FIELD OF THE INVENTION

The present invention relates to a premix for feeding animals, comprising a carrier, a beta agonist and a surfactant, said premix being formulated with smaller particles than usual, therefore presenting a better uniformity when mixed with food.

BACKGROUND

Premixes comprising active principles are currently used for orally delivering active compounds to animals, in particular to farm animals. These premixes are generally composed of a premix carrier of mineral or vegetal origin, a small quantity of active principles, and additives and excipients. They are easily incorporated to food, according to specific instructions of dosage, which allow the oral deliverance of the right dose of the active principle to the animals.

Drawbacks of this method are mainly the lack of homogeneity when the premix is incorporated into the animal food. Indeed, even if the blending is performed carefully, obtaining a homogeneous mix is challenging, due to the huge differences between the premix and the food ingredients, in terms of particles size, weight, hydrophilic and lipophilic features. Moreover, many active principles have electrostatic characteristics that make it difficult to homogenize the blend, since these compounds tend to accumulate in dead areas, on the walls of the blenders and of food containers. These drawbacks are of particular relevance when the active components of the premix are beta agonists. Beta agonists are used to increase the rate of weight gain, to improve feed efficiency and increase carcass leanness during the last days on feed of breeding animals. Among them, the compound zilpaterol hydrochloride is widely used under the form of a premix, sold under its trade name Zilmax®. Zilpaterol is typically fed in the last three-six weeks of cattle's lives, with a brief period before death for withdrawal, which allows the drug to leave the animal's tissues. Process for its preparation is described in patent US 5,731,028.

For these pharmaceutical ingredients, the recommended dose is about 5 to 20 grams per ton of animal food, and these extreme levels are very challenging in terms of homogeneity of the final mix. The lack of homogeneity in the animal food can induce a variation of consumptions of the active principles. If the proper dose is not administered in an uniform way in all animals, there will be a lack of uniformity in the benefits expected by the farmer. Various strategies have been developed for obtaining more homogeneous premix and final mix.

US 5,624,710 describes a premix wherein the granules supporting the active substances are coated with a water-soluble substance. It also describes the process for preparing said granular premix with particles having a diameter about the same size than the particles of animal feed, ranging between about 100 and 1,000 microns (μιη).

The patent application EP 197 188 suggests the use of a non- ionic, physiologically compatible surfactant, to be sprayed during the blending of the premix, in order to limit the dust formation during the blending. These surfactants are for example monoesters of propylene glycol and of alimentary fatty acids. Moreover, this premix is characterized with a specific particle size of the support, between 300 and 800 microns, and a specific particle size of the active ingredient, between 50 to 200 microns.

The commercialized product Zilmax® is produced by a process wherein the zilpaterol is dissolved in a fluid and sprayed onto the premix carrier (i.e. corn particles). The mixture is then dried and sieved. This long, expensive and environmental unfriendly process does not enable a suitable homogeneity of the mixture.

The patent US 8,343,956 describes a process for making a crystalline zilpaterol salt, in order to obtain zilpaterol particles that have a smaller size than the support particles. The use of small zilpaterol particles allows the homogeneous adhesion of said particles to the support, to ease the formulation of the final mix. However, when this premix is blended with the animal feed, the variation coefficient is of about 20%, and in all cases stays above 10% in the final mix, which should be improved.

In the present invention, zilpaterol salt is not dissolved in a fluid, but is mixed in solid form with the premix carrier. Moreover, the particles sizes of the premix carrier, and of the zilpaterol, have been improved and are as described hereunder. The present invention provides a premix composition, characterized by the allowance of a very good homogeneity of the final mixture, while being easier, faster and cheaper to put into practice.

BRIEF DESCRIPTION OF THE INVENTION

The present invention concerns a premix composition for administering zilpaterol to cattle, comprising: a) a premix carrier having an overall particle size comprised between 300 and 400 μπι, b) zilpaterol, and c) a surface agent.

The present invention is also related to a method of increasing the rate of weight gain in cattle, a method of increasing quality of meat in cattle, and a method of increasing carcass leanness in cattle, comprising the administration to the cattle of feed additives consisting of a premix composition according to the invention, over a period of at least two weeks.

FIGURES

Figure 1. Visual aspect of the Zilmax® premix (upper tube) and the premix according to the invention (lower tube). Figure 2. Process for the preparation of the premix and samples.

Figure 3. Particule size distribution (size is expressed in μηι) of the premix composition according to the invention, and of the Zilmax® premix.

(A) Table of the size distribution of the particles of the premix according to the invention; - (B) Graph showing the size distribution of the particles of the premix according to the invention; (C) Graph showing the size distribution of the particles of the commercially available premix Zilmax®;

- (D) Table of the size distribution of the particles of the commercially available premix Zilmax®. Figure 4. Measure of the coefficient of variation at 1 minute, 2 minutes and 3 minutes in a mineral mix, prepared with Zilmax® (Reference) or with the premix composition according to the invention (Invention).

Figure 5. Mesure of the Coefficient of variation (C.V.) in the final mix (food + premix), in function of the mixing time. A) C.V. of the mix Zilmax® and food;

B) C.V. of the mix "premix of the invention" and food.

DETAILLED DESCRIPTION OF THE INVENTION

The present invention is related to a premix composition comprising: a) a premix carrier having an overall particle size comprised between 300 and

400 μιη, b) zilpaterol, and c) a surface agent.

In a specific aspect of the invention, the particles of the premix carrier are defined as follow:

• at least 90% of the particles have a size equal to or below 730 μπι (D90);

• at least 50% of the particles have a size equal to or below 315 μηι (D50);

• at least 10% of the particles have a size equal to or below 60 μπι (D10). The following terms are defined for a better understanding of the invention. The term "premix carrier" designates edible, non-toxic compositions suitable for incorporation in livestock feed, that are the support for active principles. This support is chemically inert and is generally constituted of meals or flours, such as calcium carbonate, soybean mill run, rice mill hulls, wheat middlings, wheat bran, corn gluten, corn gluten meal, and other mill run byproducts. The term "support" designates the same object and is used indifferently in the application.

The term "particle size" refers to the size of the particles in μπι, as measured with an appropriate apparatus, such as a Beckman Coulter-SVM particle size analyzer or a Mastersizer 2000 particle size analyzer. Such apparatus use a technique of laser diffraction to measure the size of particles. It operates by measuring the intensity of light scattered, as a laser beam passes through a dispersed particles sample. This data is then analyzed to calculate the size of the particles that created the scattering pattern.

In the framework of the invention, "a particle size of x μητ", can be interpreted as "a particle size of about x μπι", wherein "about" means a value comprised within more or less 10%, or more or less 5% of the given value.

The term D10 refers to a value where at least 10% of the particles have a size less or equal to the given value. For instance a D10 value of 15μπι, means that at least 10% of the particles have a size equal to or below 15μπι.

The term D50 refers to a value where at least 50% of the particles have a size less or equal to the given value.

The term D90 refers to a value where at least 90% of the particles have a size less or equal to the given value.

The term "overall particle size" refers to the D50 value.

The phrase "the overall particle size is comprised between 300 and 400 μπι" means that the D50 is from about 300 μπι to about 400 μιη, and in particular the specific embodiments wherein the premix carrier has a D50 =300 μηι or a D50 = 400μπι belong to the scope of the invention. Naturally the given percentages of particles are not to be comprised strictly, but means "about at least 90%", "about at least 50%" and "about at least 10%". In particular a slight derivation, of less than 5%, of the number is tolerated. Therefore, for example, a premix composition comprising 89% of the particles having a size equal to or below 730 μπι is comprised in the invention.

Ziloaterol

The terms "zilpaterol" "zilpaterol hydrochloride" and "zilpaterol HCL", used interchangeably, refer to a beta-adrenergic agonist, of CAS number 117827-79-9, and showing the chemical formula:

Zilpaterol is usually presented under crystallized form, a process of preparation is described in US 5,731,028. Zilpaterol can be recrystallised to obtain the suitable granulometry. Such crystallisation is performed using usual solvents such as:

- polar aprotic solvent such as ethyl acetate, dichloromethane, acetone, or

- polar protic solvent, such as methanol, ethanol, isopropanol...

The term "surface agent" or "surfactant", used interchangeably, refers to compounds that lower the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Non-ionic surfactants that do not ionize in water are preferred. Various types can be distinguished according to their chemical nature: ester bond (R-CO-O-R'), ether bond (R-O-R') or amide bond.

In a specific aspect of the invention, the premix composition comprises zilpaterol, wherein the particles of zilpaterol present the following size features:

- at least 90% of the particles have a size equal to or below 177 μπι;

- at least 50% of particles have a size equal to or below 101 μπι; - at least 10% of particles have a size equal to or below 15 μπι.

Said specific crystallized anhydrous zilpaterol is obtained from crystallisation using usual solvents such as described above. Zilpaterol can also be micronized with a suitable apparatus to obtain the desired size distribution. In a particular aspect of the invention, the particles of zilpaterol can be much smaller, and in particular can be defined as follow:

- at least 90% of the particles have a size equal to or below 15 μην,

- at least 50% of particles have a size equal to or below 5 μπι;

- at least 10% of particles have a size equal to or below 2 μπι. Zilpaterol is present in the premix composition under a crystallized form.

According to a specific embodiment of the invention, zilpaterol is present in the premix composition at a concentration ranging from about 2% to about 7%, more preferentially from about 4% to about 5% in weight, related to the total weight of the composition.

Premix carrier As presented above, the premix carrier is the inert support for the active principle zilpaterol. In a specific embodiment of the invention, the premix carrier is of mineral or vegetal origin. Preferably, the premix carrier is obtained from corn, and most preferably from corn cobs. Indeed, a corn cob has the ability to absorb up to seven times its own weight, which confers him excellent properties as a carrier. Furthermore, it is safe for animals, ecologically friendly and biodegradable.

According to a specific embodiment of the invention, the premix carrier is present in the premix composition at a concentration ranging from about 70% to about 90%, preferentially from about 80% to about 85% in weight, related to the total weight of the premix composition. Surface aeents

It has been shown in EP 197 188 that addition of physiologically compatible, non-ionic surface agents to premix composition allows a better homogenization of the blend. Typically, the surface agent is sprayed during the process of blending the premix carrier and zilpaterol.

In particular, a process for preparing the premix composition comprises at least the following steps: o blending the premix carrier and zilpaterol for about 5 to 20 minutes; o spraying over the mixture the surface agent.

According to a first aspect of the invention, the surface agent is an ionic surface agent; the man skilled in the art knows various ionic surface agents that can be sprayed over the premix composition, in particular: · Among the anionic surfactants, such as sulfate, sulfonate, phosphate esters, and carboxylates, the man skilled in the art will choose for instance ammonium lauryl sulfate, sodium lauryl sulfate, sodium dodecyl sulfate (SDS), alkyl-ether sulfates such as sodium laureth sulfate, sodium myreth sulfate, docusates such as dioctyl sodium, sulfosuccinate, perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate, linear alkylbenzene sulfonates (LABs), carboxylates such as sodium stearate, sodium lauroyl sarcosinate, perfluorononanoate, and perfluorooctanoate (PFOA or PFO).

• Among the cationic surfactants, such as primary, secondary, or tertiary amines, the man skilled in the art will choose for instance octenidine dihydrochloride, quaternary ammonium cation such as alkyltrimethylammonium salts, cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide, cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, 5-bromo-5-nitro-l,3-dioxane, dimethyldioctadecylammonium chloride, cetrimonium bromide, and dioctadecyldimethylammonium bromide. · Among the zwitterionic surfactants, the man skilled in the art will choose for instance (3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate) or lecithin.

According to a specific embodiment of the invention, the surface agent is non- ionic, and is chosen from the group consisting of: ricin oil; polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives (usually named 'polyoxyl') which comprises agents such as Polyoxyl 5 castor oil, Polyoxyl 9 castor oil, Polyoxyl 15 castor oil, Polyoxyl 35 castor oil, Polyoxyl 40 castor oil, Polyoxyl 40 hydrogenated castor oil, Polyoxyl 60 castor oil, Polyoxyl 60 hydrogenated castor oil, monoesters of propylene glycol; monoesters of alimentary fatty acids; stearyl-2-lactyl acid; acetic, lactic, citric, tartaric, monoacetyl tartaric esters of mono- and -diglycerides of alimentary fatty acids; glycerol-polyethylene glycol ricinoleate; polyethylene glycol esters of soybean oil fatty acids; sorbitan esters such as sorbitanmonostearate; sorbitantristearate; sorbitanmonolaurate; sorbitanmonooleate, and sorbitanmonopalmitate; propylene glycol alginate, polyoxyethylene stearates, polyoxyethyleneglycol esters, glycerol esters, glycerylmonooleate, polyoxyethylenesorbitan fatty esters, polysorbates, and combinations thereof.

In a preferred embodiment, the non-ionic surface agent is ricin oil. In another preferred embodiment, the non-ionic surface agent is polyoxyl.

In another preferred embodiment, the surface agent is present in the premix composition at a concentration ranging from about 2% to about 20% in weight, preferably from 5% to 15%, and more preferably from 8 to 10%, related to the total weight of the composition.

In a specific embodiment, the density (mass per unit of volume) of the premix composition is comprised between 200 g/1 to 400 g/1. In a preferred embodiment of the invention, the premix composition comprises at least the following relative amounts in weight of components:

• from about 80% to 85% of premix carrier;

• from about 4% to 5% of zilpaterol; and

• from about 2% to 20% of surface agent. In a specific embodiment, the premix composition may also include optional ingredients such as tackifiers (compounds used in formulation to increase the tack, the stickiness), anti-oxidants, vitamins, preservatives, colorings, flavorings, minerals, other dietary supplements, and the like. In a preferred embodiment, the premix composition comprises: a) from 80% to 85% of corn cobs; b) from 4% to 5% of zilpaterol; and c) from 2% to 20% of Polyoxyl, and the particles of the premix composition are defined as follow: at least 90% of the particles have a size equal to or below 730 μπι; at least 50% of the particles have a size equal to or below 315 μηι; at least 10% of the particles have a size equal to or below 60 μπι.

In another preferred embodiment, the premix composition comprises: a) from 80% to 85% of corn cobs; b) from 4% to 5% of zilpaterol; and c) from 2% to 20% of Polyoxyl, and the particles of zilpaterol present the following size features:

- at least 90% of the particles have a size equal to or below 15 μπι;

- at least 50% of particles have a size equal to or below 5 μπι;

- at least 10% of particles have a size equal to or below 2 μπι.

In another preferred embodiment, the particles size of the premix composition and of the zilpaterol are as presented above.

In another preferred embodiment, the premix composition comprises: a) from 80% to 85% of corn cobs; b) from 4% to 5% of zilpaterol; and c) from 2% to 20% of Polyoxyl, and the particles of the premix composition are defined as follow: at least 90% of the particles have a size equal to or below 730 μ ^η ι; at least 50% of the particles have a size equal to or below 315 μιη; at least 10% of the particles have a size equal to or below 60 μπι. and the particles of the zilpaterol are defined as follow:

- at least 90% of the particles have a size equal to or below 177 μιη;

- at least 50% of particles have a size equal to or below 101 μπι;

- at least 10% of particles have a size equal to or below 15 μπι.

Uses of the premix composition The premix composition of the invention is intended to be administered orally to cattle.

Preferentially, the premix composition of the invention is intended to be mixed with food for cattle, in order to administer the right daily dose of zilpaterol to said cattle. The recommended final concentration of zilpaterol in the finished food is about 6 ppm (parts-per-million, 10 ^~6 ).

In the sense of the invention, "cattle" designates the most common type of domesticated ungulates, Bos taurus, and include cows, veals, and bulls.

Food for cattle is mainly composed of high grains and roughage. Presence of roughage is important in particular in the introductory period, then grains can be increased slowly so that the gastro-intestinal tract has an opportunity to adapt to the increased starch content of the diet. Examples of high grains include, in a non-limitative manner, cottonseed meal, soybean meal, sunflower meal barley corn, oats, sorghum, and wheat.

The usual process for blending the premix composition and the food comprises generally two successive steps: - a first dilution comprising the addition of 125g of premix to 99,875 kg of grounded grain or a mineral powder, and - a second dilution of 100 kg of the "first step mix" with 900 kg of food.

Examples 2 and 3 below present a process for blending the premix composition and the food in two successive steps.

Advantageously, the premix composition of the invention is mixed homogeneously with the food, and animals get the right dose of zilpaterol, when instructions of mixing are carefully followed. Typically, the amount of zilpaterol consumed daily will range from 60 to 90 mg by head.

The present invention is related to a method of increasing the rate of weight gain in cattle, comprising the administration to the cattle of feed additives consisting of any premix composition as described above, over a period of at least two weeks.

The gain in weight is linked to a gain of body mass, mainly a gain in muscle and fat mass.

The present invention is also related to a method of increasing quality of meat in cattle, comprising the administration to the cattle of feed additives consisting of any premix composition as described above, over a period of at least two weeks.

The "quality of meat" designates the tenderness, juiciness, and appetizing color of the meat.

The present invention is also related to a method of increasing carcass leanness in cattle, comprising the administration to the cattle of feed additives consisting of any premix composition as described above, over a period of at least two weeks.

The "carcass leanness" designates the fat-free consumable mass in the animal. It can be estimated in live animals by different methods, well-known by the man skilled in the art, such as real-time ultrasounds mesurements.

In a specific embodiment, the administration of the premix composition is interrupted at least three days before death, allowing the drug to leave the animal's tissues before slaughter. EXAMPLES

Example 1: Preparation of the premix composition

In an horizontal mixer, 210 Kg of corn cobs were added along with 12.06Kg of zilpaterol HC1, and the mixing process was started, during 10 minutes, once a proper visual uniformity was reached.

The used zilpaterol salt presents the following features of granulometry: D10: 12,51 μηι; D25: 44,52 μηι; D50: 93,75 μπι; D75: 136 μπι; and D90: 173,3 μπι, as measured with a LS particle size analyser (Beckman Coulter).

25 Kg of Polyoxyl 35 were added by spraying over the premix, while the mixer is running. Then the mixing process was stopped and the appearance of the premix was checked, once approved, the mixer was started again, and 3.8 Kg of corn cob was added to fit with the desired batch size.

Once verified the aspect of the premix, it was sampled and bottled in 5 Kg bags.

Figure 1 shows the visual aspect of the premix composition such as obtained (lower tube), compared to the commercially available Zilmax® (upper tube). The premix composition of the invention presents particles of small size, although the commercially available premix has bigger particles.

Figure 2 presents the process of preparation, as described above, and the sampling to test the coefficient of variation.

Figures 3A and 3B show the granulometry of the premix composition, i.e. the size distribution of the particles, as measured with a Mastersizer 2000 particle size analyser. As can be calculated from the table shown in figure 3A:

- 89,86% of particles have a size less or equal to 724,436 μπι;

- 50,6% of particles have a size less or equal to 316,228 μπι;

- 11,08% of particles have a size less or equal to 60,256 μηι. Figures 3C and 3D show the granulometry of the premix Zilmax®. For this commercially available support, and as can be calculated from the table shown in figure 3D, column 5:

- 100% of particles have a size less or equal to 954,993 μπι; - 63,25% of particles have a size less or equal to 831 ,764 μπι;

- 11,52% of particles have a size less or equal to 630,957 μπι.

It appears, from this granulametry measures, that the D10 value of Zilmax® is ten times superior to the D10 value of the premix according to the invention. D50/D60 values are also significatively different. Example 2: First step of blending with a mineral powder

Using samples from the premix composition prepared as presented in example 1, a mixing test was made, comparing the formula of this invention with the formula of the premix Zilmax®, during the mixing process in an horizontal blender, mixing zilpaterol HC1 premixes with mineral salts. 84 g of the premix were mixed with 19.916 kg of a mineral mix, in a Lodige mixer with a working capacity of 30 kg.

4 samples in different points of the blender were taken at 1, 2 or 3 minutes.

Surprisingly, the premix of example 1 gave quite better results than the referenced product, measured as the Variation Coefficient (VC), as expressed in the graphic shown in figure 4.

Variation coefficient is obtained by the following formula:

VC = Standard Deviation of samples concentration X 100 / average of samples concentration

"Samples concentration" designates the concentration of zilpaterol HC1, obtained when performing a UPLC analysis with a validated analytical method. It can be observed that the mixing profile of the premix composition is better than the one of Zilmax®, reaching outstanding Variation Coefficients, 10% being considered as a good mixing and 5% as excellent. The Variation Coefficient reached at 3 minutes is remarkably good for the premix of the invention. Example 3: Second step of blending with food

20 kg of the mineral powder loaded with premix compositions were mixed with 280 kg of food.

Three samples in different points of the blender were taken at 1, 2 or 4 minutes. The coefficient of variation was measured as shown above, and results are shown in figures 5 A and 5B; it appears that after one or two minutes of mixing, the final mixes have comparable qualities - 40% then 20% of coefficient of variation. However, after four minutes of mixing, the final mix composed of food and premix of example 1 reaches a coefficient of variation of 10% only, although the final mix composed of food and Zilmax® presents a coefficient of variation of 40%. Example 4:

Using product manufactured as explained in the Example 1, a stability study was carried out, under three conditions:

30°C and 65% of relative humidity during a long term period of at least two years, - 40°C and 75% of relative humidity during 6 months, and

50°C and 90% of relative humidity during a period of three months. Even under the most severe condition, the product remained stable. REFERENCES

EP O 197 188 US 5,624,710 US 5,731,028 US 8,343,956