# Patent

## Title
Veterinary antiprolactinic composition for ruminants

## Abstract

This present invention relates to an antiprolactinic veterinary composition to be administered to ruminants. Said composition comprises at least one antiprolactinic compound which is an agonist of dopamine receptors, and is particularly useful for reducing udder engorgement and/or the reduction of pain after drying off and/or of increasing welfare, and/or of reducing milk leakage in ruminants as well as for preventing new mammary microbial infections, secondary infection, or co-infections in ruminants and mastitis.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation-in-part of U.S. Ser. No. 13/081,671, filed Apr. 7, 2011, which is a Continuation-in-part of International Application PCT/EP2009/063004, filed Oct. 7, 2009, which claims priority of French Application No. 0805544, filed Oct. 7, 2008. The entire contents of the preceding applications are hereby incorporated by reference into this application.


FIELD OF THE INVENTION
This invention concerns antiprolactinic veterinary compositions to be administered to ruminants. The veterinary compositions according to the present invention comprise of at least one antiprolactin agonist of dopamine receptors and are particularly useful in promoting mammary involution, regeneration of the mammary tissue and the conditions of the reduction of the lactation. They are also effective for reducing of milk leakage, udder engorgement and/or the reduction of pain after drying off in ruminants as well as for the prevention of new mammary microbial infections, secondary infection, co-infections, or intra-mammary infections by major pathogens in ruminants. In addition, the compositions according to this invention can be administered during periods of gestation as they do not present any deleterious or abortive effects on the gestation of ruminants.
In ruminants, lactation in general lasts between 5 to 20 months, and lasts approximately about 10 months in dairy cows. After this lactation period, the milking is generally stopped suddenly and the cow is put to rest during a period of time that traditionally lasts about 60 days (known as dry period) up to the calving, which is the starting point of the subsequent lactation. In dairy farming, this drying period is known to substantially improve the health and welfare of the ruminants. This kind of physiological rest provided to the udder is necessary for the regeneration of the mammary glands and preparation of subsequent lactations. It also limits the emergence of health-related troubles caused during the milking and calving, such as the emergence of intra-mammary inflammations and intra-mammary infections that have a significant economic impact.
Antiprolactinic compounds are prescribed in medicines for humans to treat hyperprolactinemia, pituitary adenomas, Parkinson's disease or even to provoke the stopping of lactation in certain women who cannot breast-feed due to medical reasons. Such medicines contain inhibitors of prolactin hypophyseal as the active principal, a hormone that activates milk secretion. It concerns medicines such as Parlodel® (bromocriptine, Novartis), Dostinex® or Cabaser® (cabergoline, Pfizer) for which usage is explicitly prohibited during pregnancy because of their deleterious effects on the fetus. At the moment of the prescription for stopping the milking, it is recommended to administer them right after the delivery in order to stop the rise of the milk within twenty-four hours.
In veterinary medicine, compositions are based of prolactin inhibitors such as Galastop® (cabergoline, Ceva Santé Animale) can be further administered as treatments for lactations of pseudo gestations in female cats and dogs. They are also prescribed following early weaning or abortions or during the immediate withdrawal after birth or parturition. In animals, as in human patients, prolactin secreting inhibitors have always been prescribed after gestation or pregnancy because of the toxicity, risks of malformed fetus and the risks of spontaneous abortions.
In ruminant mammals, it has been established that prolactin plays an important role for the maturing of the mammary glands and the start up of the lactation after weaning, but does not affect the production of milk during the established lactation (Karg H. & Shams D., J. Reprod Fertil., 1974 August; 39(2); 463-72—Shams et al., Experentia, 1972; (28): 697-699—Akers et al., Endocrinology, 1981; (109): 23-30—Plant K. et al., Domest Anim Endocrinol, 1987; (4); 279-290—Knight, Livestock Production Science, 2001, (70); 87-93—Dalh et al, J; Anim. Sci., 2003; (81), supl 3; 11-17).
Contrary to many publications indicating that the diminishing of the prolactin has very weak or no effect on the production of milk during the lactation established in principal dairy animals, such as cows and goat, the applicant surprisingly discovered that a single dose of an antiprolactinic compound agonist of dopamine receptors led to a substantial reduction of lactation and allowed the efficient induction of the initial phase of drying up which consists of mammary involution.
Similarly and against all expectations, the applicant found out that the veterinary compositions comprising antiprolactinic compounds that are agonists of dopamine receptors can be administered to ruminants in order to induce a reduction of lactation, promote mammary involution and the conditions of drying of the tissue and this, during gestation without creating any deleterious or abortive effects.
The administration of these veterinary compositions to ruminants has revealed itself to be particularly advantageous as it allows a significant reduction of lactation and promotes mammary involution and this even during the gestation period, resulting in an improvement of drying conditions. Besides, veterinary compositions ensure a significant improvement of the health of the udder, a better regeneration of damaged secretory tissues of the udder, as well as a reduction of the frequency of intra-mammary diseases and/or infections and for example the mastitis of the ruminants during the subsequent lactations.
SUMMARY OF THE INVENTION
This invention relates to veterinary compositions comprising at least one antiprolactinic compound agonist of dopamine receptors to be administered to ruminants. The antiprolactin compounds of veterinary compositions according to the invention are chosen among the ergot-derived dopamine receptor agonists or non ergot-derived dopamine receptor agonist.
According to the invention, these compositions are administered in therapeutically effective amounts, particularly as single treatment, in order to induce a reduction of lactation. Also, these compositions can also be administered to gestating ruminants without causing any deleterious or abortive effects on the gestation. They provide a significant welfare of the ruminants, and are particularly useful in mammary involution in ruminants, in the treatment and/or the prevention of intra-mammary inflammations and intra-mammary infections of ruminants. The present invention further relates to methods of inducing mammary involution in ruminants, as well as methods of prevention and/or treatment of intra-mammary infections of ruminants, such as for example mastitis. The present invention finally relates to kits for promoting mammary involution, and reduction of milk production in the ruminants, as well as kits for the treatment and/or the prevention of mastitis in the said ruminants.
In one aspect the present invention provides a method of reducing of milk leakage in ruminants comprising administering at least one antiprolactinic compound agonist of dopamine receptors wherein the antiprolactinic compound agonist of dopamine receptors is selected from ergoline-derived dopamine receptor agonists chosen among cabergoline, metergoline, lisurdine, bromocriptine, ergometrine and/or derivatives thereof and/or said non ergoline-derived dopamine receptor agonists chosen among ropinirol, pramipexole, rotigotine, quinagolide, and/or derivatives thereof.
In another aspect the present invention provides a method for the reduction of udder engorgement and/or the reduction of pain after drying off in ruminants comprising administering at least one antiprolactinic compound agonist of dopamine receptors wherein the antiprolactinic compound agonist of dopamine receptors is selected from ergoline-derived dopamine receptor agonists chosen among cabergoline, metergoline, lisurdine, bromocriptine, ergometrine and/or derivatives thereof and/or said non ergoline-derived dopamine receptor agonists chosen among ropinirol, pramipexole, rotigotine, quinagolide, and/or derivatives thereof.
In still another aspect the present invention provides a method for the prevention of new mammary microbial infections, secondary infection, co-infections, or intra-mammary infections by major pathogens in ruminants comprising administering at least one antiprolactinic compound agonist of dopamine receptors wherein the antiprolactinic compound agonist of dopamine receptors is selected from ergoline-derived dopamine receptor agonists chosen among cabergoline, metergoline, lisurdine, bromocriptine, ergometrine and/or derivatives thereof and/or said non ergoline-derived dopamine receptor agonists chosen among ropinirol, pramipexole, rotigotine, quinagolide, and/or derivatives thereof.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: is a graph showing the variation of the percentage of the milk production in the morning and in the evening day D-7 and day D7 (in comparison to a baseline of 100) following a single injection of 5 mcg/kg of cabergoline on day D0, in a group of placebo and in a group of treated animals (C646)
FIG. 2: is a graph that shows the variation in percentage of the prolactin rate observed every 24 hours during the period I (D-2 and D-1) and the period II (between D0 and D7), in comparison to a baseline of 100, following a single injection of 5 mcg/kg of Cabergoline on day D0, in a placebo group and in a group of treated animals (C646).
Fig.3: is Table 1 showing how biopsies and mammary secretion sample were performed.



DETAILED DESCRIPTION OF THE INVENTION
This invention relates to a veterinary composition consisting of at least one anti-prolactinic compound agonist of dopamine receptors administered in a single dose in order to induce the reduction of the lactation in the treated animal as of the first milking after treatment and next milking. This invention relates to the use of the said veterinary composition administered in sufficient therapeutic quantities in one single dose, to induce a substantial reduction of the lactation.
Prolactin is a hypophyseal hormone that has mammotropic and lactogenic effects, which means that it activates the growth of mammary glands and milk secretion. The release of prolactin is stimulated by the prolactoliberine and inhibited by the dopamine. The inhibiting action of the dopamine on the pituitary gland is mediated by the post-synaptic receptors of dopamine, such as in particular D2 receptor.
The compounds are known as antiprolactinic when they inhibit the release of prolactin. It may concern dopaminergic compounds or serotoninergic compounds. The dopaminergic compounds are agonist of dopamine receptors which bind to dopamine receptors present in particular on prolactin secretory cells on the adenohypophysis in order to inhibit the prolactin secretion.
Preferably, these antiprolactinic dopaminergic compounds bind to dopamine receptors. Most preferably, these compounds specifically bind to the D2 dopamine receptors. These antiprolactinic antiserotoninergic compounds act by stimulating dopamine release in the hypothalamus, resulting in the inhibition of prolactin secretion. The veterinary compositions according to the present invention preferably comprise antiprolactinic dopaminergic compounds.
These antiprolactinic compounds may be chosen among ergoline-derived dopamine receptor agonists and/or derivatives thereof. These ergoline derivatives are well known in the public domain and have the following general structure:




Examples of ergoline-derived antiprolactinic agonists are cabergoline, metergoline, lisuride, bromocriptine, ergometrine, as well as all the derivatives of the compounds sharing an antiprolactinic activity.
Cabergoline whose chemical name is N-[3-(Dimethylamino)propyl]-N-[(ethylamino)carbonyl]-6-(2-propenyl)-8 g-ergoline-8-carboxamide, is an anti-prolactinic agonist specific of D2 dopamine receptors. In particular, it is described in the U.S. Pat. No. 4,526,892. Its developed chemical formula is as follows:




Cabergoline constitutes the active principle of drugs for humans marketed under denominations such as Dostinex® and Cabaser®. Also, this is the basic active principle for veterinary compositions marketed under the denomination Galastop® intended for female dogs prone to lactations during pseudo-gestation. Whether it is Dostinex® or Galastop®, these compositions made up of a cabergoline base are never administered during pregnancy or gestation.
Metergoline is a synthetic compound known under the chemical name of benzyl 1 ((6aR,9S,10aR)-4,7-dimethyl-4,6,6a,7,8,9,10,10a-octahydroindolo-[4,3-fg]quinolin-9-yl) methyl carbamate. This is an antiprolactinic compound that binds to dopamine receptors, as well as to serotonin receptor 5HT, and therefore activates the release of dopamine and the inhibition of the prolactin secretion. Metergoline has the following chemical structure:




Lisuride, also known by its chemical name 1,1-diethyl-3-((6aR,9S)-7-methyl-4,6,6a,7,8,9-hexahydroindolo[4,3-fg]quinolin-9-yl) urea present in the developed chemical formula is as follows:




This is an agonist compound that is a dopaminergic inhibitor of prolactin. It is marketed under the name of Arolac® for the treatment of amenorrhea and hyperprolactemenia in human patients.
Bromocriptine is known under the name of bromo-2 ergocryptine and under the chemical name ergotaman-3′,6′, 18-trione, 2-bromo-12′-hydroxy-2′-(1-methylethyl)-5′ alpha-(2-methylpropyl). Its chemical formula is as follows:




Compositions such as Parlodel® and Bromo-Kin® used in particular for the treatment of tumors of the pituitary gland and hyperprolactinemias, or in gynecology for inhibiting lactation after delivery, comprise of a therapeutically effective amount of bromocriptine which is an ergoline-derived dopaminergic agonists.
Ergometrine or ergonovine, denonimated further as d-lysergic beta-propanolamide can also be incorporated into veterinary compositions as antiprolactinic dopaminergic agonist. Its chemical formula is as follows:




Other antiprolactinic compounds that are agonist of dopamine receptors can be used in compositions of the present invention and can be administered to ruminants, particularly during the gestation period. These can be chosen from antiprolactinic compounds that are non-ergoline-derived dopamine receptor agonists. Examples of such compounds are ropinirol, pramipexole, rotigotine, and quinagolide, as well as derivatives thereof having antiprolactinic activity.
Ropinirol whose chemical name is 4-(2-dipropylaminoethyl)-1,3-dihydroindol-2-one, acts as an agonist of dopamine receptors D2 and D3. Its chemical formula is as follows:




Ropinirol is found in compositions of products such as Requip® and Ropark® prescribed in medicines for humans for the treatment of Parkinson's disease.
Pramipexole represents another dopaminergic agonist which binds in particular to the receptors D2 and
D3 of dopamine. Its chemical name is (6S)—N6-propyl-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine and its formula is as follows:




Pramipexole is well known in the pharmaceutical field since it is marketed under the names Mirapex®, Mirapexin® or Sifrol® for the treatment of Parkinson's disease and the syndrome of restless legs.
Rotigotine which is equally known under the chemical denomination of 6-(propyl-(2-thiophen-2-ylethyl)amino)tetralin-1-ol. Its chemical formula is as follows:




Rotigotine was recently approved for the treatment of Parkinson's disease by transdermal administration in the form of a patch under the designated name of Neupro®.
Quinagolide or (3R,4aR,10aS)-3-(diethylsulfamoylamino)-6-hydroxy-1-propyl-3,4,4a,5,10,10a-hexahydro-2H-benzo[g]quinoline is an inhibitor of prolactin secretion as well. This is a specific agonist of D2 dopamine receptors. Its chemical formula is as follows:




Quinagolide is marketed under the commercial name of Norprolac® (Ferring Pharmaceuticals) for the treatment of macroprolactinoma or hyperprolactinemia.
These series of compounds have always been administered up to now, outside of pregnancy or gestation.
The applicant however surprisingly discovered that these compounds, when administered to ruminants, in a therapeutically effective amount and in a single administration, in fact induced a significant reduction of the lactation, promoting mammary involution and improving the reduction of lactation. Contrary to what was known until now, these compounds were revealed by the applicant to be particularly beneficial as they allow effective reduction of milk production, particularly during the gestation period of ruminants without provoking any deleterious or abortive effects. Finally, the compositions according to this invention ensured a significant decrease in the risks of mastitis in the treated ruminants.
As demonstrated in the Examples below, one single dose can be administered to non-human animals in order to promote mammary involution and reduction of the lactation, whether in periods of gestation or after calving. The amounts or therapeutically effective doses may vary depending on the ruminants to be treated and the mode of administration of the compositions. These doses can be easily determined by systematic trials based on the below Examples that can be readily used by skilled persons in the art. Examples of therapeutically effective doses according to the present invention comprise between 5 and 50 mcg/kg, or between 5 and 25 mcg/kg.
In one aspect the present invention provides a method of reducing of milk leakage in ruminants comprising administering at least one antiprolactinic compound agonist of dopamine receptors wherein the antiprolactinic compound agonist of dopamine receptors is selected from ergoline-derived dopamine receptor agonists chosen among cabergoline, metergoline, lisurdine, bromocriptine, ergometrine and/or derivatives thereof and/or said non ergoline-derived dopamine receptor agonists chosen among ropinirol, pramipexole, rotigotine, quinagolide, and/or derivatives thereof.
As further demonstrated in the Examples below, one single dose of the present veterinary composition can be administered to non-human animals in order to effectively reduce udder engorgement and/or pain after drying off in ruminants. It has also been demonstrated that the above described veterinary composition is effective for preventing new mammary microbial infections, secondary infection, co-infections, or intra-mammary infections by major pathogens in ruminants.
Preferably, according to the present invention, the compositions are administered in single doses per treatment and animal so as to obtain a substantial reduction of lactation comprising between 5 and 60%, between 20 and 50%, or even between 25 and 35%, over a period from 1 day, 2 days, 3 days, 4 days, 5 days or 6 days, and up to 7 days after the administration of the composition. As demonstrated in Example 2, the reduction of the lactation is significant as of the first milking, but even during the following milking.
It is possible to administer compositions according to the present invention to the ruminants in gestation and more precisely during the latter two thirds of the gestation, without causing any abortive or deleterious effects. As examples, the veterinary compositions can be administered from the 3rd month or more during the period of gestation of dairy cows. They are preferably be administered to gestating ruminants between the 3rd and the 8th months of gestation.
According to the present invention, ruminants are intended to mean herbivorous mammals such as bovines, ovines, goats, or camelids. The compositions of the present invention are administered to milk-producing mammary ruminants such as preferably dairy cows and sheep.
According to the present invention, the compositions can be administered during the gestation period or outside of the gestation period. They promote the reduction of the lactation and act on successive hormonal, physiological or morphological changes that affect the udders during the drying period, and particularly from the initial phase of the drying up, mammary involution during which the secretory tissue is disrupted and atrophied. Surprisingly, no deleterious or toxic effect, malformation of the fetus or even any abortive effect was reported when they were administered during the gestation period of the ruminants.
Veterinary compositions according to the present invention can be administered according to many ways of administration that are well known in the field and adapted to the treatment of each of these animals. They are preferably administered cutaneously, orally or parenterally. It is even more preferential to administer them parenterally, and particularly through intramuscular or subcutaneous injection. They are in appropriate forms for the selected mode of administration. They can therefore be in the form of a solution or an oral or injectable suspension liquid, or in a solid or semi-solid form, in powder form, capsule, granule, sugar coated tablet, soft capsule, sprays, caplet, pills, tablets, or pastes.
Advantageously, the veterinary composition is administered in one single injectable dose.
Depending on the formulations and drugs, the veterinary compositions may further comprise pharmaceutical conventional ingredients for the preparation of liquid or solid formulations to be administered orally or parenterally. Moreover, in the case of oral formulations, these can be administered directly to the ruminants or can be mixed into the food.
Furthermore, the compositions according to the invention may comprise depending on the type of formulation, a solvent, a flowing agent, a lubricant and any suitable mass excipient such as lactose, cellulose or starch. As lubricant, one can use stearic acid, magnesium stearate, L-leucine or for example, glyceryl tribehenate. As a disintegrating agent, one can use sodium carboxymethyl starch, or reticulated sodium carboxymethyl starch. As a flowing agent, one can use pure silicon or silicon dioxide colloidal. Oral solid forms can be in the form of tablets covered with coating.
The injectable preparations are prepared by mixing effective therapeutic quantities of at least one anti prolactinic compound as described previously with a solvent, a pH regulator, a buffer agent, a suspending agent, a solubilization agent, a stabilizer, an agent of tonicity and/or a preservative, and by transforming the mix with a traditional process for cutaneous or intramuscular injection. Examples of solvents are oily solvents such as medium chain triglycerides in C8-C10, or a mix of capric acid, caprylic acid, and triglycerides, such as those marketed under the name of Mygliol®812. Injectable preparations can be freeze-dried according to a traditional process.
Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethyl cellulose, xanthan gum, sodium carboxymethyl cellulose, sorbitane monolaureate polyethylene. Examples of solubilization agents include ricin oil solidified by polyoxyethylene, polysorbate 80, nicotinamide, sorbitane monolaureate polyethoxylated, macrogol and ester ethyl fatty ricin acid. Besides, the stabilizing agent includes sodium sulfate, sodium metasulfate and ester, while the preservative includes methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, benzyl alcohol, phenol, cresol and chlorocresol. One example of the agent of tonicity is mannitol. During the preparation of solutions and injectable suspensions, it is better to ensure that they are isotonic with blood.
Advantageously, the veterinary compositions of the present invention can be administered in association with standard treatments of mastitis of ruminants. Examples of standard care or prophylactic compositions of mastitis are local disinfectants for udders, antibiotics such as penicillins of group M, cephalosporine, gentamycin or colistine or even enzymes such as lysozymes or muramidase.
This present invention further relates to the use therapeutically effective amount of at least one antiprolactinic compound acting as an agonist of dopamine receptors as described previously for the preparation of veterinary compositions to be administered to ruminants during or outside of gestation period for improving their well being and their health, for promoting the reduction of the milk production, for promoting mammary involution, and for treating and/or preventing intra-mammary inflammations and intra-mammary infections. When these veterinary compositions are administered during the gestation period in therapeutic doses that are suitable for the maintenance of gestation, they do not cause any deleterious or abortive effects.
This invention further relates to methods of reducing milk production as well as to methods for the treatment and/or the prevention of mastitis comprising administering the veterinary compositions as previously described in therapeutically effective amount by cutaneous, oral or parenteral administration to the ruminants. The methods according to this invention are particularly advantageous as they promote mammary involution and the reduction of the lactation of the ruminants, even during gestation without causing any deleterious and abortive effects.
This present invention in addition relates a veterinary kit useful to promote the reduction of milk production of the ruminants, to improve the welfare and health of the ruminants, as well as promote mammary involution, treat and/or prevent intra-mammary diseases and/or infections and particularly of mastitis. The kits according to this invention consists of at least one compartment for an eventually sterile conditioning comprising a therapeutically effective amount of at least one antiprolactinic compound agonist of dopamine receptors as described previously for administration to ruminants. The kit comprises means of administering the compositions cutaneously, orally or by parenterally as well as an instruction sheet concerning the mode of administration of the veterinary compositions of the present invention.
EXAMPLES
Example 1
Preparation of Veterinary Compositions with a cabergoline base
Preparation of 9 liters of an injectable solution containing 500 mcg/ml of cabergoline.
Formula:


    Active principle: 4.53 g of cabergoline (titrating 100%)
    Excipient: triglycerides of a medium chain q. s. 9 l.


Step 1: 4.53 g of cabergoline and 1.5 kg medium chain triglycerides were measured in a recipient of adequate capacity, and then a mix was shaken with a magnetic shaker (500 turns per minute) for at least 60 minutes for complete dissolving.
Step 2: In a dry recipient, 5 kg medium chain triglycerides were measured, the flow of nitrogen and the mixer were set and then the cabergoline in a concentrated solution, obtained at step 1, was added to the recipient. The shaking was maintained for 30 minutes and then the volume was brought to a final volume of 9 liters by adding medium chain triglycerides. The shaking was still maintained for another 30 minutes and then solution was filtered under pressurized nitrogen through a calibrated cartridge of 0.45 microns. The filtrate was collected in an appropriate flask previously disinfected by vapor and dried with a flow of nitrogen. The solution obtained was sterilized at 121° C. during 15 minutes, and then was put into sterilized, non-pyrogenic flasks shut with rubber stoppers and aluminum capsules, washed and passed through the autoclave.
Example 2
Clinical Study of the Efficiency of the Cabergoline Based Veterinary Compositions in Ruminants
This study aimed to evaluate the reduction of milk production in 8 multipare cows of the Prim Holstein family in lactation for 6 months. These cows were aged between 3 and 9 years and have a level of production varying between 20 and 31 kg per day at the start of this study. The study was organized according to a design of cross-over (2 treatments×2 periods).
The formulation was described in Example 1, containing 500 mcg/ml of cabergoline in injectable solution that was used in this study.
Treatment Test (C646): Injectable solution of cabergoline of 500 mcg/ml, 1 single intramuscular injection of a dose of 5 mcg/kg that is 1 ml for 100 kg of body weight.
Treatment Control (Placebo): Water for the preparation of injectable, 1 intramuscular injection of a volume identical to that of the treatment test of about 1 ml for 100 kg.
The sequence of the treatment was randomized (A-B or B-A) with a wash-out period of 3 weeks between 2 treatments. The individual milk production was observed at each milking during a period of 7 days prior to the treatment and 7 days after the treatment. Blood samples were taken daily in order to monitor the evolution of prolactin plasmatic concentrations, during a period lasting from 2 days before the treatment to 7 days after the treatment.
The results shown in FIG. 1, show that the injection of the treatment Test of a dose of 5 mcg/kg caused a reduction in milk production:
From the 1st milking following the injection of the treatment Test (12 h), the reduction of the milk production was approximately of a 27%, and could go up to 42% according to the individuals/subjects.
During the follow up period of the 7 days after the treatment Test, the total reduction of the milk production was approximately 15%, and could go up to 27% according to individuals/subjects.
While generalizing the results on a population of cows, these results provided an estimate a reduction of the milk production from the 1st milking between 5 and 60% depending on the animals. The reduction in milk production after the injection of the treatment Test is presumably between 25 and 35% as the following milking. This decrease was maintained during for approximately 2 days. A progressive resumption of the milk production was then observed, nonetheless however without regaining its initial level after a week.
In parallel, the prolactin plasmatic concentrations decreased as shown in FIG. 2. The decrease in the plasmatic concentration in prolactin was very rapid, at 24 hrs following the injection, and was still present one week after the treatment. This prolactin decrease varies between 10 and 80% depending on the animals.
The single administration of cabergoline demonstrated the correlation between the significant reduction of the milk production to the reduction of the plasmatic prolactin.
Example 3
Clinical Study of the Safety of Cabergoline-Based Veterinary Compositions in Ruminants
This study aims to show the absence of risks such as abortions and fetus toxicity during the use of cabergoline in gestating ruminants even in the case of an overdose of cabergoline based veterinary compositions according to the regimen prescribed in the present invention. Groups of cows at different gestating stages receive the treatment at different doses:

    3 months of gestation (6 animals), dose of 15 mcg/kg—2 injections at an interval of 48 hours
    7 to 8 months of gestation: (5 animals), dose of 15 mcg/kg—2 injections at an interval of 48 hours
    6.5 to 7.5 months of gestation: (11 animals), dose of 25 mcg/kg—2 injections an interval of 48 hours


The formulation described in the Example 1, containing 500 mcg/ml of cabergoline in injectable solution is used in this study.
Regular clinical exams were conducted during a period lasting from 7 days before the treatment to 15 days after the treatment:
Daily general inspection and abortions,
Clinical exam of the genital tract including transrectal palpation and an ultrasound, in order to evaluate the suffering and fetal viability.
Blood samplings are conducted one day before the treatment and 15 days after the treatment to follow the plasmatic concentrations of different hormones and proteins indicating the health of the placenta and fetus, such as progesterone, estrone sulfate and the PAG (Pregnancy Associated Glycoprotein). The animals are followed until birth in order to evaluate the health of the newborn calves.
The results show that the two cabergoline injections at intervals of 48 hours to the dosage of 25 mcg/kg do not provoke any abortions; neither do they have any harmful effect on the gestation, or the viability and the health of the new born calves. No variation of the hormones indicating placental or fetal toxicity in connection with the treatments of different doses was observed.
In addition, animals generally tolerated the treatments well. In conclusion, a unique or repeated administration of cabergoline of duration of 48 hours of up to 25 mcg/kg is at least not risky in gestating cows.
Example 4
Clinical Study of the Efficiency and the Safety from Abortions of Quinagolide-Based Veterinary Compositions in Ruminants
9 multiparous cows of the Prim'Holstein race (approximately 600 kg) gestating for about approximately 7 months with a level of milk production of approximately 18 kg are included in this study. The study is organized as a <<cross-over>>: 2 treatments×2 periods, with a <<wash-out>>period of 3 weeks.
The animals receive the treatment Test via intramuscular injectable formulations.
Treatment Test: Injectable solution of 0.5 mg/ml of quinagolide, 1 single intramuscular injection of a dose of 1 to 3 mg/per cow of 1 ml for 100 kg of body weight
Treatment Control (Placebo): Water for the injectable preparation, 1 single intramuscular injection of a volume identical to that of the treatment Test, i.e. 1 ml for 100 kg.
The follow up of the milk production is organized 7 days before and after the treatment day for each period, as well as the inspection of the abortions similar to Example 3 is conducted during the entire duration of the study.
Example 5
Clinical Study of the Efficiency and the Safety Against Abortions of the Bromocriptine Based Veterinary Solutions in Ruminants
9 multiparous cows of the Prim ′Holstein race (approximately 600 kg) gestating for about 7 months with a level of milk production of about 18 kg are included in the study. The study is organized as a <<cross-over>>: 2 treatments×2 periods, with a <<wash-out>>period of 3 weeks.
The animals receive the treatment Test via intramuscular injectable formulations.
Treatment Test (Bromocriptine): Injectable solution of 10 mg/ml of bromocriptine, 1 single intramuscular injection of a dose of 100 to 200 mcg/kg that is 1 ml for 100 to 50 kg of corporal weight.
Treatment Control (Placebo): Water for the injectable preparation, 1 single intramuscular injection of a volume that is identical to that of the treatment Test, i.e. 1 ml for 100 kg.
The follow up of the milk production is organized 7 days before and after the treatment day for each period, as well as the inspection of the abortions similar to Example 3 is conducted during the entire duration of the study.
Example 6
Clinical Study of the Efficiency of Cabergoline-Based Veterinary Compositions for the Prevention of New Mammary Infections
596 dairy cows (298 in each group) with proved healthy udders (i.e. no major pathogen and SCC less than 200 000 cells/ml) and at least 13 kg daily milk yield at drying off were enrolled in the study.
The treatment injection was performed within 4 hours after the last milking. No other concomitant treatment with antibiotic or anti-inflammatory product was authorized.
Up to 72 hours after calving, milk samples of all four quarters were collected and assessed for microbial growth. Six to eight days after, a second milk sample from each quarter was collected and assessed for microbial growth.
The statistical unit for the analysis of the new IMI by major pathogen(s) was the quarter and only healthy quarters with no bacterial growth at drying off were analyzed i.e. 551 in C646 group and 566 in Placebo group.
Results:
The difference between C646 and Placebo regarding the rate of new IMIs was statistically significant: 20.51% (113/551) and 25.97% (147/566) in C646and Placebo groups respectively in ITT population (p<0.025). A single C646injection administered at drying-off was efficient in preventing new IMIs caused by major pathogens.
These results clearly show the efficacy and safety of a single intramuscularly C646 injection (5.6 mg/cow) after the last milking at drying off in the prevention of new intra-mammary infections by major pathogens compared to a Placebo.
Example 7
Clinical Study of the Efficiency of Cabergoline-Based Veterinary Compositions for the Reduction of Clinical Mastitis During the Subsequent Lactation.
263 dairy cows (130 and 133 in C646 and Placebo group respectively) with healthy udders and at least 16 kg daily milk yield at drying off were enrolled in the study.
The treatment injection was performed within 4 hours after the last milking. Cows were treated with intramammary antibiotic at drying in both treatment groups.
Clinical mastitis were recorded during the subsequent lactation.
Results:
The percentage of cow which presented at least one clinical mastitis during the 6 first months of the subsequent lactation was lower in C646 group than in Placebo: 18.0% (22/122) and 26.4% (33/125) in C646 and Placebo group respectively.
Results on all the subsequent lactation showed also a lower percentage of clinical mastitis in C646 group (31.4% (39/124)) compared to Placebo group (37.6% (47/125)).
These results clearly show the efficacy of a single intramuscularly C646 injection (5.6 mg/cow) after the last milking at drying off in reduction of clinical mastitis during the subsequent lactation compared to a Placebo
Example 8
Efficacy of Cabergoline-Based Veterinary Compositions for the Reduction of the Milk Leakages
263 dairy cows (130 and 133 in C646 and Placebo group respectively) with healthy udders (no clinical mastitis) and at least 16 kg daily milk yield at drying off were enrolled in the study.
The treatment injection was performed within 4 hours after the last milking.
Milk leakages were recorded after the drying off (until 7 days).
Results:
The percentage of cows with milk leakages was significantly lower in C646 group compared to Placebo group (p=0.0292): 10.2% (13/128) and 19.8% (26/131) in C646 and Placebo groups, respectively. The efficacy of C646 in reduction of milk leakages was observed on the first week of the dry period.
These results clearly demonstrate the efficacy of a single intramuscularly C646 injection (5.6 mg/cow) after the last milking at drying off in reduction of milk leakages after drying off compared to a Placebo
Example 9
Efficacy of Cabergoline-Based Veterinary Compositions for the Reduction of the Milk Leakages
917 dairy cows (298, 298 and 304 in C646, Placebo and Intramammary AB group respectively) with proved healthy udders and at least 13 kg daily milk yield at drying off were enrolled in the study and allocated to either drying off with the test product C646, Placebo or intramammary antibiotic.
Milk leakages were recorded the day after drying off.
Results:
The percentage of cows presenting milk leakages observed in the C646 group (2.0% (6/298)) was significantly lower than in the Placebo group (10.7% (32/298)) and even more considerably compared to the intramammary antibiotic group (14.8% (45/304)). A single C646 injection after the last milking at drying off was efficient to reduce significantly the percentage of milk leakage on the day following drying off.
These results demonstrate the efficacy of a single intramuscularly C646 injection (5.6 mg/cow) after the last milking at drying off in the reduction of milk leakages compared to a Placebo and an intra-mammary antibiotic referent in an international, double blind, randomised, three-arm, multi centre, clinical study.
Example 10
Efficacy of Cabergoline-Based Veterinary Compositions for the Reduction of Udder Engorgement
263 dairy cows (130 and 133 in C646 and Placebo group respectively) with healthy udders (no clinical mastitis) and at least 16 kg daily milk yield at drying off were enrolled in the study.
The treatment injection was performed within 4 hours after the last milking.
The udder pressure was assessed using a score called “UE. Score” adapted from Leitner (2007) and Gleeson (2007) which ranges from 0 (no pressure) to 3 (high pressure) [Error! Reference source not found., Error! Reference source not found.].

    0: light pressure, the udder is flabby,
    1: medium pressure, the udder yields significantly to gentle pressure from the fingers,
    2: high pressure, the udder yields slightly to gentle pressure from the finger tips, (corresponding to the physiologic engorgement of udder just before a morning milking),
    3: very high pressure, the udder tissue yields with difficulty to gentle pressure from the finger tips.


It was determined by pressing fingers into the tissue and assessing its resistance to pressure in the area of milk cistern i.e. 10 cm above the teats.
Its assessments were performed during the 3 days after drying off after drying off D1, D2, D3.
Results:
The percentage of cows presenting a high udder pressure was lower in C646 group compared to Placebo on the three days after the drying off whereas the percentage of cows presenting a light or medium udder pressure was higher. It can be concluded that C646 injection after the last milking is efficient in reduction of udder pressure during all the engorgement phase of the dry period.
On D1, D2 and D3, the distribution of cows according to their UE score was statistically different between treatment groups: p values <0.0001, 0.0018 and 0.0122 on D1, D2 and D3 respectively). The percentage of cows presenting a high udder pressure was lower in C646 group compared to Placebo on the three days after the drying off whereas the percentage of cows presenting a light or medium udder pressure was higher.
Indeed, the percentages of cows with a high udder pressure (UE score equal to 3) were:

    27.9% (36/129) vs. 67.2% (88/131) in C646 and Placebo group respectively on D1.
    41.5% (54/130) vs. 54.9% (73/133) in C646 and Placebo group respectively on D2.
    27.1% (35/129) vs. 42.1% (56/133) in C646 and Placebo group respectively on D3.


Moreover, the percentages of cow with medium or light udder pressure (UE score equal to 1 or 0) were:

    23.3% (30/129) vs. 5.3% (7/131) in C646 and Placebo group respectively on D1.
    21.6% (28/130) vs. 6.1% (8/133) in C646 and Placebo group respectively on D2.
    21.8% (28/129) vs. 11.3% (15/133) in C646 and Placebo group respectively on D3.


It can be concluded that C646 injection after the last milking is efficient in reduction of udder pressure during all the engorgement phase of the dry period compared to a Placebo.
Example 11
Use of Cabergoline-Based Veterinary Compositions for Increasing Welfare and Reducing Pain after Drying Off
263 dairy cows (130 and 133 in C646 and Placebo group respectively) with healthy udders (no clinical mastitis) and at least 16 kg daily milk yield at drying off were enrolled in the study.
The treatment injection was performed within 4 hours after the last milking.
Udder pain were recorded the day after the drying off (until 7 days)
Results
C646 decreased significantly the percentage of cows with signs of udder pain the day after drying off: 1.6% (2/129) in C646 group vs. 11.5% (15/131) in Placebo.
These results demonstrate the efficacy of a single intramuscularly C646 injection (5.6 mg/cow) after the last milking at drying off in reduction of pain after drying off compared to a Placebo.
Example 12
Use of Cabergoline-Based Veterinary Compositions for Promoting and Seeding the Mammary Gland Involution
Fourteen Holstein cows were included and randomised the day on drying off: 7 cows in each treatment group. The milk yield just before the drying off (mean of the two previous days) was 15.1 kg in mean ranged from 8.3 kg to 20.1 kg.
Biopsies and mammary secretion sample were performed according to Table 1 in Fig. 3.
Laboratory Analysis:
The mammary tissue was used for zymography. Mammary gelatinase activity was determined by using gelatin zymography and gave information on the degradation of the extracellular matrix occurring during the involution of the mammary gland and so on the tissue remodeling.
The mammary secretion sample used for:

    Lactose assay using an infrared method.
    Somatic cell count analysis by fluoro-opto-électronic method.
    Na+ and K+ assays by flame spectrometer emission.
    Lactoferrin assay using radial immunodiffusion.
    Gelatinase activity assay by zymography.

Statistical Method



The efficacy of C646 was assessed for each part of involution process and their consequences on milk composition and clinical udder evolution. Speeding up and amplification of involution processes by C646 was assessed by comparison with evolution of criteria in Placebo group. ANOVA using the MIXED procedure of the SAS software program with REPEATED statement. Time was used as a repeated effect, and cow (treatment) was used as the subject.
Results
C646 amplified the lactoferrin increase in mammary secretion occurring from D4 onwards in both treatment groups (treatment effect p=0.1028). The lactoferrin level was higher in C646than in Placebo group on D4, D8 and D14 with a trend toward significance (p=0.0589, p=0.1266 and p=0.0900 respectively).
C646 tended to hasten the tight junction opening. Indeed Na+ in milk was lower in C646 group than in Placebo group on D1 whereas K+ was higher inversely. C646 hasten the tight junction opening allowing a larger passage of sodium from blood to milk and of potassium from milk to blood on D1. Moreover, the lactose level at D1 was lower in C646group compared to Placebo with significant difference between groups on D1 (p=0.0268) and a trend was also observed on D2 (p=0.1301).
C646 induced a faster decrease of lactose concentration on D1. C646 hastened significantly the decrease of lactose in mammary secretion on the day after drying off.
C646 hastened the extracellular matrix remodelling after the drying off by increasing the activity of MMP in tissue (MMP2) and in mammary secretion (MMP9):
In mammary tissue, C646 increased significantly the activity of MMP total after drying off (significant treatment effect; p=0.0492) in particular the activity of MMP2 total (significant treatment effect; p=0.0138). The MMP2 total activity was significantly higher in C646 group than in Placebo on D1 (p=0.0111) and a trend was observed on D8 (p=0.1153).
In mammary secretion, C646 increased significantly the activity of MMP total in mammary secretion (significant treatment effect; p=0.0165) in particular the activity of MMP9 total (trend of significance; p=0.0521). The MMP9 total activity was significantly higher in C646 group than in Placebo on D1 (p=0.0374), D2 (p=0.0050) and on D3 (trend of significance; p=1036).
C646 hastened also the migration of SCC in the mammary gland after the drying off (significant treatment effect: p=0.0084). The SCC was significantly higher in C646group than in Placebo group on D1, D2 and D3 (p=0.0033, p=0.0021 and p=0.0337 respectively) and a trend was observed on D4 (p=0.0715).
C646 hastened the mammary gland involution; this speeding up concerned the process of extracellular matrix remodelling, SCC migration and tight junction opening (little amplitude).
These results demonstrate the efficacy of one injection of C646at drying off on the involution process of the mammary gland in dairy cows via analysing the mammary tissue remodelling, the mammary cell activity and tight junction opening during the two first weeks of the dry period.

## Claims

1. A method of reducing udder engorgement, and/or reducing pain after drying off, and/or increasing welfare, and/or reducing milk leakage in a gestating cow, the method comprising administering to said gestating cow a veterinary composition comprising cabergoline at a dose ranging from 5 μg/kg to 50 μg/kg body weight, wherein the veterinary composition is administered via a single intramuscular injection per treatment.

2. The method of claim 1 for further preventing new mammary microbial infections, secondary infection, or co-infections.

3. The method of claim 1 for further preventing mastitis during subsequent lactations.

4. The method of claim 1, wherein said veterinary composition is administered to the gestating cow without causing any deleterious or abortive effects.

5. The method of claim 1, where cabergoline is administered at a dose ranging from 5 μg/kg to 25 μg/kg.

6. The method of claim 1, wherein cabergoline is administered at a dose of about 5 μg/kg.