Applicant: MOHAMED ARIF

FIELD OF INVENTION

The present invention relates a pharmaceutical composition for intramammary administration to a non-human mammal, particularly an infusional composition for intramammary mastitis.

BACKGROUND OF THE INVENTION

In general, bovine mastitis is an inflammatory response of the udder tissue in the mammary gland caused due to physical trauma or micro-organism infections. It is considered the most common disease leading to economic loss in dairy industries due to reduced yield and poor quality of milk. Further, it is a condition typified by the persistent and inflammatory reaction of the udder tissue due to either physical trauma or infections caused by microorganisms. It is a potentially fatal mammary gland infection, that is most common in dairy cattle worldwide. It is a disease that is known to cause the greatest loss to the dairy industry in addition to the pain and ill health the animal has to undergo.

There is a large cohort of microorganism species that are known to cause mastitis. These range from viruses, mycoplasma, fungus, and bacteria. Bacterial organisms known to cause mastitis are Pasteurella multocida, Staphylococcus aureus; Str. zooepidemicus; Str. agalactiae; Str. pyogenes; Str. faecalis; Mycobacterium bovis; Klebsiella spp; Brucella abortus; Pseudomonas pyocyanin; E.coli; Leptospira Pomona, etc. Fungal entities responsible for mastitis are Aspergillus fumigatus; A. midulus; Candida spp; Trichosporonspp, etc. Physical injury to the mammary region, poor hygiene, and/or trauma, also cause this condition. The clear sign of mastitis is inflammation of the udder that turns into a red and hard mass. The swollen mammary gland is hot and the mere touching causes pain and discomfort to the animal. Animals do not allow touching of the udder even kicking to prevent milking. If milked the milk is usually tainted with blood clots, foul-smelling brown discharge, and milk clots.

The milk yield stops or is severely restricted. Body temperature of the animal increases. Other symptoms are lack of appetite, hindrance in mobility due to the swollen udder, and pain. The dairy animal develops sunken eyes, suffers from digestive disorders and diarrhea. Infected cattle are severely dehydrated and suffer from weight loss. In cases of severe infection, there is the formation of pus in the infected udder. Mastitis can degenerate into Toxaemia or Bacteraemia and even cause death as a result of acute infection. Mastitis can be detected at an early stage (subclinical) before the symptoms appear, through California Mastitis Test (CMT). It is a quick test that can be performed on small milk samples. Early detection helps in preventing the progress of the disease into clinical stages and causing heavy losses to dairy farmers.

There are some treatment measures like vaccines that have been designed to combat mastitis, but many are of limited protection against coliform infections. Studies have shown that the J5 core antigen vaccine is efficacious in reducing the incidence of clinical mastitis caused by E. coli, especially during early lactation, but did not reduce the prevalence of infection. Vaccines can be valuable in reducing the duration and severity of IMI. The benefits of the use of the J5 vaccine have been proven worthwhile since the mid-1990s.

NSAIDs are widely used for the treatment of acute mastitis. Aspirin, flunixin meglumine, flurbiprofen, carprofen, ibuprofen, and ketoprofen have been studied as treatments for experimental coliform mastitis or endotoxin-induced mastitis. Orally administered aspirin should be used with caution in acute coliform mastitis because it may lead to severe rumen atony.

Presently this condition is treated with the use of antibiotics in the form of injection (systemic) or intramammary infusion. The commonly used antibiotics are Penicillin, Amoxycillin, Tetracyclines or Cephalosporins and even colistin, to treat the infection and Anti-inflammatory drugs to address the inflammation.

The major problem with this treatment is the emergence of multidrug-resistant bacteria and other pathogens and so the problem not getting resolved in time. Another major problem with this treatment option is the passing of the endotoxins produced by the bacteria and other organisms in the milk and more importantly, the multidrug resistance of the bacteria being passed on to human beings through the food chain (through Milk and the Dairy products). This leads to many health care problems for humanity

There are many more inventions that are bound to address the disease mastitis, US 2009/0317364 A1 , here A novel stable synergistic composition used for the prevention and/or treatment of mastitis and metritis in mammals comprising a combination of a therapeutically effective amount of Serratiopeptidase, Lysozyme, Ocimum sanctum, and Azadirachta indica are disclosed in the present invention. The present invention further discloses a method of treatment and/or prevention of an infective condition in a fluidcontaining organ having a natural exterior orifice, such as the udder of a milkproducing animal.

An article from the URL https://indianexpress.com/article/india/india- others/herbal-cure-for-cattle-diseases/ says Mr. GADVASU V K Taneja said, “In India, due to mastitis dairy farmers incur losses estimated at over Rs 7,000 crore annually. There are several medicines available but they all are chemical-based. A variety of germicides such as iodine, chlorhexidine, sodium hypochlorite, etc have been tried successfully as teat dips in

95 preventing these infections. But one major concern that remains with these is the possible passing on of disinfectant residues into the milk. Hence, herbal drug as teat dip may be the best answer. Even WHO has emphasized the use of medicinal plants for curing mastitis as they are safer than synthetic drugs.

100 IN 3754/MUM/2014, The present invention relates to herbal compositions comprising therapeutically effective amounts of extracts of Clerodendrumphlomidis, Azadirachta indica, Vitex negun Tephrosia purpurea, and Achyranthes aspera for use in treatment or prevention of mastitis in animals in need thereof. The present invention further relates to a process of

105 preparation and various forms of administration of said compositions thereof.

In the article “Ethnomedicinal survey among Kani Tribes and traditional healers in selected Tribal populations of Trivandrum district” by Parvathy RS1*, Sivakkumar S2, Meenakumari R3, Indian tribes constitute around 8.3% no of the total population. The health problems of tribal communities are influenced by the interaction of various socioeconomic and political factors. An attempt is made to document the traditional knowledge of the Kani tribes in the floristically rich Peringamala panchayath of Thiruvananthapuram district. The investigation revealed that the Kani tribes were using 127 species

115 of ethnomedicinal plants, belonging to 57 families. The most cited family was Fabaceae and leaves were the most frequently used plant parts for the treatment of various diseases. Some medicinal plants used by Kani tribes are included in the list of rare and endangered categories, such as AristolochiaTagala, Holostemmaadakodien, Entada scandens,

120 Anaphyllumbeddomeii, decalepsisarayalpathra which are vanishing from the tribal settlement areas. So special attention should be given to cultivating and conserving these rare medicinal plants by making them available for mass cultivation, either through conventional methods or other non-conventional methods like micropropagation. The study showed that the Kani tribes have good knowledge about medicinal plants that have been passed orally from generation to generation. Current healers may be the final generation of Kani tribal healers. Due to rapid socio-economic and cultural changes, there is a possibility of losing the treasure of tribal knowledge in near future. The wealth of this tribal knowledge would lead to a greater potential for the Indian system of medicine toward new drug discovery and development.

In US 6579543B1 , A composition for topical application to an animal's skin for relief from a variety of symptoms caused by medical conditions or physical injuries. The composition includes at least one compound having analgesic activity, at least one compound having anti-inflammatory activity, at least one compound having antioxidant activity, at least one compound having anti- neuralgic activity, at least one compound having blood circulation promotion activity, and at least one compound having antidepressant activity. A method for relieving pain by topical application of the composition is also provided.

The above prior arts provides composition of medicines as either vaccines or topical medicines over skins. There is a requirement for a novel infusionalcomposition at udder and a need of preparation for the same.

OBJECT OF THE INVENTION

The primary objective of the present invention is to provide a novel composition of a broad-spectrum antibiotic, antifungal and antiviral and antiinflammatory which is safe, efficacy proven and does not cause drug resistance. medicine and that can be loaded on a vehicle/camer/base that dissolves the composition as a lipid in liquid form.

Another objective of the present invention is to provide the composition with a novel proportion that can be loaded on a vehicle/camer/base that dissolves the composition as a lipid in liquid form against the disease mastitis.

Yet the other objective of the present invention is to provide a novel preparation method of composition that can be loaded on a vehicle/carrier/base that dissolves the composition as a lipid in liquid form against the disease mastitis.

DETAILED DESCRIPTION OF THE DRAWINGS

Fig 1 illustrates a chart for the progress in the health condition of cattle/bovine udder under multiple infusions at multiple periods of the proposed composition.

When the non-human mammal or a cow presented to the clinic with a history of pain in the udder and change in milk color and consistency. On examination, hind right quarter found to be having mastitis with yellow watery milk with hard swollen udder having lump size categorized 12 and systemically affected.

Milk sample was collected from affected cows and sent to the lab for culture and further identification of causative organisms. Treatment was started with injectable antibiotic (Amoxycillinn+Caluvulinic acid given for 5 days and anti-inflammatory (Flunexine meglumine) and was given fluid therapy (Normal Saline) to stabilize and dilute the toxin and the Intramammary tube of the new drug was infused into the affected quarter and continued for 5 days twice daily.

Before treatment, milk sample from affected quarter was cultured (streaked) in EMB agar and incubated for 24 hours at 32 degrees Celcius. After 24 hours the inoculate agar was read for growth and pattern of bacterial colonies. The culture plate had colony pattern suggestive of E.coli. Colony from the plate was gram stained and observed under microscope (100x) and found to be gram-negative cocci suggestive of E Coli which further corroborated with diagnosis.

After the 5 ^th day of treatment animal responded well to the treatment and completely recovered from mastitis and the underlying infection which could be observed by the resolved swelling of the affected quarter of udder. On 5 ^th day post-treatment again the milk sample was collected and cultured for microbial growth and we have found no growth which is indicative that the sample was cleared of infection.

The animal has responded well to treatment.

Culture plate Post Treatment - No growth (Image supplied by Al Safwah Dairy farms, Salalah, Oman)

DETAILED DESCRIPTION OF THE INVENTION

As per Biodiversity related provisions from The Indian Patents Act 1970 and Rules 2003: According to Section 10 (4) (d) (ii)2 of the Patents Act, 1970, hereby the applicant discloses the source and geographical origin of the biological material used in this invention below)

1. Asiaticoside/ Extract of Centellaasiaitica containing a specified percentage of Asiaticoside: Purchased from All Season herbs, Bengaluru

2. Curcuminoids I Extract of Curcuma longa rhizome containing specified percentage of Curcuminoids: Purchased from All season Herbs, Bengaluru

3. Andrographolide I Extract of Andrographis paniculata containing specified percentage of Androgrspholide: Purchased from HerboNutra, NOIDA, New Delhi.

The proposed compositions for the treatment of non-human mammal mastitis are formed of three components, in various concentrations which are combined to form a dose. Wherein the compositions are

1. Asiaticoside or Asiatic acid or their analogues or derivatives or Centella asiatica extract containing Asiaticoside or Asiatic acid or both.

2. Curumin or Curcuminoids or Extract of Curcuma longa Rhizome containing Curcuminoids. It can also be their derivatives or analogs.

3. Andrographolides or extract of Andrographis paniculata containing andrographolides or their derivatives or their analogs.

The need for choosing the above-proposed compositions is that Mastitis is a tough complication for which only treatment option available is use of Antibiotics and NSAIDs (Non-steroidal Anti-inflammatory Drugs) which are used for a prolonged period. This leads to loss of productivity (No Milk) for almost 10 to 12 days and more importantly, even after recovery the antibiotic is passed on in the milk which gets into the Human food chain which leads to large scale antibiotic resistance and paves way for emergence of Multidrugresistant bacteria (Micro-Organisms) in human too.

A thorough understanding of the pathophysiology of mastitis in cows and other milking animals and the current treatments in practice and options available to manage this problem and the side effects it causes to animal health, economic loss, and above all the ill effects through the human food chain. This has created a deep urge to solve this problem through safe herbal treatment which is a Phytoactive principle-based formulation in human healthcare.

Yet the other important embodiment in the invention is to provide the composition with a novel proportion that can be loaded on a vehicle/camer/base that dissolves the composition as a lipid in liquid form against the disease mastitis.

EXAMPLE 1 :

OPTIMAL COMPOSITION

Table: 1 Result:

From Table 1 , the composition in the proportion reaction to the disease is 1. Discomfort in the udder.

2. Slowed healing process.

3. Inflammation resolving time is extended to more than 10 days.

Cows' metabolism gets disturbed and that affects the whole health of the cow.

Example 2

Table: 2

Result:

From Table 2, the composition in the proportion reaction to the disease is 1. Discomfort in the udder.

2. Slowed healing process.

3. Inflammation resolving time is extended to more than 10 days.

Cows' metabolism gets disturbed and that affects the whole health of the cow.

Example 3:

Table 3

Result:

From Table 3, the composition in the proportion reaction to the disease is 1. There is no discomfort in the udder.

2. Healing process is enhanced.

3. Inflammation is resolved and the infection cleared in 5 days.

Animal’s metabolism is improved and returning to good health within a week at the maximum

[Andrographolide:1.5% (it can be from 0.5% to 6%. Beyond this it will cause ill effects) Asiaticoside:3% (it can be from 0.5% to 9%. Beyond this it will be counterproductive) Curcuminoid: 3%(from 0.5 % to 15%. Beyond this it will be unpleasant to animals) Base: Sufficient Quantity to make up the volume]

Table 3, and Fig 1 exhibits the arrival of the present invention, and evidence the proof of curation of disease mastitis in such a way that

1. The inflammation is resolved in short period the pain relieved and the animal is feeling comfortable.

2. The infection is cleared totally in 5 days and

3. the metabolism is improved and at the maximum of one week the cow becomes normal.

All these we achieve without any side effects of the currently available treatment option like Nephrotoxicity of the NSAIDs (Non-SteroidalAnti inflammatory Drugs) and the Multidrug resistance arising out of usage of large amounts of antibiotics. Yet another important embodiment in the invention is to provide a preparation method for the medicine having the proposed composition and proportion, where All the three ingredients andrographolide, asiaticoside, and curcuminoid are taken in the required quantity 1.5%, 3%, 3% repectively of and mixed in a lipid base either of vegetable or origin or synthetic, preferably, PEG (Poly Ethelene Glycol) and stirred well for some time for getting homogenous material. This medication is filled in sterile empty Intramammary syringes (tip caped) in a sterile environment under controlled temperature and humidity. Then these filled individual syringes are sleeved in Polyethylene sleeves and sealed. 10 or 15 such sleeved individual syringes are packed in printed cartons.

The following procedure is followed to prepare the abovesaid composition along with its proportion/

1. The required amount (based on the total batch quantum being produced) of the ingredients namely, Andrographolide, Asiaticoside and Curcuminoids are weighed and kept separately.

2. The required amount of preservative

3. The required amount of Lipid liquid (either vegetable oil or PEG (polyethylene Glycol, Preferbly PEG 400) is weighed and kept separately.

4. All the drug ingredients are dissolved in the Liquid lipid in a stainless steel tank with a motorized stirrer which is allowed to run for about 3 hours, to get a uniform mixture.

5. Empty sterilized (autoclaved) intramammary syringes which are nozzle closed with a lid(Cover) are taken and the pistons are pulled out.

6. Empty syringes are filled with a required quantum of the mixed drug (15 grams or 30grams for eg.) with the use of cream filling machine. 7. Filled in syringes are loaded with pistons and these syringes are sent for PVC sealing/wrapping and sealed.

8. All these activities take place in a temperature and humidity-controlled Noncontaminated and sterilized room.

9. Each batch of sample tubes is sent to Q.C. for analysis for microbial and Heavy metal content and ensured they are within the prescribed limit.

10. Sealed syringes are arranged in 30s or 50s individual boxes and sealed.

11. Sealed such boxes are arranged in bogger cardboard boxes sealed and sent to storage area to be stacked.

There is a synergistic effect arising out of this combination: treating Mastitis (inflammation of the udder) as well as the underlying infection (which is already becoming resistant to majority of the antibiotics available today) by this combination. Treatment of Mastitis is becoming complicated today, especially on large dairy farms.

Together these ingredients work synergistically:

Reduces the pain and resolves the inflammation. Inflammation is the body’s immune response to injury and there there is a disharmony in the immune response. Enhanced secretion of inflammatory chemokines like TNF Alfa and IL 6. This formulation restores immune response and inhibits the secretion of TNF Alpha and IL6.

There is an accumulation of large amounts of free radicals at the cellular level which hinders the normal cellular activity. This formulation scavenges accumulated free radicals at the cellular level and promotes secretion of indigenous antioxidants and restores normal cellular activity which helps to fight against infection. In the presence of Inflammation, the circulation to the affected area in the body is affected which also hinders the normal functioning of the cells and tissue. This formulation improves microcirculation to the affected areas of tissue and helps the body to resolve the inflammation and enhance the process of healing.

The formulation has a broad spectrum of antimicrobial activity and effectively clears the pathogens (microbes) responsible for causing the inflammation, especially the E.coli infection which is growing becoming resistant to many of the antibiotic treatments available today.

Therefore this formulation effectively works in resolving the inflammation and pain as well as clearing the infection in the short period in treating mastitis in cows and other milking animals which leads to good health for animals, Humans and prevents loss of productivity and economy.

Advantages a. Safe (without causing any side effects to the health of animals) and highly effective. b. Drastic cut in Number of days’ treatment - Animals recover in 5 to 7 days’ treatment. Results in savings in the cost of medicine and restoring productivity -milk production to normal in a short time. Economy.

C. Considerable reduction in the use of antibiotics

Above all quality of the milk produced, without contamination of antibiotics and endotoxins in milk leading human health complications and developing antibiotic resistance in humans. Avoid emergence of Multidrug-resistant microbes.

It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any combinations of any two or more of said steps or features.