OA19425A - Veterinary compositions and the uses thereof for preventing and/or treating parasitic infections in non-human mammals. - Google Patents

Abstract

The present invention relates to a veterinary composition comprising diminazene and levamisole or salts thereof, wherein the weight ratio of diminazene or its salts to levamisole or its salts ranges from 0.50 to 2.00. The present invention also relates to such veterinary composition for use for preventing and/or treating a parasitic infestation in a non-human mammal, preferably a bovine. <img file="OA19425A_A0001.tif"/>

Description

The présent invention herein provides a veterinary composition comprising diminazene and levamisole or salts thereof, wherein the weight ratio of diminazene or its salts to levamisole or its salts ranges from 0.10 to 5.00, preferably from 0.50 to 3.00, more preferably from 0.50 to 2.00. In a preferred embodiment, the weight ratio of diminazene or its salts to levamisole or its salts ranges from 0.50 to 1.00, preferably from 0.60 to 0.80, more preferably about 0.70.

As disclosed herein, weight ratios are calculated on the basis of the présent diminazene or its salts, for instance diminazene diaceturate 4H₂O sait.

The présent invention also provides a veterinary composition comprising:

- from 5 to 50%, preferably from 10 to 30%, more preferably from 15 to 25%, by weight of diminazene or its salts; and

- from 10 to 50%, preferably from 20 to 40%, more preferably from 25 to 40%, by weight of levamisole or its salts;

relative to the total weight of the composition.

Diminazene (also called 4-[2-(4-carbamimidoylphenyl)iminohydrazinyl] benzenecarboximidamide), is a di-amidine dérivative having the following formula:

H /N. /N.

F y n γη ^hnaAaA \Az^nh2 nh₂ nh

Diminazene is currently used as anti-infective drug and is effective against protozoa such as Babesia, Trypanosoma, and Cytauxzoon.

Levamisole (also called (S)-6-phenyl-2,3,5,6-tetrahydroimidazo[2,l-h][l,3]thiazole) has the following formula:

OCD and was discovered in 1966 by Janssen Pharmaceutica. Levamisole is a drug used to treat parasitic worm infestations, particularly as a dewormer in veterinary medicine. However and as reported in the introduction part, levamisole is known to hâve deleterious or négative effect for treating Trypanosoma infestations.

Within the context of the invention, the salts of the active compounds of the combination of the invention or the compositions comprising such combination correspond to its pharmaceutically acceptable salts. A pharmaceutically acceptable sait includes inorganic as well as organic acids salts. Représentative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric and the like. For instance, a preferred sait of levamisole is levamisole hydrochloride. Représentative examples of suitable organic acids include formic, acetic, trichloroacetic, propionic, benzoic, cinnamic, fumaric, maleic, methanesulfonic and the like. For instance, a preferred sait of diminazene is diminazene diaceturate.

As used herein, the term “about” will be understood by a person of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 20 %, preferably 10 % of the particular tenu.

In a preferred embodiment, the veterinary composition further comprises phenazone. Phenazone (also called l,2-dihydro-l,5-dimethyl-2-phenyl-3/f-pyrazol-3-one) of the following formula:

is an analgésie, a non-steroidal anti-inflammatory drug and an antipyretic, currently used to relieve pain and fever.

In this preferred embodiment, phenazone stabilizes the compositions of the invention. Indeed, the inventors hâve demonstrated that compositions comprising a weight ratio phenazone/diminazene or its salts superior to 1.6 avoid any précipitation and/or allow to maintain the solution as a clear yellow solution after 24 hours storage at room température.

In a more preferred embodiment, the veterinary composition further comprises phenazone, in which the weight ratio of phenazone to diminazene or its salts is greater than 1.6, preferably between 1.6 and 4.8, more preferably between 1.6 and 2.0, even more preferably between 1.7 and 1.9.

The veterinary compositions of the invention optionally comprises an additional active agent such as an antibiotic, an anti-inflammatory agent, an anthelmintic, an endectocide, a minerai, or a vitamin. In a preferred embodiment, the additional active agent is a vitamin, preferably vitamin B1 to B12, more preferably vitamin B 12a (cyanocobalamin) or vitamin B 12b (hydroxocobalamin).

The veterinary composition of the invention may also comprise acceptable excipients or carriers such as hydrophilic solvents. Such solvents are semi-polar to polar solvents and are typically used in granulation processes. For instance, hydrophilic solvents include without limitation water, alcohols such as methanol, éthanol, propanol, butanol, and glycerol, and ethers such as diethyl ether, polyethylene glycol, propylene glycol, and diethylene glycol monoethyl ether. In a preferred embodiment, the veterinary composition of the invention comprises éthanol.

In a particular embodiment, the composition as disclosed herein is a powder. This powder can be prepared according to a process comprising the steps of mixing solid active ingrédients to form a blend which is then granulated by continuously adding a hydrophilic solvent such as éthanol, and finally dried to form the powder.

A preferred composition of the invention is thus a veterinary composition, preferably in a powder form, comprising:

- from 15 to 25%, preferably from 20 to 25% by weight of diminazene or its salts;

- from 25 to 40%, preferably from 30 to 40% by weight of levamisole or its salts;

- from 30 to 50%, preferably from 35 to 45%, more preferably about 40% by weight of phenazone;

relative to the total weight of the composition.

In a further preferred embodiment, the veterinary composition, preferably in a powder form, comprises:

between 1.10 and 1.20 g of diminazene or its salts, preferably about 1.16 g of diminazene diaceturate sait, between 1.50 and 2.00 g, levamisole or its salts, preferably about 1.75 g of levamisole hydrochloride sait, and between 1.90 and 2.30 g, preferably about 1.95 g of phenazone.

In the veterinary compositions as disclosed herein, the ingrédients as above detailed are présent in the same composition.

Application

The présent invention also provides a veterinary composition as disclosed herein for use for preventing and/or treating a parasitic infestation in a non-human mammal.

Another object of the invention is a use of a veterinary composition as disclosed herein for the manufacture of a drug for preventing and/or treating a parasitic infestation in a non-human mammal.

A further objet of the invention is a method for preventing and/or treating a parasitic infestation in a non-human mammal comprising administering an effective amount or an effective dose of a veterinary composition as disclosed herein in a non-human mammal in need thereof.

Within the context of the invention, the expression “effective dose” or “effective amount” means the dose or the quanti ty suffi ci eut for preventing and/or treating a parasitic infestation. In a particular embodiment, the expression “effective dose” or “effective amount” allows to kill at least 50%, 60%, 70%, 80%, 90% and preferably 100% of the parasites infesting or having infested the non-human mammal.

As used herein, the term “treatment” or “treating” refers to any act intended to ameliorate the health status of said non-human mammal such as therapy, prévention, prophylaxis and retardation of a parasitic infestation as disclosed herein. In certain embodiments, such term refers to the amelioration or éradication of a disease due to a parasitic infestation or symptoms associated with such disease. In other embodiments, this term refers to minimizing the spread or worsening of the disease due to the parasitic infestation resulting from the administration of compositions as disclosed herein to an infested non-human.

The term “treatment” or “treating” also includes the préventive treatment of a non-human mammal against a disease due to a parasite infestation, which désignâtes a treatment performed before the non-human mammal has been exposed to or in contact with the causative agent of the disease such as a parasite, or after said exposure/contact but before development of the symptoms or at an early stage of development of the disease due to a parasite infestation.

In a particular embodiment, the term “treatment” or “treating” désignâtes the protection of a nonhuman mammal against a disease, e.g., against the effect of an exposure to the causative agent, or against the development of the disease in exposed non-human mammals. The invention particularly suits to protect non-human mammals against an infectious disease due to a parasitic infection or infestation. An object of the invention is therefore a method for protecting a nonhuman mammal against at least one parasitic infestation, comprising administering an effective amount or an effective dose of a veterinary composition as disclosed herein in a non-human mammal in need thereof.

Such term also includes an increase in the welfare of the treated non-human mammal, for instance in increasing appetite, the production of méat, milk, bodyweight, etc., and in reducing fever or restoring body température.

The term “treatment” or “treating” also includes the alleviation of the symptoms such as a decrease of body weight, fever, anémia, as well as a delay, réduction, or cure of an existing infestation.

A further object of the invention is a method for restoring body température and/or body weight, and/or supressing parasitemia, and/or treating anémia in a non-human mammal comprising administering an effective amount or an effective dose of a veterinary composition as disclosed herein in a non-human mammal in need thereof.

A further object of the invention is a use of a composition or a composition for use as disclosed herein for restoring body température and/or body weight, and/or supressing parasitemia, and/or treating anémia in a non-human mammal.

Within the context of the invention, a “non-human mammal” comprises without limitation, canidae, for instance dogs, foxes, coyotes, and wolfs, felidae, for instance cats, lions, and panthers, bovidae, for instance cattle and dairy cow, equidae, for instance horses, and camelidae, for instance camel. In a preferred embodiment of the invention, a non-human mammal is cattle or a bovine.

Particularly, the veterinary composition as disclosed herein are used for treating a parasitic infestation such as trypanosomosis, babesiosis and/or gastro-intestinal parasites, preferably a parasitic infestation with Trypanosoma spp and/or Babesia spp and/or with gastro-intestinal and respiratory parasites, in a non-human mammal as above defined.

Trypanosomes can infect ail domesticated animais and clinical cases hâve been reported in cattle, water buffalo, sheep, goats, camels, horses, donkeys, alpacas, Hamas, pigs, dogs, cats and other species. Trypanosomes include different species such as Trypanosoma brucei, T. vivax, T. simiae, T. gambiense, T. rhodesiense, T. congolense, T. cruzi, T. evansi, or T. equinum. The host preferences of each trypanosome species may differ but Trypanosoma congolense, T. vivax, T. brucei, and T. simiae hâve a wide host range among domesticated animais such as pigs, camels, horses and cattle. In a particular embodiment, a Trypanosoma infestation according to the invention results from an invasion of Trypanosoma congolense, Trypanosoma vivax, Trypanosoma brucei, and/or Trypanosoma evansi, preferably Trypanosoma congolense.

Babesia spp can also infect ail domestic animais and cause babesiosis), toxoplasmosis and cryptosporidiosis. Particularly in bovines, it causes hemolytic anémia. As an example of Babesia spp, B. duncani, B. divergens, B. venatorum, B. microti, B. duncan, B. bovis, B. bigemina, and B. major may be cited without limitation. In a particular embodiment, a Babesia infestation according to the invention results from an invasion of Babesia bovis, Babesia bigemina, Babesia divergens, and/or Babesia major.

Gastro-intestinal parasites infect the gastro-intestinal tract of human and non-human mammals. They can live throughout the body but most preferably within the gastro-intestinal tract. Respiratory parasites infects airways and lungs of humans and non-human mammals. Gastrointestinal and respiratory parasites include without limitation helminths and protozoa. In a preferred embodiment, gastro-intestinal and respiratory parasites are nematodes and are, more particularly, Ostertagia spp, Haemonchus spp, Trichostrongylus spp, Cooperia spp, Nematodirus spp, Bunostomum spp, Oesophagostomum spp, and Dictyocaulus spp. In a further particular embodiment, a gastro-intestinal parasite infestation results from an invasion of Ostertagia spp, Haemonchus spp, Trichostrongylus spp, Cooperia spp, Nematodirus spp, Bunostomum spp, Oesophagostomum spp, and/or Dictyocaulus spp.

The veterinary compositions of the invention are thus suitable for régions where such parasites can be currently found such as Africa, Asia and South America. The compositions of the invention hâve thus the major advantage to exhibit parasiticide effect against both trypanosomes, gastro-intestinal, and respiratory parasites. A preferred object of the invention is therefore a veterinary composition as disclosed herein for use for treating and/or preventing an infestation with Trypanosoma spp, preferably Trypanosoma congolense, Trypanosoma vivax, Trypanosoma brucei, and/or Trypanosoma evansi; and/or with Babesia spp, preferably with Babesia bovis, Babesia bigemina, Babesia divergens, and/or Babesia major, and/or an infestation with gastrointestinal and respiratory parasites, preferably with Ostertagia spp, Haemonchus spp, Trichostrongylus spp, Cooperia spp, Nematodirus spp, Bunostomum spp, Oesophagostomum spp, and/or Dictyocaulus

The compositions of the invention as disclosed herein can be administrated in a non-human mammal by any route known from a skilled person such as an oral route, a topical route or a parentéral route such as injection. In a preferred embodiment, the compositions of the invention are administered in a non-human mammal by intramuscular or subcutaneous injection, preferably by intramuscular injection. The results as illustrated by examples show that a single intramuscular injection is suffi ci ent to prevent and/or treat a parasitic infestation in a non-human mammal. In a further preferred embodiment, the compositions of the invention are administered in a single take.

The compositions of the invention administered by intramuscular injection use techniques and/or devices known per se in the art. In this regard, intramuscular injection can be performed with a syringe, a gun, a micro-needle injection device, a needle-ffee injection device, a puise device, etc. In a preferred embodiment, injection is performed with a needle injector or a syringe. In another particular embodiment, injection is performed with a needle-ffee injection device such as a puise needle-ffee System, more particularly a spring-powered, battery-powered, or compressedgas-powered device. Intramuscular injection may be made in any muscle. For livestock, such as cattle or bovines, intramuscular injection is preferably made in the area of the neck, or behind the ear, preferably as a deep intramuscular injection in the neck.

In this context, the composition of the invention is formulated as a solution or suspension, or any form suitable for intramuscular or subcutaneous injection. The composition may further comprise veterinary acceptable excipient(s) such as solvents, solubilisers, surfactants, antioxidants, thickeners, preservatives, anti-foaming agents, etc. Suitable solvents include, without limitation, physiologically acceptable water, alcohols, esters, vegetable oils, and the mixtures thereof, in isotonie solutions. Solubilisers include, for instance polyvinylpyrrolidone. Examples of suitable antioxidants include ascorbic acid, gallic acid esters, and sulphates. Suitable preservatives include, without limitation, benzyl alcohol, n-butanol, benzalkonium chloride, benzoic acid or citric acid. Anti-foaming agents include without limitation oil carrier such as minerai oil, vegetable oil, white oil or any other oil that is insoluble in the foaming medium, silicone oil, simethicone émulsion, wax and/or hydrophobie silica such as ethylene bisstearamide (EBS), paraffinic waxes, ester waxes, silica, fatty alcohol, fatty acid soaps or esters, silicone compound, polyethylene glycol and polypropylene glycol copolymers and alkyl polyacrylates.

In a particular embodiment, the composition of the invention is an injectable solution. This solution can be prepared by adding water, preferably stérile water, to a veterinary composition in a powder form as above described.

For instance, an injectable solution of the invention comprises:

about 1.16 g of diminazene or its salts, about 1.75 g of levamisole or its salts, about 1.95 g of phenazone, said ingrédients are dissolved in 20-30 mL of stérile water.

The dose of the active ingrédients, diminazene and levamisole or salts thereof, may vary depending on the non-human animal species. Conventional dosages rates from 1 to 50 mg per kg bodyweight (mg/kg) of the non-human animal may be used, preferably from 1 to 20 mg/kg, more preferably from 1 to 10 mg/kg. A preferred dosage for an intramuscular injection in a bovine comprises about 3.5 mg/kg diminazene diaceturate and about 5.0 mg/kg levamisole.

Treatment can be applied as a unique injection or can be repeated several times, for instance every three weeks, once a month, once every two months or once every three months.

The invention further provides a kit comprising (a) one compartment comprising diminazene or its salts and (b) a second compartment comprising levamisole or its salts, wherein the weight ratio of diminazene or its salts to levamisole or its salts ranges from 0.10 to 5.00, preferably from 0.50 to 2.00, from 0.50 to 1.00, more preferably about 0.70, as a combined préparation for simultaneous, separate or sequential use for preventing and/or treating a parasitic infestation in a bovine.

As used herein, the terms “separate” or “sequential” mean that diminazene and levamisole are administered successively. For instance, diminazene is firstly administered in a bovine, and levamisole is secondly administered in the same non-human mammal, or vice versa. In this context, diminazene and levamisole are physically sufficiently distinct from being separately or sequentially administrable.

A further object of the invention is a kit as defined above, further comprising a package leaflet or user instructions including the information that said préparation is to be used for preventing and/or treating a parasitic infestation, preferably Trypanosoma infestation and/or gastrointestinal and respiratory parasite infestation in a bovine.

Further aspects and advantages of the invention will be disclosed in the following experimental section. The examples below illustrate the présent invention and are given as non-limiting illustrations.

EXAMPLES

Example 1: Compositions of the invention and the process of préparation thereof.

Powder

Ingrédients	% (w/w)
Diminazene diaceturate	24
Levamisole hydrochloride	36
Phenazone	40

Diminazene diaceturate, levamisole hydrochloride, and phenazone hâve been 1 mm sieved and mixed during 2 minutes at 150 tr/min. The blending has been granulated by continuously adding anhydrous éthanol during 2 minutes, and then densified for 3 minutes. Granules hâve been dried during 4 hours at 37 °C in a stove, and then 1 mm sieved.

Injectable solutions

The injectable solution of the invention (IVP 1) has been prepared by adding 26 mL of stérile water to 4.9 g of the powder composition as above defined.

The injectable solution IVP2 has been prepared by adding 15 mL of stérile water to 2.36 g Veriben (Diminazene).

Example 2: Biological studies

I. Materials and methods

The schedule of the study is illustrated in the table 1 below.

Table 1:

Acclimatisation	Ranking and allocation to groups	Pathogen inoculation	IVP administration	Blood collection for pharmacokinetics (Groups 2 and 3 only)
Days -14 to-1	Day -2 ± -1 day	Day -7	Day 0	Days -8 (ait animais), -1, 0, 1,2,4. 6. 8,10,12 and 14

Blood collection for PCR	Faecal collection, EPGs and coproculture	Blood collection for and performing of PCV and buffy coats
Day-14	Days -3 and 7	Once during Days -14 to -10 and on Days -8, -4, -2, -1,0,1,2, 7,14 and 21

1. Acclimatisation (Pays -14 to -1) bovines (Nguni) were acclimimatised for at least 14 days before IVP (Investigational Veterinary Product) administration. No médication was given during the acclimatization period.

2. Ranking and allocation to groups (Day -2 ± -1 day)

Following clinical examination performed during acclimatisation period, 20 bovines were included and randomly allocated to the three groups (G1 : n = 4; G2: n = 8; G3: n = 8).

3. Pathogen inoculation (Day -7)

Fresh cattle blood containing the pathogen T. congolense, 02J strain from donner cattle, was used to induce an infestation by intra venous inoculation on Day -7 for each bovine. Such pathogen was of a strain reported to be sensitive in cattle to diminazene.

4, IVP administration (Day 0)

The IVPs (Investigational Veterinary products) were administered by intra-muscular injection in the neck of the bovine once at day 0 at the dose rates indicated in table 2 below. 3.5 mg diminazene diaceturate per kg bodyweight corresponds to an injectable solution of 5.8 mL per 100 kg body weight (IVP2) for group 2. 3.5 mg diminazene diaceturate and 5.0 mg levamisole correspond to an injectable solution of 10 mL per 100 kg body weight (IVP1) for group 3. The animais in control group (Group 1) received a rescue treatment (Veriben®) at Day 7.

Table 2:

Group	Sample size	IVP	Active ingredient(s)	Dose rate
1	4	Not applicable
2	8	IVP2 (Veriben®)	Diminazene	3.5 mg diminazene diaceturate per kg bodyweight
3	8	IVP 1 (C753)	Diminazene and levamisole	3.5 mg diminazene diaceturate and 5.0 mg levamisole per kg bodyweight

5. Blood collection for PCR (Day -14)

Blood specimens of at least 3 mL were collected in EDTA tubes for PCR (Polymerase Chain Reaction) analysis once for each animal on Day -14. A strain spécifie PCR analysis of the pathogen strain used was also done prior to inoculation of donor animais to confirm the strain had not been contaminated by other Trypanosoma strains.

6. Faecal collection. EPGs and coproculture

Faecal egg counts were performed as a general screening to détermine the possible presence of worm infections, if any. Faecal specimens were collected from each animais on Days -3 and 7. Animais were housed individually and fresh faeces were collected from the floor or collected from the rectum of the animal. Collected faeces were broken and mixed well for each animal, individually. A 4 g sample of faeces, taken from the well-mixed specimen, was suspended with the aid of a magnetic stirrer in 56 mL of a sucrose solution [sugar 400 g, H2O 600 mL], While stirring the suspension, a Pasteur pipette was used to fill the counting chambers of a MacMaster slide. The slide consisted of two persplex slides, separated by strips of Perspex 1.5 mm thick and with a 1.0 cm2 area ruled over each chamber. Eggs in the suspension floated upwards in the counting chamber and could then be counted under a microscope within the 1.0 cm2 area. Two or three chambers were counted depending on slide type used and the mean number calculated. Egg numbers were then expressed as eggs per gram of faeces (EPG) as follows:

Mean number of eggs counted in the squares X 100

Coprocultures were performed as part of a general screening to détermine the possible presence of worm infections, if any. Nematode species infecting bovines could not always be accurately identified on egg morphology alone. Faecal cultures were prepared, incubated and harvested as per study site SOP. Faces from individual animal were mixed with vermiculite to attain a moist granular consistency. Small amounts of bore-hole water were added when the mixture was too dry. The mixture was transferred to a suitable container and lightly compressed to form a cake in the bottom. It was compressed just sufficiently so that it did not fall out when the container was tumed over and were not too wet. The inside of the container was wiped clean above the level of the compressed mixture. The container was covered in such a way that some air circulation was possible and marked with at least the date of collection and culture préparation, study number and animal ID. The prepared culture was incubated at approximately 27 °C. After approximately 3 days, the inside of the container was aerated by lifting the lid and sides wetted lightly if the content appeared too dry. Between 5 to 7 days, the containers could be removed from the incubator; lightly wetted and left outside the incubator. After approximately an hour, hatched larvae crawled up in the film of moisture on the inside of the container. They could be washed out with dechlorinated water. Larvae were stored in shallow water in a properly parked container at 8 °C to 10 °C. Each individual suspension was mixed to allow a homogeneous distribution of larvae. From each suspended solution, the first 100 larvae encountered were identified. Species composition was then expressed as a percentage to provide an indication of prevalence.

7. Blood collection for PCV and parasitemia/buffy coats

Blood specimens of approximately 3 mL for détermination of packed cell volume (PCV) and parasitemia were collected for ail animais in heparinized collection tubes from a nominated vein on Day -14 for enrolment and on Days -8, -4, -3, -2, -1, 0, 1, 2, 7, 14 and 21.

II. Results

1. Local and general tolérance of a single intra-muscular injection of IVP 1 and UVP 2. Intramuscular IVP1 and IVP2 were well tolerated. No pain for animal and local reaction (Edema) has been observed.

2. Body température.

Body températures were measured on Days -14 and daily form Days -8 to Day 21. As reported by figure 1, control group 1 (infected and not treated) had the highest body températures on Day 0 and the body température both decreased for Group 2 and 3 after IVP administration, thereby demonstrating a fever réduction with IVP 1 and IVP 2 treatments.

3. Bodyweight.

The animais were weighted on a calibrated and verified electronic scale on Days -14, -2, and 21. As reported by figure 2, body weight gain has been observed after IVP 1 and IVP 2 administrations, thereby demonstrating a biological effect with IVP 1 and IVP 2 treatments. Body weight gain observed for control group is due to the rescue treatment with Veriben . On overall, the bodyweight development of animais in control group (Group 1 ) was lower.

4. Parasitemia (Trypanosoma congolenseY

IVPs were fully effective in curing the animais. No parasite was observed in treated animais during the follow up period on Days 0, 1,2, 7, 14, and 21 (Table 3). Fisher’s exact test was used to compare the IVP groups and the control group together (Table 4). The différence between treated and control groups was highly significant. There was no significant différence between IVPs (IVP1 and IVP2).

Table 3:

Summary of parasitaemia

Group	Absent / N (%)
Day 1	Day 2	Day 7	Day 14	Day 21
Group 1	0/4	0/4	0/4	4/4(100%)	4/4 (100%)
Group 2	7/8 (87.5%)	8/8 (100%)	8/8 (100%)	8/8 (100%)	8/8 (100%)
Group 3	7/8 (87.5%)	8/8 (100%)	8/8 (100%)	8/8(100%)	8/8 (100%)

Group 1 : Négative control

Group 2: Cattle received IVP 2, Veriben®, as a deep intra-muscular injection in the neck.

Group 3: Cattle received IVP 1, C753, as a deep intra-muscular injection in the neck.

Table 4:

Comparison	Day 1	Day 2	Day 7
Group 2 - Group 1	0.0101	0.0020	0.0020
Group 3 - Group 1	0.0101	0.0020	0.0020
Group 3 - Group 2	1.0000

p-value: fishers exact test

Group 1: Négative control

Group 2: Cattle received IVP 2, Veriben®, as a deep intra-muscular injection in the neck.

Group 3: Cattle received IVP 1, C753, as a deep intra-muscular injection in the neck.

For the control group 1, ail animais had parasitemia on Day 1, 2 and 7 and ail animais had an absence of parasitemia on Days 14 and 21 after reçue treatment administered on Day 8.

For both IVP groups (Groups 2 and 3), only one animal had parasitemia on Day 1 and 100% of animais were cured 2 days following treatment.

The number of animais with an absence of parasitemia in the IVP groups (Groups 2 and 3) compared to the control group 1 was statistically significant (p < 0.05). Also, comparison Group 3-Group 2 (p = 1.0000) has demonstrated that there was no négative effect due to the levamisole.

5. Trypanosoma PCV.

Mean PCV for each group for Days -14, -7, 0, 7, 14, and 21 were calculated and compared using an ANOVA test. Results are reported in Figures 3A-3C. As it has been observed, the mean PCV values in the control group 1 (29.7% at D7) was less than the IVP groups (38.2% for Groups 2 and 35% for Group 3 at D7) after Day 0, thereby demonstrating full recovery of hematocrit levels (PCV) with IVP 1 and IVP 2 treatments. Of note that the Control group (Group 1) received a rescue treatment on Day 8, explaining the increase of the hematocrit on Days 14 and 21.

6. Faecal egg counts and coproculture.

Parasite Eggs per gram of faeces (EPC) for each group is summarized in Table 5 below.

Table 5:

Group	Statistic	Day -3	Day 7
Group 1	n	4	4
	Mean	158.33	133.33
	SD	206.155	81.650
	Médian	100.00	133.33
	Minimum	0.0	33.3
	Maximum	433.3	233.3
Group 2	n	8	8
	Mean	95.83	41.67
	SD	114.694	46.291
	Médian	33.33	33.33
	Minimum	0.0	0.0
	Maximum	300.0	133.3
Group 3	n	8	8
	Mean	37.50	0.00
	SD	45.207	0.000
	Médian	16.67	0.00
	Minimum	0.0	0.0
	Maximum	100.0	0.0

Ail animais in group 3 (IVP 1) had no eggs per gram, while group 1 (control) and group 2 (IVP 2) still had some eggs (133.33 and 4.291 per gram, respectively) after IVP administration on Day 0.

Larval identification and énumération following coproculture is reported in the table 6 below. Harvesting of faecal cultures and subséquent énumération and identification yielded two nematode species, namely Ostertagia ostertagi and Haemonchus placei.

Coproculture results support the trend identified when using faecal egg counts, namely little réduction in worms number between Days -3 and 7 for groups 1 and 2 (Control and IVP 2 groups), with very few worms présent in group 3 on Day 7. For group 3 prevalence decreased from 100% (ail 8 animais infected with at least one of the worm species on Day -3) to 50% (4 animais infected with at least one of the worm species on Day 7). In addition, intensity of infection (total number of worms) decreased from 100 worms (100 larvae examined for identification purposes) to 2 worms between Day -3 and Day 7. The overall curative effîcacy of the combination was 98%.

Table 6:

Group	SD faeces collected	Animal ID	Ostertagia ostertagi	Haemonchus place!	Prevalence	Intensity
NA	-3	CVB 440	88	12	100	100.0
CVB 446	96	4
CVB 457	83	17
1	CVB 436	100	0
CVB 438	86	14
CVB 448	84	16
CVB 452	72	28
7	CVB 436	96	4	100	100.0
CVB 438	96	4
CVB 448	98	2
CVB 452	94	6
2	-3	CVB 437	94	6	100	99.9
CVB 445	77	23
CVB 449	75	25
CVB 451	74	26
CVB 453	100	0
CVB 454	89	10
CVB 455	100	0
CVB 456	83	17
7	CVB 437	97	3	100	100.0
CVB 445	93	7
CVB 449	81	19
CVB 451	88	12
CVB 453	99	1
CVB 454	96	4
CVB 455	100	0
CVB 456	93	7
3	-3	CVB 441	100	0	100	100.0
CVB 442	95	5
CVB 443	94	6
CVB 444	93	7
CVB 447	93	7
CVB 450	84	16
CVB 458	84	16
CVB 459	62	38
7	CVB 441	1	0	50	2.0
CVB 442	0	0
CVB 443	0	0
CVB 444	1	0
CVB 447	3	1
CVB 450	0	0
CVB 458	2	0
CVB 459	0	0

Note: Total number of worms (combined for both species) used for calculation of intensity Prevalence = (Number of animais infected/Total number of animais assessed) x 100 Intensity = Total number of parasites/Number of infected animais

Group 1 : Négative control

Group 2: Cattle received IVP 2, Veriben*, as a deep intra-muscular injection in the neck.

Group 3: Cattle received IVP 1, C753, as a deep intra-muscular injection in the neck

7. Conclusions

In conclusion, the inventors hâve demonstrated that a composition of the invention comprising 5 diminazene and levamisole in the spécifie weight ratio as defined herein had a similar efficient activity against trypanosomes compared to the commercially available formulation Veriben® (Diminazene) when applied in bovines, thereby showing no négative effect or négative interaction of levamisole on Diminazene.

In addition, the inventors hâve demonstrated an additional efficient activity against gastrointestinal parasites, such as nematodes, with the compositions of the invention.

Example 3: Stability studies

I. Materials and methods

Powder formulations of the invention hâve been prepared by the process of example 1 and were 10 dissolved in 21 ml of stérile water. The composition for one dose is detailed by the following table 7.

Table 7:

Composition for one dose	A	B	C	D	Reference Powder for injection	Reference Solution for injection
Diminazene Diacéturate 4H20 (g)	1.16	1.16	1.16	1.16	1.16	1.16
Levamisole HCl (g)	1.75	1.75	1.75	1.75	0	0
Phenazone (g)	1.20	1.65	1.95	2.21	1.31	5.6
Total one dose (g)	4.11	4.50	4.90	5.12	2.47	15 mL
Ratio Phenazone/Diminazene diaceturate	1.0	1.4	1.7	1.9	1.1	4.8

II. Results

Stability of compositions A, B, C, and D are presented in the following table 8. The appearance of the obtained solution were noted immediately after reconstitution and after 24 hours storage at room température.

Table 8:

Composition for one dose	A	B	C	D
Appearance of solution atTO	Clear yellow solution

Appearance of solution after 24 hours storage at room température

Précipitation

Précipitation

Clear yellow solution

Clear yellow solution

As shown by table 8, précipitation of diminazene occurs if the weight ratio phenazone/ diminazene diaceturate 4H₂O is less than 1.6. Therefore, the weight ratio Phenazone/Diminazene 4H₂O must be superior to 1.6 to avoid any précipitation of the complex for solubilization in 5 water when volume is comprised between 20 ml and 30 ml. This amount of phenazone in the current formulation is higher than the amounts known and used in existing diminazene-based powder formulation (ratio = 1.2). It is lower when compared to the commercial ready to use solutions for injection (Ratio = 4.8).

Claims (24)

1. A veterinary composition comprising diminazene and levamisole or salts thereof, wherein the weight ratio of diminazene or its salts to levamisole or its salts ranges from 0.50 to 2.00.

2. The veterinary composition according to claim 1, wherein the weight ratio of diminazene or its salts to levamisole or its salts ranges from 0.50 to 1.00.

3. The veterinary composition according to claim 1 or 2, wherein the weight ratio of diminazene or its salts to levamisole or its salts ranges from 0.60 to 0.80.

4. The veterinary composition according to any one of daims 1 to 3, wherein the weight ratio of diminazene or its salts to levamisole or its salts is about 0.70.

5. The veterinary composition according to any one of daims 1 to 4, comprising:

- from 5 to 50%, by weight of diminazene or its salts; and

- from 10 to 50%, by weight of levamisole or its salts; relative to the total weight of the composition.

6. The veterinary composition according to any one of daims 1 to 5, comprising: - from 10 to 30%, by weight of diminazene or its salts; and

- from 20 to 40%, by weight of levamisole or its salts; relative to the total weight of the composition.

7. The veterinary composition according to any one of daims 1 to 6, comprising: - from from 15 to 25%, by weight of diminazene or its salts; and

- from 25 to 40%, by weight of levamisole or its salts;

relative to the total weight of the composition.

8. The veterinary composition according to any one of daims 1 to 7, further comprising phenazone.

9. The veterinary composition according to claim 8, wherein the weight ratio of phenazone to diminazene or its salts is greater than 1.6.

10. The veterinary composition according to claim 9, wherein the weight ratio of phenazone to diminazene or its salts ranges from 1.6 to 4.8.

11. The veterinary composition according to claim 9, wherein the weight ratio of phenazone to diminazene or its salts ranges from 1.6 and 2.0.

12. The veterinary composition according to claim 9, wherein the weight ratio of phenazone to diminazene or its salts ranges from 1.7 to 1.9.

13. The veterinary composition according to any one of daims 1 to 12, comprising:

- from 15 to 25%;

- from 25 to 40%; and

- from 30 to 50%;

relative to the total weight of the composition.

14. The veterinary composition according to any one of daims 1 to 13, comprising:

- from 20 to 25% by weight of diminazene or its salts;

- from 30 to 40% by weight of levamisole or its salts; and

- from 35 to 45%, by weight of phenazone;

relative to the total weight of the composition.

15. The veterinary composition according to any one of daims 1 to 14, comprising an additional active agent including an antibiotic, an anti-inflammatory agent, an anthelmintic, an endectocide, a minerai or a vitamin.

16. The veterinary composition according to claim 7, wherein the additional active agent is a vitaminincluding vitamin B1 to B12.

17. The veterinary composition according to any one of claims 1 to 16, wherein said composition is a powder.

18. The veterinary composition according to any one of claims 1 to 17, wherein said composition is an injectable solution.

19. A veterinary composition according to any one of claims 1 to 18, for use in preventing and/or treating a parasitic infestation in a non-human mammal.

20. The veterinary composition for use according to claim 19, wherein the parasitic infestation is an infestation with Trypanosoma spp including Trypanosoma congolense, Trypanosoma vivax, Trypanosoma brucei, and/or Trypanosoma evansi; and/or with Babesia spp including Babesia bovis, Babesia bigemina, Babesia divergens, and/or Babesia major-, and/or an infestation with gastro-intestinal and respiratory parasites including Ostertagia spp, Haemonchus spp, Trichostrongylus spp, Cooperia spp, Nematodirus spp, Bunostomum spp, Oesophagostomum spp, and/or Dictyocaulus spp.

21. The veterinary composition for use according to claim 19, wherein the non-human animal is a bovine.

22. The veterinary composition for use according to any one of claims 19 to 21, wherein said composition is administered by intramuscular or subeutaneous injection, including intramuscular injection.

23. A kit comprising (a) one compartment comprising diminazene or its salts and (b) a second compartment comprising levamisole or its salts, wherein the weight ratio of diminazene or its salts to levamisole or its salts ranges from 0.50 to 2.00as a combined préparation for simultaneous, separate or sequential use for preventing and/or treating a parasitic infestation in a bovine.

24. The kit according to claim 23, wherein it further comprises a package leaflet or user instructions including the information that said préparation is to be used for preventing and/or treating a parasitic infestation including infestation with Trypanosoma spp and/or

5 Babesia spp, and/or gastro-intestinal and respiratory parasite infestation in a bovine.