Injectable veterinary composition with anti-prolactin activity

The present invention relates to an injectable veterinary composition, with anti-prolactin activity containing cabergoline as an active ingredient.

Cabergoline{l-[-((6-allylergoline-8β-yl)carbonyl)]-l-[-3 -(dimethyl amino)propyl] -3- ethylurea} is a drug which is known both for its strong anti-prolactin activity, and for its prolonged duration of action and low toxicity.

Its activity in inhibiting prolactin (PRL) secretion has been shown both in vitro, and in vivo on different animal models. The anti-prolactin action of carbergoline appears as a consequence of a direct stimulation of dopaminergic receptors which are present on the prolacting-secreting pituitary cells.

Cabergoline has a wide margin of safety: the therapeutic index (DL 50 and DE 50 ratio) thereof in the rat is 13,000.

A veterinary drug for oral administration is commercially available, which contains cabergoline in a concentration of 50 mcg/ml in a solvent composed of medium chain triglycerides. Such medicament, sold under the trade name GALASTOP®, can be either administered directly into the mouth of the animal, or mixed with food. GALASTOP® finds its reason for use in those conditions governed by prolactin, such as pseudopregnancy and postpartum lactation in female dogs and cats.

However, treatment of pseudopregnancy in the female dog requires a prolonged administration. It has been reported in the literature that the use of cabergoline by oral administration, in doses ranging between 1.5 and 5 mcg/kg/day for two-eight days, determines a clinical response in the decrease of the pseudopregnancy symptoms within three-four days, with the disappearance of manifestations within seven days after treatment. Furthermore, the literature reports that the use of a 5 mcg/kg/day dose orally for five-seven days causes an improvement of the clinical conditions in 80% of the subjects within seven days.

The authorized dosage regimen of Galastop for oral use in the dog and cat is 5 mcg/kg/day for 4 - 6 days.

The object of the present invention is to identify a new administration form of cabergoline which, within the scope of the therapeutic treatments indicated above, is suitable in order to achieve a substantial decrease of the active ingredient which has to be administered, and further suitable in order to alleviate treatment through a reduced number of administrations.

The present invention thus relates to an injectable composition containing cabergoline, having the characteristics defined in the following claims. Such Injectable composition comprises a therapeutically effective amount of cabergoline in an oily solvent and is intented for parenteral administration to non human animal.

In particular, a preferred composition which is the object of the invention is an injectable veterinary composition, intended to the administration via subcutaneous route, comprising a therapeutically effective amount of cabergoline ranging from 10 to 40 mcg/ml, 10 to 30 mcg/ml, preferably from 10 to 20 mcg/ml, and most preferably around 15mcg/ml in an oily solvent. Such concentration range allows administrating a volume of inoculum which is optimal for veterinary use. Most preferred compositions are in the form of a sterile composition or solution for parenteral administration.

As indicated above, the injectable formulation which is the object of the invention has important therapeutic advantages compared to the commercially available anti-prolactinic drug-based formulations for oral administration; in particular, the advantage of reducing the number of administrations and treatment time. This means that in order to achieve the same therapeutic results changing the administration route to the parenteral route has substantially modified the pharmacodynamics of the molecule, resulting in a quicker response associated to a lesser number of administrations. Therefore, injectable compositions and route of administration according to the present invention allow using substantially lesser amounts of the active ingredient cabergoline, thereby alleviating treatment through a reduced number of administrations. Use of injectable compositions

according to the present invention allow for a remarkable reduction of the number of administration and treatment time when compared with existing oral forms, Le _. ., one or two injections are prescribed versus four to eight oral administration once or twice a day. Also, injections of lesser amount of the active ingredient cabergoline, from 10 to 40 mcg/ml, 10 to 30 mcg/ml, preferably from 10 to 20 mcg/ml, and most preferably around 15mcg/ml are only required. Most preferably, two injections of the pharmaceutical composition are performed with an interval ranging between 48 and 72 hours, in an amount of 1.5 mcg/kg p.v. cabergoline.

The oily solvent can be one oil from almond oil, wheat germ oil, peanut oil, sesame oil or, particularly, a triglyceride or a mixture of triglycerides comprising medium chain (C ₈ -C i ₀ ) triglycerides which are liquid at room temperature. A preferred solvent is a mixed capric acid/caprilic acid triglyceride, obtained from fractionated coconut oil, commercially available under the commercial name MIGLYOL® 812. Such oil has a capric acid percentage from 30 to 45% total fatty acids, and a caprilic acid percentage from 50% to 65% total fatty acids, ensuring an excellent oxidative stability (peroxides indexes in mEq O/kg below 1 and a low viscosity, ranging between 27-33 mPa.s). Oily solvent comprising selected triglycerides as described are suitable and adapted to a parenteral administration. More importantly selected oily solvent have been evidenced as being compatible with the active ingredient cabergoline and ensure the chemical physical and microbiological stability of the pharmaceutical compositions of cabergoline. In effect, performed tests have evidenced a surprisingly high solubility and stability of cabergoline in the oily solvent comprising medium chain triglycerides as described above. Pharmaceutical compositions of cabergoline advantageously present an excellent stability when stored for long periods of time, up to 36 months at room temperature. Also, pharmaceutical compositions present excellent characteristics of safety, fluidity and syringeability ensuring easy administration via the parenteral route.

In addition to the oily solvent, the compositions according to the present invention may also include conventional ingredients of pharmaceutical formulations.

The present invention also relates to a method of treating disorders associated with an

increase of prolactin, comprising administering via the parenteral route, a therapeutically effective dose of the above-described pharmaceutical compositions to a patient, such as a non human animal.

Therapeutically effective dose of cabergoline composition is intended to mean a dose which is effective in alleviating disorders associated with a prolactin increase, for example by inhibiting prolactin secretion.

Generally, in the animal therapy, the veterinary and/or farmer may easily determine the dosing depending on the disease, age, weight of the animals, and other parameters.

Non human animals are intended to mean all mammals, with the exception of human beings, including inter alia domestic or companion animals such as cats, dogs, pigs, camels, ovine, bovine, horses, and rodents.

The present invention further relates to a method of preparation of the parenteral compositions of cabergoline. Such method of preparation comprises the solubilization of the active ingredient in the oily solvent in order to obtain a concentrated solution, thereby performing the final dilution with the same solvent. The solution is then subject to sterilization, Le_., the solution being for example autoclaved, preferably at 121 ⁰ C for 15 minutes, then packed or dosed in sterilized and eventually depyrogenated bottles equipped with closing means such as for example washed and autoclaved rubber stoppers and aluminium caps. The obtained solutions may be stored and remains clear and stable for 36 months from the physical, chemical, and microbiological point of view when in its intact package.

The present invention further relates to a pharmaceutical or veterinary product comprising a container or a bottle which has been specifically designated to allow storage of the cabergoline composition under stable conditions, without any degradation or oxidation, for long periods of time, even when the containers are stored upside down. Such containers or bottles are filled with the composition as described above and are equipped with a closure rubber stopper in nitrile or chlorobutyl rubber coated with a fluoropolymeric film. In

effect, rubber stopper made of in nitrile or chlorobutyl rubber coated with a fluoropolymeric film have surprisingly showed no adsorption of cabergoline, allowing to store the containers in all positions for long period of time, even in the upside down position.

The in-use stability study has further shown, by assessment of chemical, physical, and microbiological parameters, the formulation stability for 28 days from the first withdrawal from rubber stopper of the bottle holding the solution. In the stability after first withdrawal and the intact package stability (upside down bottle), it has been shown that there are no interferences or adsorption by the same rubber stopper on the active ingredient cabergoline. This object has been achieved by the nature of the used material: nitrile rubber or chlorobutyl rubber coated only with a fluoropolymeric film. The use of other traditional stoppers causes the adsorption of the active ingredient and the resulting loss of the same in the end product.

Finally, the present invention relates to kits intended for veterinary use and for treatment of non human animals affected by disorders associated to an increase of prolactin. Such kits may the container and the pharmaceutical compositions of cabergoline as previously described, as well as instructions on the mode of administration of the composition to a subject.

The present invention will be better understood from the Examples herein below. EXAMPLES

Example 1 : Preparative example

Preparation of 50 L injectable solution containing 15 mcg/ml cabergoline.

Formula

Active ingredient: 0.75 g cabergoline (substance with a 100% titer).

Excipients: medium chain triglyceride q.s. to 50 L.

Production process:

Step 1 : 0.75 g cabergoline and 2.25 kg medium chain triglycerides is dosed in a vessel having a suitable capacity; then, it is stirred under a nitrogen flow for at least 60 minutes to complete dissolution;

Step 2: in a dry solution vessel, 30 kg medium chain triglycerides is dosed; the nitrogen flow and stirring is initiated, then the cabergoline concentrated solution which has been obtained in step 1 is poured in the solution vessel; it is kept under stirring for 30 minutes, then it is brought up to the end volume of 50 L with medium chain triglycerides; then, stirring is resumed for additional 30 minutes; it is filtered under nitrogen pressure through a 0.45 μm cartridge; filtrate is collected in an storage tank already sanitized in a flow of vapour, and dried in a nitrogen stream; the obtained solution is sterilized at 121 °C for 15 minutes; then, the solution is dosed into sterilized and depyrogenated bottles equipped with rubber stoppers and aluminium caps, also washed and autoclaved.

Clinical study

Efficacy of cabergoline in the injectable preparation which is the object of the invention has been verified in particular in the treatment of the spontaneous pseudopregnancy of the female dog. In the test, the formulation obtained in the previous preparative example containing 15 mcg/ml cabergoline has been used, herein below designed as injectable formulation VEGA.

Experimental protocol

The test has been divided in three STEPS.

70 female dogs of different age, weight, and breed have been included in the test under examination, exhibiting overt clinical signs of pseudopregnancy.

In STEPS I and II, the female dogs have been assigned to the different treatment groups by a randomization protocol for parallel groups.

STEP III provided the inclusion of the subjects in a single treatment group.

Only in STEP I the control group was represented by female dogs treated with GALASTOP® (cabergoline for oral use) at the expected dose of 5 mcg/kg p.v.

STEP I: 20 female dogs received injectable VEGA (0.1 ml/kg, equal to 1.5 mcg/kg p.v.) subcutaneously at day 0 and after 48 (group A) or 72 (group B) hours. Ten subjects received GALASTOP® per os (group C).

STEP II: 30 female dogs received injectable VEGA (0.1 ml/kg, equal to 1.5 mcg/kg p.v.) subcutaneously at day 0 and after 48 (group A, 15 female dogs) or 72 (group B, 15 female dogs) hours.

STEP III: 10 female dogs received a single administration of injectable VEGA at the dose of 0.2 ml/kg, equal to 3.0 mcg/kg p.v.

The doses used were 1.5 mcg/kg p.v. for two administrations at 0 and 48 hours, or 0 and 72 hours in the STEPS I and II, or 3.0 mcg/kg p.v. in a single administration in STEP III.

Results

Results showed that the injectable formulation VEGA administered subcutaneously at a dose of 1.5 mcg/kg/day at 0 hours and after 48 or 72 hours, determines a remission of the pseudopregnancy symptoms in the female dog. In particular, the test showed how the remission of symptoms is produced in a lesser period of time in the subject treated with VEGA, which in a high (70-90%) percentage were completely recovered already at the sixth day after the administration, unlike 20% subjects treated with oral preparation.

The advantage of the injectable preparation resides in the lower total dose of administration and in the higher ease of use (two injections) compared to the oral administration prolonged for 4-6 days.

The evaluation of side-effects allowed verifying tolerability for this preparation, which has caused vomiting phenomena in a low percentage of subjects.