Claims:WE CLAIM
1. A composition comprising granules of copper salt of alpha-ketoglutarate, wherein the granules comprise an amount of copper alpha-ketoglutarate together with acceptable excipients.

2. The composition according to claim 1, wherein copper alpha-ketoglutarate granules comprise:
- copper alpha-ketoglutarate: 50 – 100 mg;
- diluent: 3.27 – 4.61 gm;
- binder: 2.5% - 30%;
- lubricant: 1% - 7.5%

3. The composition according to claim 2, wherein the diluent is chosen in the group consisting of guar gum, cellulose, microcrystalline cellulose, calcium phosphate, starch, kaolin, dehydrate calcium sulphate, calcium carbonate, lactose, saccharose, glucose, sorbitol, dextrose, mannitol or their mixtures.

4. The composition according to claim 2, wherein the binder is chosen in the group consisting of corn starch, acacia, tragacanth, gelatin, starch paste, pregelatinized starch, alginic acid, cellulose, starch 1500, methocel, walocel, luvicross, luvicaprolactam or their mixtures.

5. The composition according to claim 2, wherein the lubricant is chosen in the group consisting of magnesium stearate, silica, magnesium or calcium stearate, sodium stearyl fumarate, hydrogenated vegetable oils, mineral oils, polyethylene glycols, sodium lauryl sulphate, glycerides, glyceryl dibehenate, glycerol stearate or their mixture.

6. The composition according to claim 2, wherein copper alpha-ketoglutarate granules comprise:
- copper alpha-ketoglutarate: 70 - 90 mg;
- guar gum: 3.7 - 4.5 gm;
- corn starch: 6 – 10 %;
- magnesium stearate: 1 – 3 %

7. The composition comprising copper alpha-ketoglutarate granules according to claim 1, comprising one or more pharmaceutically acceptable nutritional ingredients.

8. The composition according to claim 4, wherein the nutritional agent is chosen in the group consisting of thiamine, folic acid, pantothenic acid or their mixtures.

9. The composition comprising copper alpha-ketoglutarate granules according to claim 1 in form of tablets, capsules, or granulate in bags.

10. A process for the preparation of copper alpha-ketoglutarate granules comprising an amount of copper alpha-ketoglutarate between 50 mg to 100 mg comprising the step of:
- Dry mixing copper alpha-ketoglutarate in an amount comprised between 50 mg to 100 mg with a diluent in an amount comprised between 3.27 – 4.61 gm;
- Preparing binder paste with binder in an amount comprised between 2.5% to 30% at 90°C
- Using binder paste to prepare dough of copper alpha-ketoglutarate and diluent followed by screening through a sieve
- Drying the granules at temperature comprised between 40°C and 60°C in a time range higher than 12 hours
- Screening the granules for uniform size followed by adding lubricant in an amount comprised between 1% to 7.5% for a time comprised between 5 minutes to 15 minutes

Dated this on 28th Day of November 2020
, Description:FIELD OF THE INVENTION
The present invention relates to the oral drug delivery system comprising copper salt of alpha-ketoglutarate (Cu-AKG) as a drug for treating mastitis. More particularly, the invention relates to composition comprising granules of copper salt of alpha-ketoglutarate (Cu-AKG) as a drug for treating mastitis. The invention also describes processes for their preparation and their use in the treatment and prevention of pathologies directly or indirectly related to mastitis.
BACKGROUND OF THE INVENTION
Conditions caused by mastitis are among the most common cases of illness and death and death in cattle, and have a great influence on dairy farmers. It is a creates a direct impact on dairy industry in terms of the production of raw milk and related profitability, providing high quality milk to consumers, and above all health of the cattle. Commercial products for the treatment of mastitis include tablet, capsules and injectables.
Mastitis occurs when the leucocytes (WBC) are released into the mammary glands in response to bacteria invading the teat canal or thermal trauma on the udder. A condition of oxidative stress is produced when the production of oxidants exceeds the capacity of antioxidant defense, resulting in oxidative damage to macromolecules such as lipids, DNA and proteins. During the early lactation period of dairy cows mastitis induces the increase of free radicals formation in milk and leading to oxidative stress. Supplementation of antioxidant vitamins and trace elements stabilize the highly reactive free radicals generated as a result of oxidative stress during mastitis thereby they may reduce inflammatory response and; maintain the structural and functional integrity of cells.
Copper (Cu) is an integral component of ceruloplasmin, which facilitates iron absorption and transport. Its deficiency in cattle is generally due to the presence of dietary antagonists, such as sulfur, molybdenum and iron that reduce Cu bioavailability. In clinical mastitis there is significant decrease in blood SOD and catalase activities, reduced glutathione (GSH) concentration and an increase in erythrocytic lipid peroxides occurs. The enzyme SOD, present in the cytosol of cells and extracellularly, is Cu and Zn dependent.
Alpha-ketoglutarate (AKG) is involved in the Krebs cycle: it is a nitrogen acceptor, and therefore an amine acceptor, generating glutamine by means of transamination or amination reactions through glutamate-dehydrogenase in perivenous hepatocytes. The amidation of glutamate to glutamine by means of glutamine-synthetase takes place in several tissues and organs, such as muscles, lungs, liver and brain. Alpha-ketoglutarate is typically used for kidney disease; intestinal and stomach disorders, including bacterial infections; liver problems; cataracts; and recurring yeast infection.
The current treatments of mastitis utilize tablets, capsules, injectables of target drugs. At the moment there is no treatment that directly targets the relief of oxidative stress using copper and provide additional immunity boosting properties. The present invention reports use of copper alpha-ketoglutarate (Cu-AKG) – a novel conjugate. Cu-AKG helps in the prevention, treatment, and management of mastitis. In addition, it addresses the problem of deficiency of copper in cattle is generally due to the presence of dietary antagonists, such as sulfur, molybdenum and iron that reduce Cu bioavailability. Further, the present invention reports synthesis of Cu-AKG granules for oral route of administration. The excipients used in the present invention helping the boosting of immunity in cattle and increases the milk production as well. Furthermore, additional nutritional substances have been incorporated in the present invention to boost the cattle immunity with specific focus on preventing, treating, and managing any pathologies directly or indirectly related to mastitis.
SUMMARY OF THE INVENTION
In view of the foregoing, for overcoming the shortcomings of the prior art, the object of the invention is to provide a composition comprising granules of copper alpha-ketoglutarate (Cu-AKG). Yet another object of the present invention is to provide a method to prepare a composition comprising granules of copper alpha-ketoglutarate (Cu-AKG).
An aspect of the invention is a composition comprising granules of copper salt of alpha-ketoglutarate together with acceptable excipients.

In one embodiment, the composition of copper alpha-ketoglutarate granules comprises of copper alpha-ketoglutarate in amounts between 50 – 100 mg; a diluent in amount between 3.27 – 4.61 gm; a binder in amount between 2.5% - 30%; a lubricant in amount 1% - 7.5%.

In one embodiment, the diluent is chosen in the group consisting of guar gum, cellulose, microcrystalline cellulose, calcium phosphate, starch, kaolin, dehydrate calcium sulphate, calcium carbonate, lactose, saccharose, glucose, sorbitol, dextrose, mannitol or their mixtures.

In one embodiment, the binder is chosen in the group consisting of corn starch, acacia, tragacanth, gelatin, starch paste, pregelatinized starch, alginic acid, cellulose, starch 1500, methocel, walocel, luvicross, luvicaprolactam or their mixtures.

In one embodiment, the lubricant is chosen in the group consisting of magnesium stearate, silica, magnesium or calcium stearate, sodium stearyl fumarate, hydrogenated vegetable oils, mineral oils, polyethylene glycols, sodium lauryl sulphate, glycerides, glyceryl dibehenate, glycerol stearate or their mixture.

In a preferred embodiment, the composition of copper alpha-ketoglutarate granules comprises of copper alpha-ketoglutarate in amounts between 50 – 100 mg; guar gum in amount between 3.27 – 4.61 gm; corn starch in amount between 2.5% - 30%; a magnesium stearate in amount 1% - 7.5%.

In the most preferred embodiment, the composition of copper alpha-ketoglutarate granules comprises of copper alpha-ketoglutarate in amounts between 70 - 90 mg; guar gum in amount between 3.7 - 4.5 gm; corn starch in amount between 6% - 10%; a magnesium stearate in amount 1% - 3%.

In another embodiment, the composition comprising copper alpha-ketoglutarate granules additionally comprises of one or more pharmaceutically acceptable nutritional ingredients, wherein the nutritional agent is chosen in the group consisting of thiamine, folic acid, pantothenic acid or their mixtures.

In an alternate embodiment, the composition comprising copper alpha-ketoglutarate granules shall be finalized in form of tablets, capsules, or granulate in bags.

In a preferred embodiment, the composition comprising of copper alpha-ketoglutarate granules shall be made using the wet granulation technique. In one embodiment, the process for the preparation of copper alpha-ketoglutarate granules shall comprise the followings steps. The first step shall be dry mixing copper alpha-ketoglutarate in an amount comprised between 50 mg to 100 mg with a diluent in an amount comprised between 3.27 – 4.61 gm. The second step shall be preparing binder paste with binder in an amount comprised between 2.5% to 30% at 90°C. The third step shall be using binder paste to prepare dough of copper alpha-ketoglutarate and diluent followed by screening through a sieve. The fourth step shall be drying the granules at temperature comprised between 40°C and 60°C in a time range higher than 12 hours. The final step shall be screening the granules for uniform size followed by adding lubricant in an amount comprised between 1% to 7.5% for a time comprised between 5 minutes to 15 minutes.
DETAILED DESCRIPTION OF THE INVENTION
The present invention describes compositions comprising Cu-AKG granules wherein said granules comprise a Cu-AKG amount between 50 mg to 100 mg for the treatment of mastitis in cattle. The described compositions comprise Cu-AKG granules, alone or together with pharmaceutically acceptable excipients, active and/or nutritional agents, such as, in an illustrative but not limitative way, amino acids, vitamins, proteins, antioxidants. The present invention also describes processes for obtaining Cu-AKG and processes for obtaining pharmaceutical compositions comprising said Cu-AKG granules.

To clarify the above and other purposes, features, and advantages of this invention, specific embodiment of this invention is especially listed and described in detail with the examples as follows. The principal and mode of operation of this invention have been described and illustrated in its embodiment. At the outset, a person skilled in the art will appreciate that this invention may be practiced otherwise than is specifically described and illustrated. The invention should not be limited by the above described embodiment, method, and examples, but by all embodiments and methods within the scope and spirit of the invention. Also, in the following description of the invention, certain terminology may be used for the purpose of reference only, and is not intended to be limiting.
Definitions and Use of Terms
As used in this specification and in the claims, which follow, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an ingredient" includes mixtures of ingredients; reference to "an active pharmaceutical agent" includes more than one active pharmaceutical agent, and the like. "Treating" or "treatment" of a disease includes (1) preventing the disease from occurring in an animal that may be predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e. arresting its development, or (3) relieving the disease, i.e. causing regression of the disease.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value and/or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

The invention sets out a chemical method that is used for preparing copper salt of alpha-ketoglutarate i.e. Cu-AKG (IN202041051386). Firstly, sodium bicarbonate dissolved in water was allowed to be added slowly in different portions into alpha-ketoglutaruic acid which was pre-dissolved in water at 0-5°C. The mixture was further stirred at 40 - 45°C for 12 hours followed by cooling it to room temperature. Next, Copper (II) Chloride Dihydrate was added gradually into the alpha-ketoglutaric acid disodium salt solution at 0 - 5°C. The mixture was allowed to come to room temperature and subjected to heat at 40 - 45°C for 24 hours. After 24 hours, the reaction mixture temperature was reduced to room temperature and the precipitate was kept at 0 - 5°C in ice bath to increase precipitation. Then, precipitate was filtered by using Buchner funnel under vacuum filtration. The solid particles were repeatedly washed with cold water to remove water miscible impurities. The solid was dried in an oven at 50°C for 3 hours and kept at 2 - 8°C in refrigerator. The copper alpha-ketoglutarate dihydrate, thus yielded, was collected and confirmed for the presence of Cu2+ ion.

Example 1
Preparation of copper alpha-ketoglutarate (10g scale)
In one illustrative embodiment of the inventions, described herein is the method for making copper alpha-ketoglutarate. Alpha-ketoglutaric acid (10 g, 68.442 mmol, 1 equiv.) was dissolved into 30 mL of water in an oven-dried magnetic bar charged 500 mL round bottom flask at 0-5 °C using ice-water bath by stirring for 5 minutes. NaHCO3 (11.5 g, 136.884 mmol, 2 equiv.) was dissolved in 200 mL water. This solution was allowed to add slowly in different portions into alpha-ketoglutaric acid solution at 0-5 °C by stirring continuously. Further, the solutions were continuously stirred at 40-45 °C for 12 hours and subsequently cooled down to room temperature. Copper(II) Chloride Dihydrate (11.668 g, 68.442 mmol, 1 equiv.) was added slowly into the alpha-ketoglutaric acid Disodium salt solution at 0-5 °C under continuous stirring. The mixture were allowed to come at room temperature and subjected to heat at 40-45 °C with continuous stirring for 24 hours. The resultant precipitate was kept at 0-5 °C in ice bath to increase precipitation. The final precipitate was collected and washed repeatedly with 250 mL water. The solid Copper alpha-ketoglutarate dihydrate was dried in an oven at 50 °C for 3 hours and kept at 2-8 °C in refrigerator to give about 37% yield.
Example 2
Preparation of copper alpha-ketoglutarate (50g scale)
In one illustrative embodiment of the inventions, described herein is the method for making copper alpha-ketoglutarate. Alpha-Ketoglutaric acid (50 g, 342.21 mmol, 1 equiv.) was dissolved into 150 mL of water in an oven-dried magnetic bar charged 2000 mL round bottom flask at 0-5 °C using ice-water bath by stirring for 5 minutes. NaHCO3 (57.50 g, 684.42 mmol, 2 equiv) was dissolved in 1000 mL water. This solution was allowed to add slowly in different portions into alpha-ketoglutaric acid solution at 0-5 °C by stirring continuously. Further, the solutions were continuously stirred at 40-45 °C for 12 hours and subsequently cooled down to room temperature. Copper(II) Chloride Dihydrate (58.34 g, 342.21 mmol, 1 equiv.) was added slowly into the alpha-ketoglutaric acid Disodium salt solution at 0-5 °C under continuous stirring. The mixture were allowed to come at room temperature and subjected to heat at 40-45 °C with continuous stirring for 24 hours. The resultant precipitate was kept at 0-5 °C in ice bath to increase precipitation. The final precipitate was collected and washed repeatedly with 500 mL water. The solid Copper alpha-ketoglutarate dihydrate was dried in an oven at 50 °C for 3 hours and kept at 2-8 °C in refrigerator to give about 41% yield.
Various routes of administration such as oral, parenteral, etc., are used for delivery of copper formulation. With respect to the administration to animals, oral route of administration is preferred due to its convenience in administration as compared to that in than other routes. Further, the oral route for drug delivery is also preferred due to its advantages like acceptability, easy preparation method and safety concerns. However, in case of many therapeutic agents, oral route of administration presents critical problem in uniform distribution of active ingredient due to poor flow property of powder. Thus, the essence of the present invention is composition comprising of granules to improve flow property and uniform distribution of Cu-AKG.
Accordingly, the present invention relates to the oral drug delivery system comprising Cu-AKG as a drug candidate for the treatment of mastitis in cattle. The purpose of the composition of the present invention is to facilitate the administration of copper treatment for mastitis in cattle through granule composition. Additionally, the antioxidant activity of the alpha-ketoglutarate in the composition shall assist in improving the general immunity in the cattle. It is desired that the composition be simple and inexpensive. Thus, an economical oral drug delivery system to facilitate the administration of copper in the treatment of mastitis in cattle is desired. Further, the composition shall ensure the uniform distribution of Cu-AKG in the granules.
Central to this invention is the object to prepare granules of Cu-AKG. Dry and wet granulation processes are well known in the art. It should be noted that while the dry granulation process may be applicable to the current composition, the present inventors prefer the wet granulation process for the purpose of preparing the present invention. Typically, wet granulation involves a starting powder mixing step followed by wetting and kneading it with a suitable amount of liquid phase. Some wet granulation processes known in the art employ kneading and granulation through nets; fluid bed granulation; granulation by extrusion - spheronization and granulation in high speed mixers-granulators. The granules obtained by means of a wet granulation process must then be dried by means of drying system in static or dynamic mode, also well known in the art.
Accordingly, in an embodiment, a wet granulation method was utilized for making granules to provide stable, with uniform particle size and spherical granules formulation containing Cu-AKG for the treatment of mastitis. In particular, the granules of the present invention comprise Cu-AKG together with one or more diluents, one or more binders, one or more lubricants, one or more nutritional substance forming a granules oral dosage form of Cu-AKG. It should be noted that one or more plasticizers and/or one or more polymers may be added to the said granule composition to further enhance its properties.
Table 1 reports composition of the granules.
Table 1
Ingredient Quantity
Cu-AKG 50 – 100 mg
Diluent 3.27 – 4.61 gm
Binder 2.5% - 30%
Lubricant 1% - 7.5%

The compositions according to the present invention can comprise a variable amount of Cu-AKG granules useful for obtaining the desired and optimal dosage of a Cu-AKG amount comprised between 50 – 100 mg.
A diluent suitable for the preparation of the granules object of the present invention is chosen in the group consisting of guar gum, cellulose, microcrystalline cellulose, calcium phosphate, starch, kaolin, dehydrate calcium sulphate, calcium carbonate, lactose, saccharose, glucose, sorbitol, dextrose, mannitol, partially pregelatinized starch or their mixture. In the present invention, an emphasis is placed on using biocompatible and biodegradable diluent like guar gum. It is also desired that the diluent of choice shall help improve the nutritive value, blood parameter and milk production in cattle.
A binder useful for the preparation of the granules is chosen in the group consisting of corn starch, acacia, tragacanth, gelatin, starch paste, pregelatinized starch, alginic acid, cellulose, starch 1500, methocel, walocel, luvicross, luvicaprolactam, cellulose, cellulose derivatives, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, potato starch, maize starch, partially gelatinized starch, gums, synthetic gum, natural gum, polyvinylpyrrolidone, polyethylene glycol, gelatin, polyols, propylene glycol, alginates, sugars or their mixtures.

A lubricant useful for the preparation of the granules is chosen in the group consisting of magnesium stearate, silica, magnesium or calcium stearate, sodium stearyl fumarate, hydrogenated vegetable oils, mineral oils, polyethylene glycols, sodium lauryl sulphate, glycerides, glyceryl dibehenate, glycerol stearate or their mixtures.
The nutritional substances comprised in the Cu-AKG granule composition are preferably chosen among vitamins like thiamine, folic acid, pantothenic acid which act as an antioxidant hence help in the treatment of mastitis. Additionally, other vitamins such as vitamin A, vitamin B complex, vitamin Bl, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B12, biotin, choline, folic acid, inositol, PABA (Para-aminobenzoic acid), vitamin C, vitamin D, vitamin E, vitamin K may also be added to the composition.
Water places itself as a most suitable granulating agent use in the present invention. Also, organic solvents such as isopropyl alcohol, ethanol, acetone, methanol also present good options due to their comparatively less toxic nature.
Cu-AKG granules constituting a preferred composition of the present invention are characterized in that they comprise a Cu-AKG amount comprised between 50 mg to 100 mg together with a diluent such as guar gum, a binder such as corn starch, a lubricant such as magnesium stearate suitable for obtaining its uniformed distribution.

The preferred composition of the granules is reported in Table 2.

Table 2
Ingredient Quantity
Cu-AKG 50 - 100 mg
Gaur gum 3.27 – 4.61 gm
Corn Starch 2.5% - 30%
Magnesium Stearate 1% - 7.5%

The process for granule preparation is reported and described in the present invention. Granules containing Cu-AKG were prepared by wet granulation method. Adequate amount of binder was added in water to make a slurry. Dough of Cu-AKG and diluent was prepared with the help of binder and screened through sieve. Granules thus obtained were dried in a tray dryer at 40°C for 12 hours. After drying the granules were screened to obtain uniform size granules. Lubricant was added to improve the flow properties. Granules were lubricated for 5 min, 10 min and 15 min to optimize the time of lubrication. Finally, the resultant granules were stored at a low relative humidity to maintain the integrity of granules. The bulk density, tapped density, compressibility index, Hausner’s ratio and angle of repose Particle size distribution were determined. The angle of repose after optimal lubrication time was used to select the optimized formulation. Particle size distribution performed by sieving method revealed data of percent particle size retain on the particular sieve, of various prepared granules formulations of Cu-AKG.
The following examples broadly illustrate the nature of this invention and the manner in which it is to be performed without limiting the nature and scope of the invention. Examples 3-14 are comparative examples useful to demonstrate that the process for the preparation of granules results from optimal combination of chosen process parameters, excipients and amount thereof. A gradual increase in the binder concentration exhibited the need for calibration of other excipients. Examples 5-7 have high binder concentrations, affecting the binding ability and swelling of the dough and consequently had excellent Compressibility index and Hausner’s ratio. Further, different lubrication times were used and thereby it is reported that a lubrication time of 15 minutes has the least angle of repose. Comparative preparations with varying amounts of excipients have been reported in the present invention to achieve the most optimal composition.
It should be noted that the composition of the present invention can also be applied to other drugs for example, but not limited to, copper sulphate, copper methionate, copper amino acid chelate complex, calcium copper edtate, copper glycinate, copper iodide, copper chloride, copper bromide, copper sulphate pentahydrate, copper trifluro methane sulphonate, copper acetate anhydrous etc.
Example 3
Preparation of granules with Binder concentration of 2.5%
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. 83 mg of Cu-AKG and 4.62 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 250 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. Then dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 5 min, 10 min and 15 min. to optimize the time of lubrication. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 1 has been reported in Table 3
Table 3
Ingredient Quantity
Cu-AKG 83 mg
Gaur gum 4.61 gm
Corn Starch 2.5%
Magnesium Stearate 1%

Example 4
Preparation of granules with Binder concentration of 5%
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then 83 mg of Cu-AKG and 4.37 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 500 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 5 min, 10 min and 15 min. to optimize the time of lubrication. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 2 has been reported in Table 4.

Table 4
Ingredient Quantity
Cu-AKG 83 mg
Gaur gum 4.37 gm
Corn Starch 5%
Magnesium Stearate 1%

Example 5
Preparation of granules with Binder concentration of 7.5%
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then, 83 mg of Cu-AKG and 4.12 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 750 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 5 min, 10 min and 15 min. to optimize the time of lubrication. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 3 has been reported in Table 5.

Table 5
Ingredient Quantity
Cu-AKG 83 mg
Gaur gum 4.12 gm
Corn Starch 750 mg (7.5%)
Magnesium Stearate 50 mg (1%)

Example 6
Preparation of granules with Binder concentration of 10%
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then 83 mg of Cu-AKG and 3.9 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 1 gm of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 5 min, 10 min and 15 min. to optimize the time of lubrication. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 4 has been reported in Table 6.

Table 6
Ingredient Quantity
Cu-AKG 83 mg
Gaur gum 3.9 gm
Corn Starch 1 gm (10%)
Magnesium Stearate 50 mg (1%)

Example 7
Preparation of granules with Binder concentration of 20%
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then, 83 mg of Cu-AKG and 3.76 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 2 gm of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#14). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 100 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 5 min, 10 min and 15 min. to optimize the time of lubrication. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 5 has been reported in Table 7.

Table 7
Ingredient Quantity
Cu-AKG 83 mg
Gaur gum 3.76 gm
Corn Starch 2 gm (20%)
Magnesium Stearate 100 mg (2%)

Example 8
Preparation of granules with Binder concentration of 25%
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then, 83 mg of Cu-AKG and 3.52 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 2.5 gm of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#14). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 125 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 5 min, 10 min and 15 min. to optimize the time of lubrication. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 6 has been reported in Table 8.

Table 8
Ingredient Quantity
Cu-AKG 83 mg
Gaur gum 3.52 gm
Corn Starch 2.5 gm (25%)
Magnesium Stearate 125 mg (2.5%)

Example 9
Preparation of granules with Binder concentration of 30%
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then 83 mg of Cu-AKG and 3.27gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 500 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#14). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 150 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 5 min, 10 min and 15 min. to optimize the time of lubrication. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 7 has been reported in Table 9.

Table 9
Ingredient Quantity
Cu-AKG 83 mg
Gaur gum 3.27 gm
Corn Starch 500 mg (30%)
Magnesium Stearate 150 mg (3%)

Example 10
Preparation of granules with optimized formulation and 50 mg of Cu-AKG
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then, 50 mg of Cu-AKG and 4.15 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 750 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 15 min.. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 8 has been reported in Table 10.

Table 10
Ingredient Quantity
Cu-AKG 50 mg
Gaur gum 4.15 gm
Corn Starch 750 mg (7.5%)
Magnesium Stearate 50 mg (1%)

Example 11
Preparation of granules with optimized formulation and 60 mg of Cu-AKG
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then, 60 mg of Cu-AKG and 4.14 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 750 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 15 min. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 9 has been reported in Table 11.

Table 11
Ingredient Quantity
Cu-AKG 60 mg
Gaur gum 4.14 gm
Corn Starch 750 mg (7.5%)
Magnesium Stearate 50 mg (1%)

Example 12
Preparation of granules with optimized formulation and 70 mg of Cu-AKG
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then, 70 mg of Cu-AKG and 4.13 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 750 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 15 min. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 10 has been reported in Table 12.

Table 12
Ingredient Quantity
Cu-AKG 70 mg
Gaur gum 4.13 gm
Corn Starch 750 mg (7.5%)
Magnesium Stearate 50 mg (1%)

Example 13
Preparation of granules with optimized formulation and 90 mg of Cu-AKG
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then, 90 mg of Cu-AKG and 4.11 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 750 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 15 min. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 11 has been reported in Table 13.

Table 13
Ingredient Quantity
Cu-AKG 90 mg
Gaur gum 4.11 gm
Corn Starch 750 mg (7.5%)
Magnesium Stearate 50 mg (1%)

Example 14
Preparation of granules with optimized formulation and 100 mg of Cu-AKG
Granules as an oral formulation of Cu-AKG was prepared by wet granulation method. All the ingredients were passed through # 30. Then, 100 mg of Cu-AKG and 4.10 gm of gaur gum was dry mixed in a mortar. Starch paste was prepared in a beaker by addition of 750 mg of corn starch in 10 ml of water to form a slurry after that placed on a heating plate at 90°C with stirring to form a corn starch paste. After that dough of Cu-AKG and gaur gum was prepared with the help of corn starch as a binder and screened through sieve (#25). The obtained granules were dried in a tray dryer at 40°C for 12 hours. After drying we have screened it to obtain uniform size and 50 mg magnesium stearate was added as lubricating agent to improve the flow properties. It was lubricated for 15 min. Stored at a low relative humidity to maintain the integrity of granules. Composition of Example 12 has been reported in Table 14.

Table 14
Ingredient Quantity
Cu-AKG 100 mg
Gaur gum 4.10 gm
Corn Starch 750 mg (7.5%)
Magnesium Stearate 50 mg (1%)

Table 15 reports the bulk density, tapped density, compressibility index, Hausner’s ratio and angle of repose for the compositions prepared in Examples 1-7. Particle size distribution was also determined. The angle of repose after optimal lubrication time was used to select the optimized formulation.
Table 15
Parameters Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7
Binder concentration 2.5% 5% 7.5% 10% 20% 25% 30%
Bulk density (gm/mL) 0.32 0.32 0.43 0.30 0.48 0.36 0.38
Tapped density (gm/mL) 0.35 0.37 0.47 0.33 0.51 0.38 0.41
Compressibility index 8.57 13.51 8.51 9.09 5.88 5.26 7.32
Hausner’s ratio 1.09 1.16 1.09 1.10 1.06 1.06 1.08
In the evaluation of particle size analysis approximately 84% of the granules of Example 3 were retained on the #40 mesh in comparison to other compositions. It is reported that the granules of the composition of Example 3 were uniform size. Results of particle size distribution is reported in Table 16.
Table 16

Mesh no. % of granules retained on a sieve
Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7
40 69.73 66.66 84.42 67.88 13.20 41.53 31.81
60 20.64 22.98 10.80 21.67 3.14 6.59 6.81
80 1.60 1.6 1 2.08 0.20 0.43 0.45
Fines 7.21 7.81 4.02 8.61 1.25 1.09 1.81

Table 17
Characterization of optimized formulations
Parameters Example
8 Example
9 Example 10 Example 11 Example 12
Binder concentration 7.5%

Bulk density (gm/mL) 0.43 0.41 0.43 0.42 0.44
Tapped density (gm/mL) 0.46 0.45 0.47 0.46 0.47
Compressibility index 6.52 8.88 8.51 8.69 6.38
Hausner’s ratio 1.07 1.10 1.09 1.10 1.07

Table 18
Particle size distribution of optimized formulations

Mesh no. % of granules retained on a sieve
Example 8 Example 9 Example 10 Example 11 Example 12
40 84.42 82.57 85.10 83.96 81.45
60 10.80 12.31 10.84 11.58 13.92
80 1.21 2.41 1.32 1.53 2.51
Fines 3.57 2.71 2.74 2.93 2.12

It should be noted that the composition comprising Cu-AKG granules shall be used as is. Alternatively, the granules composition may be finalized, but not limited to, in form of tablets, capsules, or granulate in bags which may be suitable for administering to the subject animal.

The composition of Cu-AKG granules of the present composition shall be used for the treatment and prevention of pathologies directly or indirectly related to mastitis in cattle. The composition of the present invention shall be administered at a dose and in a manner that is efficacious and does not irritate the mammary glands.