (Negative Control)

Sheep site F V F V V F L R V F

Baseline Temperature

CC) 34 34 34 34 34 34 34 34 34 34

Peak Time (hr) 2 2 2 2 2 2 1 1 2 2

Peak Temp (⁰C) 37.2 37.3 38.3 38.3 37.4 37.5 37.1 37.3 37.6 37.7

Total area above normal (°C hr) 270 263 340 265 265 188 217 190 86 92

Peak Area (°C hr) 123 129 225 164 91 99 25 28 38 48

Time over 34°C (hr) 120 122 125 123 64 64 20 23 21 23

F = Formulation including ricobendazole V = Formulation vehicle excluding ricobendazole L = Left side R= Right side

All test sheep exhibited an increase in skin temperature at the injection sites and reached a temperature maximum after approximately two hours. The only exception to this was for the saline formulation (negative control), which reached maximum temperature after approximately 1 hour.

It is understood by the inventors that manipulation of catheterization may cause an increase in body temperature hence this may explain why the negative control Sheep B also exhibited a temperature rise.

Between Formulation and Vehicle

No significant difference in skin temperature was noted between the two injection sites (formulation and vehicle) for Sheep B (negative control).

The formulation injection site for Sheep C (the positive control) reached a maximum temperature approximately 1 ^CC higher than the vehicle site (hydrochloric acid (HCI) solution).

For the complexed and micro-emulsion formulations, there was a slightly higher skin temperature and peak area observed for the test formulation sites over the vehicle injection sites. The temperature increase for the complexed site occurred at an early stage and returned to normal levels after approximately two days. The micro-emulsion formulation exhibited an approximate temperature maximum three days after administration.

Minimal differences in skin temperature were observed at the injection sites (formulation and vehicle) for the water in oil emulsion formulation. Between Formulations

Table 5 also shows the skin temperature versus time; the peak area over 34 °C (found to be the normal skin temperature); and the length of time that the temperature was elevated above the normal temperature.

As expected, across the five sheep, positive control Sheep C had the highest peak temperature (over 38°C); largest peak area; and longest duration of temperature increase (and hence peak area) lasting approximately 120 hours. The overall reaction of each formulation was found to be rankable in the following order (highest temperature to lowest temperature):

1. Low pH solution - Positive Control (38.3°C) >

2. Complexed (37.6-37.7°C) > 3. Micro-emulsion (37.4-37.5°C) >

4. Water in Oil Emulsion (37.2-37.3°C) > or =

5. Saline - Negative Control (37.1 -37.3°C).

Formulation and vehicle injection sites exhibited minimal increases in peak temperature and peak area (PAUC)₁ with the increase usually lasting for 1 to 2 days (except for the emulsion formulation). Peak area proved to be a more reliable method to indicate the inflammatory response rather than the total area. Inflammation extent caused by formulation and vehicle injections ranked by peak area were found to be in the order (highest to lowest):

1. Low pH solution - positive control (225-164°Chr) >

2. Emulsion (123-129°C hr) > 3. Micro-emulsion (91 -99°C hr) >

4. Complexed (38-48°C hr) >

5. Saline - Negative Control (25-28°C hr).

Plasma Creatine Kinase (CK) Level

Plasma CK levels measured for each sheep during the first 48 hours after injection are shown in Figures 3 and 4.

The results show an increase in CK level for all sheep to differing extents, including the negative control sheep B. Generally, CK levels less than 500i.u. are not considered clinically significant and elevation measured in this study is likely to have resulted from:

• CK rise caused by the manipulation of cannulation - there is a clear relation between the time gap between drug administration (first sampling) and cannulation;

• CK level fluctuated when the animals were able to run freely and needed to be caught for sampling i.e. Sheep C, D and E were held during the first 48 hours and sheep A and B were released on the first day; and/or,

• Assay error. Biopsy Results

No necroses or inflammatory reactions were observed for any test sheep at the biopsy site. Table 6 below shows the results for each formulation and vehicle.

Figures 5 and 6 also show the intersection of the skin at the injection sites including epidermis 10, dermis 11 , panniculus muscle 12, subcutaneous fat 13 or adipose tissue for normal tissue (Figure 5) and the positive control Sheep 3 (Figure 6).

Histological Results

Saline samples for Sheep B (negative control) (formulation and vehicle) showed no remarkable tissue damage. Sheep C skin treated with low pH ricobendazole solution (positive control) showed mild to moderate multifocal coalescing inflammation of adipose tissue (steatites). This can be seen in Figure 6. The inflammation does not extend to the deep margin.

Note that the positive control vehicle (low pH) did not cause tissue damage as severe as the formulation. No remarkable morphology change was noted in the samples for the micro-emulsion formulation (formulation and vehicle).

Minimal haemorrhage in the subcuticular fat region was observed for the water in oil emulsion.

Local haemorrhage in the hypodermis and pannicular fat was observed for the complexed formulation (formulation and vehicle).

Table 6 - Skin Tissue Pathology

Pharmacokinetics

Figures 7 and 8 show the measured blood plasma concentration of ricobendazole over time (Figure 7) and the ricobendazole metabolite, albendazole sulfone, blood plasma concentration over time (Figure 8) in sheep post administration of the ricobendazole containing formulations.

The formulations differed in the pharmacokinetic parameters as shown in Table 7 below. Table 7 - Pharmacokinetic Parameters

Pharmacokinetic

Ricobendazole Parameters Albendazole Sulfone

Sheep C A D E C A D E

Cmax (μg/ml) 1.09 2.97 1.12 1.26 0.67 0.80 0.66 0.54

Tmax (hr) 9 4 24 12.0 9 4 24 12.0 k (hr^"1) 0.074 0.153 0.089 0.076 0.025 0.154 0.107 0.128 h/2 (hr) 9.3 4.5 7.8 9.08 28.2 4.5 6.5 5.43

AUdMμg.day.mr¹) 29.03 56.51 45.57 32.38 17.61 23.29 25.69 17.26

MRT (hr) 17.8 15.3 32.5 23.2 24.7 21.4 37.0 28.2

Relative F (%) 1.00 1.94 1.55 1.11 1.00 1.32 1.45 0.98 Abbreviations Used: k is the elimination rate constant; f _1/2 is the half time;

C_max is the observed maximum plasma concentration; Tm_ax is the time at which C_max occurred;

AUC o-oc is the area under the concentration- time curve extrapolated to infinity;

MRT is the mean residence time; Relative F is the relative bioavailability compared to the low pH solution.

It was found that the low pH solution (positive control) exhibited slow absorption with a plateau concentration of 1 μg/ml between approximately 5 to 18 hours and a mean residual time of 17.8 hours.

The complexed formulation presented a rapid and (in the inventor's experience), a likely complete absorption, which resulted in the highest plasma ricobendazole concentration of 3μg/ml at T_max 4.5 hrs. The AUC is nearly double that observed for the positive control formulation. The water in oil emulsion exhibited a sustained absorption and a longer mean residence time (32.5hr) than the positive control. Ricobendazole concentration remained at a level of approximately 1 μg/ml for approximately 30 hours and the AUC was higher than the positive control.

The micro-emulsion exhibited rapid absorption compared with the positive control but, in the inventor's opinion, absorption may be incomplete. The mean residence time is slightly longer than the positive control solution.

The above results imply that for all three test formulations, the anthelmintic delivery systems used increased the bioavailability of the ricobendazole over the positive control. When ranked in order of bioavailability increase, the order is (highest to lowest, amount / % increase over the positive control): 1. Complexed Formulation (3μg/ml, 94%) >

2. Water in Oil Emulsion (1 μg/ml for approximately 30 hours, 55%) >

3. Micro-emulsion (1 μg/ml and slightly higher MRT over the positive control, 15%) >

4. Positive Control. Experiment 2 Summary

All three formulations containing ricobendazole compound modified with various formulation approaches (complexed, micro-emulsion and water in oil emulsion) were found to improve tissue tolerance over existing formulations e.g. low pH when injected into sheep.

All associated formulation vehicles were well tolerated with no observation of pain upon injection, no change in histology in skin tissue at injection sites and minimal skin temperature rise.

All three formulations were found to increase bioavailability of ricobendazole relative to a positive control (a low pH solution).

The complexed formulation was found to be absorbed rapidly and completely in the sheep bloodstream. The water in oil emulsion was observed to act with a controlled release profile.

It should be appreciated by those skilled in the art that the above experiment shows that a complexed anthelmintic formulation appears to have a high potential to prevent drug precipitation. In the case of the micro-emulsion formulation it is the inventors understanding that, when the formulation is mixed with the physiological fluid e.g. blood, a coarse emulsion forms, generating an emulsion layer that may avoid the burst release of low pH solution formulated in the micro-emulsion. fCl Experiment 3 - In Vivo Characterisation

Formulation Preparation

A complexed anthelmintic formulation ('formulation 1') was produced using the same method as that described in Experiments 1 and 2 above using ricobendazole as the anthelmintic, HCI acid, hydroxylpropyl-β-cyclodextrin as the complexing agent, benzyl alcohol and water.

A cyclodextrin vehicle formulation ('formulation 2') was produced using just cyclodextrin complexing agent and water (20% wt concentration).

A low pH control formulation ('formulation 3') was produced using ricobendazole as the anthelmintic in an HCI acid (pH ~2) solution without complexing agent.

A 'vehicle' formulation ('formulation 4') was produced using only 0.2M HCI acid.

Animals and Experimental Design

12 female adult sheep were used in Experiment 3. At the start of the trial, the sheep weighed 63.7 ± 2.2 kg on average and had no signs of disease. In addition, none of the sheep selected for experiment 3 had pre-existing visible lesions on the injection site, nor had they received any injections to either side of the back during the prior six weeks. Sheep were identified by ear tag and housed in a controlled test facility.

Sheep were randomly divided into two treatment groups (n = 6), being a control group given formulations 3 and 4 and a test formulation group given formulations 1 and 2. Tests were conducted in three phases as shown below in Table 8 below.

Table 8: Experimental design for irritation and pharmacokinetic studies of ricobendazole formulations and vehicles after subcutaneous administration in sheep (Experiment 3)

2

B 388 62 Formulation Formulation 2 Formulation 1 1 & 2

390 64

C Formulation

391 61 Formulation 3 Formulation 4 3 & 4

D Formulation

396 63 Formulation 2 Formulation 1

1 & 2

E 397 Formulation

61 Formulation 1 Formulation 2

1 & 2

F Formulation

392 65 Formulation 3 Formulation 4

3 & 4

G Formulation

395 62 Formulation 4 Formulation 3

3 & 4

H Formulation

398 67 Formulation 1 Formulation 2

1 & 2

I 394 67 Formulation Formulation 2 Formulation 1

1 & 2

J Formulation

399 64 Formulation 4 Formulation 3

3 & 4

K 393 66 Formulation Formulation 3 Formulation 4

3 & 4

*** Sheep hurt after cannulation and caused bleeding from catheter overnight. The sheep was withdrawn from the trial.

Experimental protocol Jugular Vein Cannulation

The day before injection, two areas (approximately 20*15cm) on each side of the back was shaved and marked using a permanent marker to identify the injection sites. A 14 gauge 5.1 cm long catheter was implanted in the jugular vein under ketamine/xylazine (80/8 mg/kg) anaesthesia for blood sampling. Animals were allowed to recover from surgery for 1 day before receiving test injections.

Drug administration

Injection of the formulations were made subcutaneously at a dose of 0.1 ml/kg (equivalent to 5 mg/kg ricobendazole base) into the marked injection sites using a 19 gauge needle, and the corresponding vehicle was injected on the contralateral side of the sheep back at the same dose rate of ricobendazole. Blood sampling

6 ml blood samples were withdrawn from the jugular catheter before injection and at 0.25, 0.5, 1 , 1.5, 2, 3, 4, 5, 6, 9, 12, 18, 24, 30, 36, 42 and 48 hours post dosing. This was completed to test drug and main metabolite levels for pharmacokinetic studies. 2 ml of additional blood was collected for measurement of creatine kinase concentrations at 0 (before drug administration), 2, 4, 6, 9, 12, 18, 24 hours time points from formulation administration.

Blood samples obtained were stored in heparinized tubes; plasma was separated by centrifuged at 3000 rpm for 10 minutes and stored at -20 ⁰C until analysed for drug concentration with a validated high performance liquid chromatography (HPLC) method. 0.5 ml plasma samples were stored in different tubes for analysis for creatine kinase concentration using standard analysis methods. Assay was performed within 2 days of collecting samples.

Injection site reactions - Observation of injection site reactions (a) Signs of pain on injection were recorded using the following scale:

0 = No signs of pain observed

1 = Pain signs not significant

2 = Pain signs at the end of injection, movement continued for <10 seconds.

3 = Showing pain immediately upon the injection and lasted for half minute. 4 = Showing severe pain immediately upon the injection and struggling.

(b) Injection sites were observed including appearance of swelling and the size of edema;

(c) Pain on palpation at the injection sites over the 48 hours and daily in the week.

(d) Punch biopsy and tissue histological examinations.

(e) Skin temperature change Skin temperature at the injection site was measured using an infrared non-touch thermometer before and at 0.5, 1 , 2, 4, 6, 9, 12, 18, 24, 30, 36 and 48 hours post injection. Two other sites were taken as reference points as shown on Figure 9 with the aim to eliminate the circadian changes in body temperature of the animals. Points on the skin for measurement of skin temperature are indicated by the letter X in Figure 9. Skin temperature at the injection site was measured in Fahrenheit ("F). Skin temperature changes at the injection site (T_chan_ge) were calculated as the difference relative to the two reference sites (T_ref1, T_ref2):

^•Vise ⁼ Tjψcjonsite ~ ( Vl ⁺ Λ-e/2 ) ' 2 ~ <7

where σ is the initial difference before treatment. All results were expressed as mean ± standard error (S. E.).

Punch Biopsy and Tissue Histology

After seven days, sheep were anaesthetised and skin from the sheep at the injection sites was removed by punch biopsy (12mm diameter) and placed in 10% formalin. Once haemostasis at the biopsy site was assured, the wound was closed with 2/0 sutures and the animal allowed to recover. Histopathological examination under light microscopy was performed by an experienced Pathologist, who was unaware of the given treatment, at an independent laboratory. Microscopic changes in tissue were quantified by subjective evaluation of the following criteria on the scale of 0 (none) to 4 (greatest):

1. Perivascular dermatitis (inflammation in the superficial dermis).

2. Necrosis: irreversible cells death in tissue - muscle, subcutis, dermis - the most severe change.

3. Steatitis: inflammation of the fat.

4. Haemorrhage: the presence of blood in interstitial tissue outside the blood vessels resulted from escape of erythrocytes across intact vessels or from vascular rupture, often this is simply related to the collection of the biopsy, it can also be associated with the inflammation.

Pharmacokinetic study

Quantification of Ricobendazole and Related Metabolites

HPLC methods for quantification of ricobendazole and its main metabolites were completed as per known methods.

Pharmacokinetic Data Analysis

Pharmacokinetic parameters were estimated using non-compartmental analysis based on statistical moment theory. The maximum concentration C_max and the time when it occurred, Tmax. were observed directly. The linear trapezoidal rule was used to calculate area under the curve (AUC) and the area under the first moment curve (AUMC). AUC and AUMC extrapolated beyond the last data point C_n to infinite time were obtained from C_nA and C_n IK (tn+1/λ) respectively, λ is the slope obtained from the regression of natural log concentration versus time in the terminal phase, over the last three data points. Elimination half-life (t_1/2) was calculated as t_{U 2} = 0.693/ /I . Mean residence time (MRT) was calculated as the ratio of AUMC to AUC. Relative bioavailability (F) of CD formulation was obtained by comparing AUC with that of the control formulation:

π,_{n / λ} ^Λ A T^KjTC ^ (Formulation) t (Vo) =

AUC₁ (Control)

Statistical analysis

Levels of statistical significance (PO.05) were assessed using the ANOVA program to compare the data sets.

Experiment 3 Results and Discussion Irritation Studies - Visual Observations

Table 9 below shows the visual observations made for sheep responses to subcutaneous injection of the test formulations and vehicles during 7 days of the experiment.

Table 9: Legend: 1: Formulation 1; 2: Formulation 2; 3: Formulation 3; 4: Formulation 4; M: muscle; D: dermis; S: subcutis).

Treatment Histology

Pain on Swelling/

Phase Sheep injection edema size Perivascular Hemor¬

Necrosis Steatitis dermatitis rhage

1 A 389 1 3 1 2cm (1-6h) 1 1 M 3 3

1 B 388 1 3 0 - 0 0 2 2

2 D 396 1 3 2 - 0 3D 0 1

2 E 397 1 3 3 ± (2-3h) 0 3M 1 2

3 H 398 1 3 0 - 0 0 0 2

3 I 394 1 3 1 0 0 0 1

1 A 389 1 4 0 - 0 0 3 2

1 B 388 1 4 0 - 0 0 1 1

2 D 396 1 4 0 - 0 0 0 1

2 E 397 1 4 0 - 1 0 2 2

3 H 398 1 4 0 - 0 0 1 1

3 I 394 1 4 0 - 1 0 0 1

1 C 391 2 3 0 - 1 0 2 1

2 F 392 2 3 0 - 1 0 1 1

2 G 395 2 3 1 2cm (1-6h) 2 0 1 0

3 J 399 2 3 2 3cm(1-9h) 2 1M 2 2

3 K 393 2 3 0 - 0 0 0 1

1 C 391 2 4 0 - 1 1S 2 1

2 F 392 2 4 0 - 1 0 2 1

2 G 395 2 4 1 2cm (1-4h) 2 0 1 1

3 J 399 2 4 0 - 2 0 2 1

3 K 393 2 4 0 2 0 1 1

No presence of redness at the injection site was observed in sheep even if edema was observed. Further, no sheep showed visible pain signs to palpation on the injection site during the 7 days of the experiment.

Two of the six sheep exhibited a mild pain response after receiving formulation 1 , and one sheep had slight swelling at the injection site. This swelling reduced and disappeared in a few hours. Injection of formulation 2 (complexing agent alone) did not cause pain on injection or a significant inflammation response. Both formulation 3 (low pH and ricobendazole (¹RBZ')) and formulation 4 (low pH only) caused no or slight pain on injection in sheep.

Very slight swelling in the skin was found in two sheep shortly after injection of formulation 4 and one for the formulation 3.

Formulation 2 showed great tissue tolerance in sheep without evidence causing any pain on injection or significant inflammatory responses at the injection site. Unexpectedly, the local anaesthetic effect of benzyl alcohol in formulation 1 was not obvious with the presence of hydroxylpropyl-β-cyclodextrin. This may be due to benzyl alcohol, acting as a hydrophobic compound, and forming an inclusion complex with cyclodextrin and reducing the local anaesthetic effect. In addition, addition of hydroxylpropyl-β-cyclodextrin to formulations 1 and 2 increases the formulation osmolality and may also contribute to pain on injection. Sodium chloride equivalent (E) of ricobendazole low pH solution (formulation 3) is 1.0 (tonicity equal to a 1.0% NaCI solution), nearly isotonic, whereas formulation 1 is equivalent to a 1.5% NaCI solution, which is slightly hypertonic.

Histopathology of Injection Site Microscopic histology of the tissue of injection sites using the scoring system are listed in Table 2 above. Sheep biopsies treated with the formulation 3 and formulation 4 caused minimal or mild perivascular dermatitis and steatitis as shown in Figures 10A and 10B.

Minimal necrosis in muscle tissue was observed in the tissue treated with formulations 1 and 3 and in one of five animals (Figure 10C and 10D). Biopsies treated with formulation 1 showed no perivascular dermatitis with one exception (minimal changes), and three of the six animals showed signs of steatitis in the biopsies. Minimal or mild necrosis was seen in three sheep in different tissue, but it did not extend to other tissues.

Contrary to formulation 1 , no necrosis or morphological changes occurred in the tissue samples treated with formulation 2 (complexing agent alone). Only a few samples showed minimal steatistis and local haemorrhage.

Comparing the histology of the inflammation responses described above, ANOVA shows that there was no statistically significant difference between the formulations 1 and 3 (P>0.05) except for perivascular dermatitis (PO.05) (see Table 10 below). Table 10: Two-way analysis of variation (ANOVA) for histological changes in biopsies

Perivascular

Factor dermatitis Necrosis Steatitis Hemorrhage

Formulation 1 vs 2 <0.05^* >0.05 >0.05 >0.05 Formulation 3 vs 4 >0.05 >0.05 >0.05 >0.05

Histological measurement by light microscopy was completed at 7 days after formulation (1 to 4) administration. The timing of measurement was done to accurately reflect acute inflammation responses rather than the chronic reactions, which may be caused by ricobendazole precipitate. Skin temperature at the injection sites

Local skin temperature at the injection sites and reference sites over a time period of 48 hours after subcutaneous injection of formulations 1 and 3 and vehicles (formulations 2 and 4) varied from 91-96T (see figures 11A and 11B). The temperature rise at the injection sites compared to the two reference sites is shown in Figure 11C.

Two minimum temperature measurements were observed at the 12 and 30 hour points over the 48 hours after injection between approximately 12am and 5am at night. A transient hypothermia 30 minutes after injection was observed in most of the animals, then temperature increased and became stable at a higher level. Two maximum elevation temperature TeITIm_3x were observed, the time these occurred (T_max) and the area under the temperature elevation-time curves (AUC) are compared in Table 11 below.

Table 11: Local skin temperature parameters in sheep after subcutaneous administration of formulation 1 (n=6) and formulation 3 (n=5) at a dose of 0.1 ml/kg (5 mg/kg ricobendazole) and the respective vehicles, formulations 2 and 4, at same dose (formulation and corresponding vehicle were injected on each side of sheep back).

Parameters Formulation 3 Formulation 4 Formulation 1 Formulation 2

Mean SE Mean SE Mean SE Mean SE

AUC (hr-°F) 68.9 10.8 78.1 23.3 46.0 16.2 35.2 9.4

Tmaxi (hr) 9.4 3.8 22.8 4.4 8.6 4.5 6.7 4.7

Tem _{max 1} (⁰F) 2.5 0.4 3.1 0.8 2.3 0.6 2.4 0.6

(^βC) 1.7 0.3 2.1 0.5 1.5 0.4 1.6 0.4

Tmax 2 (hr) 42 0.0 42.0 3.8 33.6 5.4 24.0 1.7

Tem _{max 2} (°F) 2.9 0.3 2.1 0.5 1.4 0.3 1.7 0.3

(⁰C) 1.9 0.2 1.4 0.3 0.9 0.2 1.1 0.2

Three types of temperature-time curves were observed at the injection sites after injection of the formulations 1 and 3 and their vehicles, formulations 2 and 4 (Figure 11 A, 11 B, 11 C). This may suggest different mechanisms of injection site reactions including:

1 ) The temperature remained at a low, stable and close to normal level for cyclodextrin complexing agent alone (formulation 2) indicating minimal inflammation response in skin temperature.

2) A second hypothermia phenomenon was observed in both formulations before the hyperthermia at about 6-9 hours and 12 hours after administration of formulation 1 and formulation 3 respectively. It is thought this may be the result of a transient cut¬ off of blood vessels due the drug deposition. Loss of function of temperature regulation at the injection site due to the tissue damage may also be the cause or part of the cause. 3) Formulation 4 (HCI alone) caused a constant hyperthermia after administration with the maximum 2.1⁰C maintained at 12-18 hours even at the night time and then returned to nearly normal after 48 hours. It has been reported that decrease in pH can stimulate nociceptors and induce pain and neurogenic inflammation. Both the area under the temperature elevation-time curves (AUC) and maximum increase in skin temperature (Tem_max) are ranked in the same order being:

Formulation 4 >

Formulation 3 >

Formulation 1 > Formulation 2

Creatine Kinase Level

Average concentration of plasma creatine kinase (CK) of sheep after receiving subcutaneous injection of formulations 1 and 3 along with the respective vehicles (formulations 2 and 4) are presented in Figure 12. Both sets of results were found to be associated with high inter-individual variability. A higher level of CK at the initial time (before injection) for both group and then a slight increase at 9 hours and then returned to for both formulations were observed. However, CK concentrations generally remained at the normal level in both groups.

CK is intracellular enzyme and primarily located in skeletal brain and heart muscle. Damage to these tissues caused the release of CK in to blood. After intramuscular injection of an irritating substance it was found the C_max of CK appeared at 6 hours in cattle. In this study CK level in sheep after receiving a dose of formulations 1 and 3 (equivalent to 0.1 ml/kg) and the same dose of vehicle (formulations 2 and 4) simultaneously did not show significant increase in CK concentration over the 24 hours in any of the individual animal. Pharmacokinetic study

Following subcutaneous injection of formulation 1 and the formulation 3 at a dose of 5 mg/kg (ricobendazole) into sheep, the concentration-time curves of ricobendazole and its main metabolite, albendazole sulfone (ABZSO₂), were determined as shown in Figures 13 and 14 respectively. Figure 15 shows the mean values for the two formulations (±standard error). Non-compartmental pharmacokinetic parameters of ricobendazole are listed in Table 12 below.

Table 12: Non-compartmental pharmacokinetic parameters of ricobendazole after subcutaneous administration of formulation 1 (n=6) and formulation 3 (n=5) in sheep at a dose of 5 mg/kg (ricobendazole base). a) Individual data of formulation 1(n=6):

Kinetic variables A B D E H I Mean s.d. CV(%)

^* max

(μg/ml) 2.80 3.74 2.31 2.94 3.90 1.95 2.9 0.77 26.2

Tmax (h) 5 6 5 5 5 4 5.0 0.63 12.6 k(Mb) 0.19 0.18 0.19 0.21 0.072 0.08 0.2 0.06 39.2

R-sq 3.66 3.93 3.62 3.38 9.61 8.58 5.5 2.84 52.0 till (h) 0.86 0.92 0.99 0.86 0.95 0.91 0.9 0.05 5.6

AUCcMSh (μg.h/ml) 43.82 62.09 42.3 55.57 76.73 40.7 53.5 14 26.4 AUC₀-- (μg.h/ml ) 43.91 62.37 42.4 55.65 80.75 42.1 54.5 15.3 28.0

AUMC 662 1060 566.3 762.4 1216 593.2 810.2 267 33.1

MRT (h) 15.09 17.00 13.4 13.70 15.07 14.1 14.7 1.3 9.0

b) Individual data of formulation 3 (n=5):

Kinetic variables C G F J K Mean s.d. CV (%)

Cmax (μg/ml) 1.58 1.045 1.26 1.2 1.72 1.34 0.31 23.3

Tmax (h) 4 12 12 12 6 9.2 3.9 42.4

/c (1/h) 0.07 0.11 0.05 0.08 0.16 0.09 0.04 44.3

R-sq 0.91 0.97 0.95 0.92 0.94 0.94 0.02 2.6

Ua (h) 10.17 6.09 12.97 8.85 4.22 8.46 3.43 40.5

AUC(_M8 (μg.h/ml) 44.99 22.48 32.43 37.2 37.97 35.01 8.32 23.8

AUC₀- (μg.h/ml) 48.55 22.93 35.60 38.21 38.21 36.70 9.20 25.0

AUMC 892.3 380 632.8 735.1 546.2 637.3 193.1 30.3

MRT (h) 18.38 16.54 17.78 18.63 14.29 17.12 1.78 10.4

T_max, C_max, and AUC₀-- for formulation 3 were 9.2 ± 3.9 hours, 1.34 ± 0.31 μg/ml, and 36.7 ± 9.2 μg.hr/ml, respectively; while the corresponding data for formulation 1 were 5.0 ± 0.6 hours, 2.9 ± 0.8 μg/ml, and 53.5 ± 14.1 μg.hr/ml. These results strongly suggest that cyclodextrin complexing agent improved both ricobendazole absorption rate and extent significantly (P<0.05). It should be noted that the pharmacokinetic results obtained in this experiment such as AUC and fi_/2 are comparable with results reported in the literature. For example, one reference reports that after intravenous administration of ricobendazole at the same dose in sheep (n=6) AUC was 52.0 ± 8.0 μg.hr/ml, which was nearly identical with the AUC of the test formulation used in this experiment. Thus, drug absorption of formulation 1 may be assumed to be complete. It is understood that this is because a higher portion of free drug dose is available for absorption in formulation 1 compared with formulation 3 which has no complexing agent.

The loss of plasma ricobendazole appears to follow first-order kinetics. The slope in the elimination phase appeared to have a significant difference in formulations 1 and 3. Formulation 1 appeared to have a shorter half-life (5.5 ± 2.8 hours) and MRT (14.7 ± 1.3 hours) than formulation 3 (8.5 ± 3.4 hours and 17.1 ± 1.8 hours respectively). It is understood that this is primarily due to complete absorption of formulation 1 and by contrast, the slow release of drug precipitate at the injection site from formulation 3.

Table 13 below shows the statistical results indicating that incorporation of complexing agent in formulation 1 significantly improves drug absorption kinetic parameters.

Table 13: Comparison of pharmacokinetic parameters of RBZ containing formulations 1 and 3 after subcutaneous injection in sheep (*P<0.05, significant difference).

Kinetics parameters P-value Group ratio (CD solution /control )

Cmax (μg/ml) 0.002* (2.94±0.77/1.34±0.31) 2.2

T_maχ (h) 0.028^* (5.0±0.63/9.2±3.9) 0.5 k(Mb) 0.103 (0.15±0.06/0.10 ±0.04) 1.6

Ua (h) 0.147 (5.47±2.84/8.46 ±3.43) 0.6

AUCtMβhfμg.h/ml) 0.030^* (53.5±14/35.0±8.3) 1.5

AUC₀.-(μg.hr/ml ) 0.049* (54.5±15.3/36.7±9.2) 1.5

MRT (h) 0.030^* (14.7±1.3/17.1±1.8) 0.8

Experiment 3 Summary Local irritation and bioavailability of a 5% ricobendazole injectable formulation at low pH with the presence of 20% cyclodextrin formulation 1 , compared with the low pH solution alone (formulation 3) was investigated in sheep after subcutaneous administration at a dose of 5 mg/kg (0.1 ml/kg). The respective vehicles (20% cyclodextrin (formulation 2) and 0.2 M hydrochloric acid (formulation 4)) were also tested in the same animals for irritation assessment at the same dose.

It was found that formulation 1 containing cyclodextrin complex and ricobendazole significantly increased drug absorption. Compared with formulation 3, AUC and C_max measurements were 1.6 and 2.2 times higher for formulation 1 containing cyclodextrin. Therefore, it is the inventors' understanding that formulation strategy to minimise precipitation is of importance for improvement of bioavailability and tissue tolerance (long term). This is partially because of inclusion formation with a complexing agent, and because of the inhibitive effect of complexing agent on anthelmintic precipitation at the injection site. Thus, using a combined approach of pH adjustment and complexation, drug absorption enhancement of at least 1.6 orders of magnitude is possible. One advantage of this is that finding is that a smaller dose could be administered to achieve the same efficacy. This in turn could significantly reduce the degree of local irritation.

The complexing agent used showed good tissue tolerance in sheep without evidence of causing any pain on injection or significant inflammatory responses at the injection site. This suggests complexing agents are a good injectable formulation carrier for poorly soluble drugs. Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

Claims

WHAT WE CLAIM IS:

An injectable formulation including at least one anthelmintic compound which is complexed with at least one complexing compound wherein the complexing compound is characterised by its ability to alter the solubility and/or dispersion properties of the anthelmintic compound.

The injectable formulation as claimed in claim 1 wherein the complexing compound is a β-cyclodextrin compound of the formula:

or,

or an analogue or derivative thereof.

3. The injectable formulation as claimed in claim 1 or claim 2 wherein the complexing compound is a cyclodextrin compound selected from:

(a) hydroxypropyl β-cyclodextrin compounds of the formula:

where R is CH₂CHOHCH₃ or H; or,

(b) sulphabutyl ether β-cyclodextrin compounds with the formula:

where R is (CH₂)₄SO₃Na or H; or,

(c) α-cyclodextrin with six sugar units in the cyclodextrin ring; or,

(d) γ-cyclodextrin with eight sugar units in the cyclodextrin ring.

4. The injectable formulation as claimed in claim 1 wherein the complexing compound is hydroxypropyl-β-cyclodextrin with the formula:

where R is CH₂CHOHCH₃ or H.

5. The injectable formulation as claimed in claim 1 wherein the complexed formulation includes approximately 20%w/v hydroxypropyl-β-cyclodextrin.

6. The injectable formulation as claimed in any one of claims 1 to 5 wherein the formulation also includes benzyl alcohol.

7. The injectable formulation as claimed in any one of claims 1 to 6 wherein the formulation releases the anthelmintic compound in a rapid manner.

8. An injectable formulation including at least one anthelmintic compound which is in the form of a micro-emulsion wherein the anthelmintic compound is characterised by having poor solubility and/or dispersion characteristics in an aqueous environment.

9. The injectable formulation as claimed in claim 8 wherein the micro-emulsion formulation includes at least one medium chain triglyceride compound and at least one emulsifier.

10. The injectable formulation as claimed in claim 8 or claim 9 wherein the micro-emulsion formulation includes 75% wt of at least one medium chain triglyceride compound and at least one emulsifier.

11. An injectable formulation including at least one anthelmintic compound which is in the form of a water in oil emulsion wherein the anthelmintic compound is characterised by having poor solubility and/or dispersion characteristics in an aqueous environment.

12. The injectable formulation as claimed in claim 11 wherein the water in oil emulsion formulation includes ethyloleate and at least one emulsifier.

13. The injectable formulation as claimed in either claim 11 or claim 12 wherein the water in oil emulsion formulation includes 42% wt ethyloleate and 3% wt emulsifiers.

14. The injectable formulation as claimed in any one of claims 11 to 13 wherein the emulsion releases the anthelmintic compound in a slow manner.

15. The injectable formulation as claimed in any one of claims 8 to 14 wherein the formulations are characterised by their ability to retain anthelmintic compounds in solution within internal aqueous droplets of the systems and as a result, alter the solubility and/or dispersion properties of the anthelmintic compound.

16. The injectable formulation as claimed in any one of the above claims wherein the anthelmintic compound has a solubility in an aqueous environment of less than 10mg of anthelmintic per ml of aqueous solution.

17. The injectable formulation as claimed in any one of the above claims wherein the anthelmintic compound is characterised by having a bioavailability of less than approximately 50% absorption of the anthelmintic compound in the blood stream.

18. The injectable formulation as claimed in any one of the above claims wherein the aqueous environment is extra-cellular fluid.

19. The injectable formulation as claimed in any one of the above claims wherein the anthelmintic is a benzimidazole compound of the formula:

20. The injectable formulation as claimed in claim 18 wherein the benzimidazole compound is albendazole:

and combinations, analogues and derivatives thereof.

21. The injectable formulation as claimed in claim 18 wherein the benzimidazole compound is ricobendazole:

and combinations, analogues and derivatives thereof. 22. The injectable formulation as claimed in claim 18 wherein the benzimidazole compound is fenbendazole:

and combinations, analogues and derivatives thereof.

23. The injectable formulation as claimed in claim 18 wherein the benzimidazole compound is oxfenbendazole:

and combinations, analogues and derivatives thereof.

24. The injectable formulation as claimed in claim 18 wherein the benzimidazole compound is parbendazole:

and combinations, analogues and derivatives thereof.

25. The injectable formulation as claimed in any one of the above claims wherein the formulation is administered by means selected from: intravenous, subcutaneously, intramuscular.

26. The injectable formulation as claimed in any one of the above claims wherein the anthelmintic compound is acidified with acid to a pH of 2 or less.

27. The injectable formulation as claimed in claim 26 wherein the acid is hydrochloric acid.

28. A method of treating a parasite infection in an animal by administration of an injectable formulation as claimed in any of claims 1 to 27.

29. A method of increasing the solubility and/or dispersion of an anthelmintic compound in an aqueous environment by complexing the anthelmintic compound with at least one complexing compound.

30. The method of claim 29 wherein the anthelmintic compound is a benzimidazole compound.

31. The method of claim 29 or claim 30 wherein the complexing agent is a cyclodextrin compound.

32. A method of increasing the solubility and/or dispersion characteristics of an anthelmintic compound in an aqueous environment by mixing the anthelmintic compound into a micro-emulsion.

33. The method of claim 32 wherein the micro-emulsion includes at least one medium chain triglyceride compound and at least one emulsifier.

34. A method of increasing the solubility and/or dispersion characteristics of an anthelmintic compound in an aqueous environment by mixing the anthelmintic compound into a water in oil emulsion.

35. The method of claim 34 wherein the water in oil emulsion includes ethyloleate and at least one emulsifier.

36. A method of increasing the bioavailability of an anthelmintic compound wherein the anthelmintic compound is characterised by having poor solubility and/or dispersion characteristics in an aqueous environment, via any one of the steps of:

(a) complexing the anthelmintic compound with at least one cyclodextrin compound;

(b) mixing the anthelmintic compound into a water in oil emulsion; or,

(c) mixing the anthelmintic compound into a micro-emulsion.

37. The method of claim 36 wherein the anthelmintic compound is a benzimidazole compound.

38. Use of a formulation as claimed in any of claims 1 to 27 in the manufacture of a medicament for the treatment of a parasite infection in an animal.

39. Use of a formulation as claimed in any of claims 1 to 27 in the manufacture of a medicament to increase the solubility and/or dispersion of an anthelmintic compound wherein the anthelmintic compound is characterised by having poor solubility and/or dispersion characteristics in an aqueous environment.