WO2018176093A1 - Berbine alkaloid formulations in the prevention and/or treatment of infectious disease - Google Patents
Berbine alkaloid formulations in the prevention and/or treatment of infectious disease Download PDFInfo
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- WO2018176093A1 WO2018176093A1 PCT/AU2018/050288 AU2018050288W WO2018176093A1 WO 2018176093 A1 WO2018176093 A1 WO 2018176093A1 AU 2018050288 W AU2018050288 W AU 2018050288W WO 2018176093 A1 WO2018176093 A1 WO 2018176093A1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/116—Heterocyclic compounds
- A23K20/132—Heterocyclic compounds containing only one nitrogen as hetero atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/116—Heterocyclic compounds
- A23K20/121—Heterocyclic compounds containing oxygen or sulfur as hetero atom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/195—Antibiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
Definitions
- This invention relates to berberine alkaloids, formulations thereof, and their use in the prevention and/or treatment of infectious disease in animals and as preservatives.
- the invention relates to berberine alkaloids, formulations thereof, and their application as
- antimicrobial agents in the prevention and/or treatment of infectious disease including bacterial, viral, parasitic or fungal infections in food-producing animals and as feed preservatives.
- Antibiotic use has been a staple in animal production worldwide for decades. It is estimated that the world uses about 63,000 tons of antibiotics each year to raise cows, chickens and pigs, which is roughly twice that of antibiotics prescribed by doctors globally to tight infections in people, with current trends suggesting world consumption of antibiotics in animals will go up by two- thirds in the next 20 years. Antibiotics have been supplemented to animal and poultry feed to not only treat and control infections, but also as growth promoters at low doses, and are considered to improve the quality of the product, resulting in a lower percentage of fat and a higher protein content in the meat.
- Antimicrobial resistance is a natural process whereby microbes evolve to be able to resist the action of drugs, making them ineffective. This leads to antibiotics becoming less effective over time and in extreme cases, ultimately useless. AMR has increasingly become a problem because the pace at which new antibiotics are discovered has slowed dramatically and consequently there are a very limited number of new drugs. Meanwhile, antibiotic use has risen exponentially increasing the development of resistance.
- AMR Antimicrobial resistance
- MRSA Methicillin-resistant Staphylococcus aureus
- Klebsiella pneumoniae are a major cause of hospital-acquired infections.
- K. pneumonia which are common intestinal bacteria, have become resistant to even last resort treatment by ⁇ -lactam carbapenem antibiotics in some countries.
- treatment of urinary tract infections caused by E. coli bacteria is now ineffective because of resistance to fluoroquinolone antibiotics.
- Use of ⁇ -lactam antibiotics and fluoroquinolones can lead to secondary infection and further complications such as overgrowth of Clostridium difficile (CD).
- CD is a bacterium that can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. Illness from CD most commonly affects older adults often in long-term care facilities and typically occurs after use of antibiotic medications. However, studies show increasing rates of CD infection among people traditionally not considered high risk, such as younger and healthy individuals without a history of antibiotic use or exposure to health care facilities. Each year in the United States, about a half million people get sick as a result of release of CD toxins, and in recent years, CD infections have become more frequent, severe and difficult to treat with the rise of antimicrobial resistance. Ironically, the standard treatment for CD is another antibiotic: metronidazole for mild to moderate infection; vancomycin for more severe infection. However, up to 20 percent of people with CD get sick again. After two or more recurrences, rates of further recurrence increase up to 65 percent.
- Antimicrobial resistance is a complex problem that affects all of society and is driven by many interconnected factors. Single, isolated interventions have limited impact. Coordinated action is required to minimize the emergence and spread of antimicrobial resistance. It is important to develop new antimicrobial drugs as alternatives to combat the world wide resistance problems facing human and animal health.
- Salmonellosis is one of the most common and widely distributed food-poisoning and is caused by the bacteria salmonella. It is estimated that tens of millions of human cases occur worldwide every year and the disease results in more than hundred thousand deaths. Antimicrobial resistance in Salmonella serotypes has been a global problem. Surveillance data demonstrated an obvious increase in overall antimicrobial resistance among salmonellae from 20%-30% in the early 1990s to as high as 70% in some countries at the turn of the century. Salmonella lives in the intestines of husbandry animals (especially chicken and cattle). It can be found in water, food, or on surfaces that have been contaminated with the feces of infected animals or humans (Figure 2 depicts aspects of Salmonella infection and food poisoning).
- Campylobacteriosis is a gastrointestinal disease caused by bacteria called Campylobacter (CB) and a major cause of foodborne illness.
- CB is mainly spread to humans by eating or drinking contaminated food (mainly poultry), water or unpasteurised milk.
- CB can also be spread through contact with infected people, or from contact with cats, dogs and farm animals that carry the bacteria.
- Figure 3 shows the epidemiology. Most people who become infected with CB will get diarrhoea, cramping, abdominal pain, and fever that lasts from one to two weeks. Symptoms usually develop within 2 to 5 days after infection. The diarrhoea may contain blood or mucous. In rare cases, CB can enter the bloodstream and cause more serious disease.
- antimicrobial agents not as antibiotics but as preservatives to avoid the spoilage of food.
- Compounds with antibiotic activity may be used as preservatives.
- antimicrobial preservatives in common use include sodium benzoate, vitamin C and sodium nitrites and nitrates. Preservatives are typically selected on the basis that they are harmless to animals and humans. However, sodium benzoate and vitamin C mixtures and sodium nitrites and nitrates have all been identified as increasing cancer risk. A mixture of sodium benzoate and vitamin C can give rise to benzene in soft drinks.
- the present disclosure relates to a method for the prevention and/or treatment of an infectious disease in an animal, wherein the method comprises administering a berberine alkaloid to the animal.
- the present disclosure also relates to a composition comprising any two or more of the following: a berberine alkaloid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatrin, andrographolide, and piceid.
- a composition comprising a berberine alkaloid and any one or more of the following: arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, piceid, honokiol and thymol.
- the present disclosure also relates to a composition
- a composition comprising a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in animal.
- the present disclosure also relates to a composition comprising berberine and one or more agents suitable for the prevention and/or treatment of an infectious disease in animal.
- the present disclosure also relates to an animal feed comprising a berberine alkaloid.
- the present disclosure also relates to an animal feed comprising a berberine alkaloid and an animal foodstuff, wherein the berberine alkaloid is in an amount of about 0.001 % w/w to 2 % w/w of the animal foodstuff.
- the present disclosure also relates to an animal feed comprising a composition described herein.
- the present disclosure also relates to an animal feed comprising a composition described herein and an animal foodstuff, wherein the composition is in an amount of about 0.001 % w/w to 1 % w/w of the animal foodstuff.
- the present disclosure also relates to a dosing regimen comprising administering a berberine alkaloid, or a composition or an animal feed as disclosed herein for 1 to 6 weeks to an animal, wherein the berberine alkaloid, or the composition or animal feed is administered for 1 to 6 weeks and in an amount effective to prevent and/or treat an infectious disease in an animal.
- the present disclosure also relates to a dosing regimen comprising administering a berberine alkaloid, or a composition or an animal feed as disclosed herein for 1 to 6 weeks to an animal, wherein the berberine alkaloid, or the composition or animal feed is administered for 1 to 6 weeks and in an amount effective to prevent and/or treat an infectious disease in an animal, wherein the infectious disease is caused by bacteria from the genus Lawsonia.
- the present disclosure also relates to a method for the reduction of feed conversion ratio in a food-producing animal, wherein the method comprises the step of administering a berberine alkaloid to said food-producing animal.
- the present disclosure also relates to a method for preventing or treating an infectious disease in an animal comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to a method for preventing or treating an infectious liver disease in an animal comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to a method for preventing or treating an infectious intestinal disease in an animal comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to a method for preventing or treating Spotty Liver Disease in a chicken comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to a method for preventing or treating an infectious disease caused by E. coli in an animal comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to a method for preventing or treating an infectious disease caused by Brachyspira in an animal comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to a method for preventing or treating an infectious disease caused by Lawsonia in an animal comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to a method for the prevention and/or treatment of an infectious disease in an animal, wherein the method comprises administering a berberine alkaloid or a composition or an animal feed disclosed herein to said animal, wherein the infectious disease is caused by bacteria from the genus Lawsonia.
- the present disclosure also relates to a method for preventing or treating an infectious disease caused by Eimeria in an animal comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to a method for preventing or treating an infectious disease caused by bacteria from the genus Clostridium in an animal comprising administering a composition or an animal feed disclosed herein.
- the present disclosure also relates to use of a berberine alkaloid in the preparation of a medicament for the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of any two or more of the following: a berberine alkaloid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, fibrauretin (palmatine), and piceid in the preparation of a medicament for the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of a berberine alkaloid and any one of more of the following: arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, piceid, honokiol and thymol in the preparation of a medicament for the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in an animal in the preparation of a medicament for the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of a berberine alkaloid in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of any two or more of the following: a berberine alkaloid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, fibrauretin (palmatine), and piceid in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of a berberine alkaloid and any one or more of the following: arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, piceid, honokiol and thymol in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in an animal in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to a berberine alkaloid for use in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to any two or more of the following: a berberine alkaloid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatnne, andrographolide, fibrauretin (palmatine), and piceid for use in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to a berberine alkaloid and any one or more of the following: arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, piceid, honokiol and thymol for use in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in an animal for use in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal; Spotty Liver Disease in a chicken; an infectious disease caused by E. coli in an animal; an infectious disease caused by Brachyspira in an animal; an infectious disease caused by Lawsonia in an animal; an infectious disease caused by Eimeria in an animal; or treating an infectious disease caused by bacteria from the genus Clostridium.
- the present disclosure also relates to an animal feed preservative comprising a berberine alkaloid.
- the present disclosure also relates to animal feed preservative comprising a berberine alkaloid wherein the feed preservative is safe.
- the present disclosure also relates to use of a berberine alkaloid in the preparation of a medicament for the prevention and/or treatment of an infectious disease caused by bacteria from the genus Lawsonia.
- the present disclosure also relates to use of a berberine alkaloid in the prevention and/or treatment of an infectious disease caused by bacteria from the genus Lawsonia.
- the present disclosure also relates to a method for the reduction of feed conversion ratio in a food-producing animal, wherein the method comprises administering a berberine alkaloid or a composition or an animal feed disclosed herein to the food-producing animal.
- the term "acceptable excipient” refers to a solid or liquid filler, carrier, diluent or encapsulating substance that may be safely used in administration.
- carriers well known in the art may be used. These carriers or excipients may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water. Excipients are discussed, for example, in Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams and Wilkins, 2005.
- acceptable salt refers to salts which are toxicologically safe for systemic administration. Acceptable salts, including acceptable acidic/anionic or basic/cationic are described in P. L. Gould, International Journal of Pharmaceutics, 1986, November, 33 (1-3), 201-217; S. M. Berge et al., Journal of Pharmaceutical Science, 1977, January, 66 (1), 1; P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts: Properties, Selection and Use, Second Revised Edition, Wiley, 2011. Acceptable salts of the acidic or basic compounds of the invention can of course be made by conventional procedures (such as reacting a free acid with the desired salt-forming base or reacting a free base with the desired salt-forming acid).
- Acceptable salts of acidic compounds include salts with cations and may be selected from alkali or alkaline earth metal salts, including, sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine and the like, and salts with organic bases.
- Suitable organic bases include N-methyl-D-glucamine, arginine, benzathine, diolamine, olamine, procaine and tromethamine.
- Acceptable salts of basic compounds include salts with anions and may be selected from organic or inorganic acids.
- Suitable anions include acetate, acylsulfates, acyl sulfonates, adipate, ascorbate, benzoate, besylate, bromide, camsylate, caprate, caproate, caprylate, chloride, citrate, docusate, edisylate, estolate, formate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mesylate, methylbromide, methyl sulfate, napsylate, nitrate, octanoate, oleate, pamoate, phosphate, polygalacturonate, salicylate, stea
- Berberine is a positively charged quaternary ammonium cation.
- Acceptable salts of beberine include without limitation chloride, hemisulfate and iodide salts.
- acceptable solvent is a solvent which for the purpose of the disclosure may not interfere with the biological activity of the solute.
- suitable solvents include, but are not limited to, water, ethanol and acetic acid, glycerol, liquid polyethylene glycols and mixtures thereof.
- a particular solvent is water.
- solvate refers to a complex of variable stoichiometry formed by a solute (for example, a berberine alkaloid) and a solvent.
- the solvent used is an "acceptable solvent” as defined herein. When water is the solvent, the molecule is referred to as a hydrate.
- antimicrobial activity is defined herein as an activity that kills or inhibits the growth of microorganisms including, but not limited to bacteria, viruses, parasites, and fungi. It would be recognised that a substance which displays antimicrobial activity may be used as a preservative to avoid the spoilage of food.
- IRP001 refers to berberine, which as described herein is a quaternary ammonium cation and plant natural product with antimicrobial activity.
- the terms “IRP001” and “berberine” are used interchangeably herein.
- IRP001 chloride or “IRP001 CI” denotes the chloride salt of berbenne; and “IRPOOl sulfate” refers to the hemisulfate salt of berberine.
- IRPOOl sulfate denotes the chloride salt of berbenne
- IRPOOl sulfate refers to the hemisulfate salt of berberine.
- IRP003 refers to piceid.
- the molecular structure of piceid is shown in Figure 14.
- berberine alkaloid(s) refers to berberine and compounds which share similar structures and characteristics to berberine and are suitable for the feed
- Such compounds include, but are not limited to the protoberberines: berberrubine, coreximine, tetrahydropalmatine, jatrorrhizine, 13-hydroxyberberine chloride, coralyne chloride, 7,8-dihydro-13-methylberberine, fibrauretin (palmatine), and 13-benzylberberine.
- Berberine alkaloids can exist in different isomers or different isomeric forms, for example, various tautomers or tautomeric forms. It will be understood that the term "berberine
- alkaloid(s) encompasses different isomeric forms in isolation from each other as well as combinations.
- Berberine alkaloids can also exist in various amorphous forms and crystalline forms (i.e.
- berine alkaloid(s) encompasses different amorphous and crystalline forms in isolation from each other as well as combinations.
- berberine alkaloid(s) encompasses acceptable salts, solvates, solvates of said salts or pro-drugs thereof.
- the term "food-producing animal” refers to an animal that is farmed for the production of food for consumption by another animal, for example, a human. It would be understood that the term “food-producing animal” includes, for example, a chicken or pig. It will be understood that the term “isomer” refers to structural or constitutional isomers, tautomers, regioisomers, geometric isomers, or stereoisomers including enantiomers or diastereisomers. Further, a racemate will be understood to comprise an equimolar mixture of a pair of enantiomers.
- prodrug refers to an inactive form of a compound which is transformed in vivo to the active form.
- Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound. Further discussion of pro-drugs may be found in Stella, V. J. et al, "Prodrugs", Drug Delivery Systems, 1985, pp. 112-176, Drugs, 1985, 29, pp. 455-473 and "Design of Prodrugs", ed. H. Bundgard, Elsevier, 1985.
- a "safe" residue level of berberine is one that poses an insignificant risk of disease, particularly cancer.
- safe feed preservative or the phrase “wherein the feed preservative is safe” and the like refer to a feed preservative that poses an insignificant risk of disease, particularly cancer.
- treatment refers to the control, healing or amelioration of a disease, disorder or condition, or a decrease in the rate of advancement of a disease, disorder or condition, or defending against or inhibiting a symptom or side effect, reducing the severity of the development of a symptom or side effect, and/or reducing the number or type of symptoms or side effects suffered by an animal subject, as compared to not administering a pharmaceutical composition comprising a compound of the invention.
- treatment encompasses use in a palliative setting.
- prevention prevent
- preventing are intended to encompass treatments that are used to delay or slow down the development of a disease, disorder or condition, or symptom or side effect thereof.
- an effective amount refers to an amount when administered to an animal, achieves a desired effect.
- an effective amount of a composition disclosed herein is an amount that prevents or treats Necrotic Enteritis in a chicken.
- the exact total effective amount of antimicrobial depends on the purpose of the treatment and other factors including the animal subject (e.g. chicken versus pig), route of administration, body weight and severity of the disease.
- range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5.5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.
- Figure 1 depicts the spread of AMR from food-producing animal to human. Figure is taken
- Figure 2 depicts aspects of Salmonella infection and food poisoning. Figure is taken from
- FIG. 3 depicts Campylobacter epidemiology. Figure is taken from
- Figure 4 depicts the molecular structure of berberine quaternary ammonium cation; berberine chloride and berberine hemisulfate.
- Figure 5 to Figure 12 depict the results of the Necrotic Enteritis pilot study in chickens described in Example 1.
- Figure 5 is a graph of bird mortality prior to autopsy for each group.
- Figure 6 is a graph depicting the median small intestinal lesion scores by treatment/challenge group.
- Figure 7 depicts Necrotic Enteritis lesion scores.
- FIG. 8 Photograph of duodenum of bird from Group 9; NE challenged, IVP/Berberine Water
- Figure 13 depicts the molecular structures and names of representative compounds of the invention:
- Figure 14 depicts the molecular structures and names of further representative compounds of the invention:
- Figure 15 depicts total individual water intake (Phase 1) for the Necrotic Enteritis pilot study described in Example 2.
- Figure 16 depicts total individual water intake (Phase 2) for the Necrotic Enteritis pilot study described in Example 2.
- Figure 17 depicts feed conversion ratio (Phase 1 and 2) for the Necrotic Enteritis pilot study described in Example 2.
- Figure 18 depicts the pen set up with day old chicks for the study described in Example 3.
- Figure 19 depicts the litter collected from seeder pens at day 14 of the study described in Example 3.
- Figure 20 depicts the 400 grams of litter allocated per pen at day 14 of the study described in Example 3.
- Figure 21 is a graph of the average daily weight gain of birds in the study described in Example 3 by treatment group: Average Daily Gain (ADG) (g/day) on y-axis versus growth period (days) on x-axis.
- Treatment group 1 control: Treatment group 2 (IVP 0.30 g/kg); Treatment group 3 (IVP 0.10 g/kg); Treatment group 4 (IVP 0.03 g/kg); Treatment group 5 (Salinomycin (Salino) 60 ppm); Treatment group 6 (Salinomycin + Zn Bacitracin 50 ppm (Salino Zn Bac)).
- ADG Average Daily Gain
- Figure 22 depicts a comparison between control finisher and the IVP used in Example 3, Treatment Group 2 (dose of IVP 0.30 g/kg).
- Figure 23 depicts faeces at 42 days from birds from Treatment Group 2 (dose of IVP 0.30 g/kg), Example 3.
- Figure 24 E. acervulina-type lesions (from outside and inside the duodenum), score +1, from Example 3.
- Figure 25 E. acervulina-type lesions, scores +2 and +3, from Example 3.
- Figure 26 E. acervulina-type lesions, score +4 from Example 3.
- Figure 27 ballooning of intestine from Example 3.
- Figure 29 Watery gut contents, including orange-coloured mucus from Example 3.
- Figure 30 depicts the correlation between corrected Feed Conversion Ratio at 42 days and total intestinal cocci diosis lesion scores at 21 days.
- the solid line shows the line of best fit; dashed lines show 95 % confidence intervals.
- the present disclosure relates to a method for the prevention and/or treatment of an infectious disease in an animal, wherein the method comprises administering a berberine alkaloid or an acceptable salt thereof to said animal.
- the animal is human.
- the animal is preferably non-human.
- the non- human animal is a food producing animal.
- the food producing animal is preferably selected from a chicken or a pig.
- the animal is an aquatic animal.
- the aquatic animal is preferably finfish.
- the aquatic animal is shellfish.
- Shellfish are preferably selected from crustaceans or molluscs.
- crustaceans are selected from the group comprising crabs, crayfish, lobsters, prawns, and shrimp.
- Molluscs are preferably selected from the group comprising clams, mussels, oysters, scallops and winkles.
- the animal is a mammal.
- the mammal preferably is a human, horse, dog, cat, sheep, cattle, pig or primate.
- the animal is a bird.
- the bird is preferably chickens, geese, turkeys or ducks. Spotty liver disease
- the infectious disease is a disease of the liver or an intestinal disease.
- the liver disease is preferably Spotty Liver Disease and the animal is a chicken.
- the chicken is an egg-laying chicken.
- the Spotty Liver Disease is preferably caused by bacteria from the genus Campylobacter.
- the Campylobacter is antibiotic resistant.
- the infectious disease is associated with food poisoning.
- the food poisoning is preferably Salmonellosis.
- Salmonellosis is caused by an antibiotic resistant strain of Salmonella.
- the infectious disease is Campylobacteriosis.
- the Campylobacteriosis is preferably caused by an antibiotic resistant strain of Campylobacter.
- the infectious disease is caused by E. coli.
- diarrhoea Of all the diseases in the sucking piglet, diarrhoea is the most common and probably the most important. In some outbreaks it is responsible for high morbidity and mortality. In a well-run herd there should be less than 3% of litters at any one time requiring treatment and piglet mortality from diarrhoea should be less than 0.5%. However, in severe outbreaks levels of mortality can rise to 7% or more and in individual untreated litters up to 100%. The main bacterial cause is E. coli. Scour in the piglet can occur at any age during sucking but there are often two peak periods, before 5 days and between 7 and 14 days.
- the infectious disease is preferably diarrhoea and the animal is a pig.
- the infectious disease is scour and the animal is a pig.
- the infectious disease is preferably dysentery and the animal is a pig.
- the infectious disease is caused by an antibiotic-resistant strain of E. coli. Swine Dysentery associated with Brachyspira
- Swine Dysentery is caused by a spirochaetal bacterium called Brachyspira including Brachyspira hyodysenteriae, Brachyspira piloscoli and Brachyspira hampsonii.
- This organism causes a severe inflammation of the large intestine with a bloody mucous diarrhoea.
- the high cost of the disease is associated with morbidity, mortality, depression of growth and feed conversion efficiency, and costs of continual in-feed medication.
- the infectious disease is caused by bacteria from the genus Brachyspira.
- the infectious disease is preferably dysentery and the animal is a pig.
- the infectious disease is caused by an antibiotic-resistant strain of Brachyspira.
- the infectious disease is preferably caused by bacteria from the genus Lawsonia.
- the infectious disease is caused by an antibiotic-resistant bacterial strain from the genus Lawsonia.
- the infectious disease is preferably caused by Lawsonia intracellularis.
- Ileitis comprises a group of conditions involving pathological changes in the small intestine associated with the bacterium Lawsonia intracellularis.
- the disease takes four different forms.
- the first form, porcine intestinal adenopathy (PIA) is an abnormal proliferation of the cells that line the intestines.
- PIA can develop into the three other forms, which are rarer: necrotic enteritis (NE), where the proliferated cells of the small intestine die and slough off with a gross thickening of the small intestine (hosepipe gut); regional ileitis (RI), inflammation of the terminal part of the small intestine and proliferative haemorrhagic enteropathy (PHE) or "bloody gut” where there is massive bleeding into the small intestine.
- NE necrotic enteritis
- RI regional ileitis
- PHE proliferative haemorrhagic enteropathy
- PHE proliferative haemorrhagic enteropathy
- the infectious disease is represented by a group of conditions selected from: porcine intestinal adenopathy, necrotic enteritis, regional ileitis and proliferative haemorrhagic enteropathy and the animal is a pig.
- porcine intestinal adenopathy necrotic enteritis
- regional ileitis regional ileitis
- proliferative haemorrhagic enteropathy and the animal is a pig.
- the infectious disease is caused by a parasite from the genus Eimeria.
- the parasite is preferably selected from E. maxima, E. acervuline, and E. brunette.
- the infectious disease is caused by an antibiotic-resistant parasite from the genus Eimeria.
- the antibiotic- resistant parasite is preferably selected from an E. maxima, E. acervuline, and E. brunette antibiotic-resistant parasite.
- the infectious disease is Coccidiosis and the animal is a chicken.
- the infectious disease is caused by bacteria from the genus Clostridium.
- the bacteria are preferably selected from the group consisting of: Clostridium difficile and Clostridium perfringens.
- the bacteria are C. difficile.
- the infectious disease is preferably diarrhoea and the animal is human.
- the infectious disease is colitis and the animal is human.
- the bacteria are C. perfringens.
- the infectious disease is preferably Necrotic enteritis and the animal is a chicken.
- the Necrotic enteritis is caused by a C. perfringens type A strain.
- the C. perfringens type A strain is preferably C. perfringens type A strain EHE-NE36.
- the C. perfringens type A strain is C. perfringens type A strain EHE-NE18.
- the Necrotic enteritis is preferably caused by a C. perfringens type C strain.
- the administration occurs via the feed or water of the chicken.
- the feed is preferably in the form of a crumble or a pellet.
- the berberine alkaloid is administered in the feed of the chicken at a dose of 0.001 g/kg to 2.0 g/kg of feed.
- the berberine alkaloid is preferably administered in the feed at a dose of 0.003 g/kg to 0.3 g/kg of feed.
- the berberine alkaloid is preferably administered in the water of the chicken at a dose of 0.001 g/L to 1 g/L of water.
- the lesion score is decreased. There is preferably a reduction in morbidity.
- TGA Therapeutic Goods Administration
- APIMVA Australian Pesticides and Veterinary Medicines Authority
- FDA Food and Drug Administration
- the FD&C Act requires that compounds intended for use in food-producing animals are shown to be safe and that food produced from animals exposed to these compounds is shown to be safe for consumption by people.
- the use in food-producing animals of any compound found to induce cancer when ingested by people or animal is prohibited by statute (21 CFR Part 500, Subpart E - Regulation of carcinogenic compounds used in food-producing animals) unless certain conditions are met (the so-called "Diethylstilbestrol (DES) Proviso").
- DES Diethylstilbestrol
- no residue refers to any residue remaining in the edible tissues that is so low that it presents an insignificant risk of cancer to consumers. More specifically, an insignificant risk of cancer is defined as a 1 in 1 million increase in risk.
- a "safe" residue level of berberine, as used herein, is one that poses an insignificant risk of disease, particularly cancer.
- the level of residue is about 10 ng of the berberine alkaloid per g of muscle tissue.
- the level of residue is preferably about 5 ng/g.
- the berberine alkaloid has been administered in the feed of the chicken at a rate of less than about 0.1 g/kg.
- the washout period is a period between 1 to 2 weeks.
- the level of residue is preferably about 13 ng of the berberine alkaloid per g of muscle tissue.
- the level of residue is preferably about 10 ng of the berberine alkaloid per g of muscle tissue.
- the level of residue is about 5 ng/g.
- the washout period is a period between 1 to 2 weeks when the berberine alkaloid has been administered in the feed of the chicken at a rate of about greater than 0.1 g/kg.
- the level of residue is preferably at about 13 ng of the berberine alkaloid per g of muscle tissue.
- the level of residue is preferably at about 10 ng of the berberine alkaloid per g of muscle tissue.
- the level of residue is about 5 ng/g.
- the residue level of a berberine alkaloid may be determined by experiment.
- An example protocol for determining the residue level of a berberine alkaloid in animal tissue using LC-MS/MS is as follows: Samples of muscle from breast, leg and thigh, and liver and kidney were excised from each bird after euthanasia. A known weight of tissue (approximately lg) was homogenized in 2 mL water. Samples were centrifuged and a known volume of the supernatant was removed for analysis of berberine by LC-MS/MS to provide the residue level of berberine in muscle tissue (ng of berberine per g of muscle tissue). Administration of berberine formulations
- the berberine alkaloid is berberine hemisulfate.
- the berberine alkaloid is preferably berberine chloride.
- the method further comprises co-administering one or more agents suitable for the prevention and/or treatment of an infectious disease.
- the one or more agents are preferably selected from the group comprising: ⁇ -lactams, macrolides, quinolones, tetracyclines, sulfonamides, aminoglycosides, glycopeptides, lincomycins, and polymxins.
- One or more ⁇ - lactams are preferably selected from the group comprising: penicillins, cephalosporins, and carbapanems.
- Agents or medications for the prevention and/or treatment of infectious disease are described, for example, in Goodman & Gilman's "The Pharmacological Basis of Therapeutics", 11 th Ed., L. L. Brunton (Ed.), J. S. Lazo and K. L. Parker (Assoc. Eds.), McGraw-Hill, 2005 (Goodman and Gilman), particularly in Section VII: Chemotherapy of Parasitic Infections, pages 1021 to 1093; and Section VIII: Chemotherapy of Microbial Disease, pages 1095 to 1314.
- the present disclosure encompasses methods for the prevention and/or treatment of an infectious disease in an animal comprising co-administration of a berberine alkaloid with an isomeric form, a racemate, an amorphous or a crystalline form, a solvate, an acceptable salt, a solvate of said salt, or a prodrug or a combination thereof of agents or medications suitable for the prevention and/or treatment of infectious disease.
- the berberine alkaloids of the present disclosure may be applied as a sole therapy or may involve, in addition, one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the prevention or treatment.
- the one or more agents are one or more additional antimicrobial compounds selected from the group consisting of: arecoline, baicalin, baicalein, anemonin, matrine, oxymatrin, andrographolide, piceid, honokiol and thymol.
- the one or more agents are one or more anti-coccidial agents.
- the one or more anticoccidial agents are preferably selected from the group comprising: amprolium, ethopabate, chlortetracycline, clopidol, meticlorpindol, decoquinate, diclazuril, dinitolmide (zoalene), halofuginone, lasalocid, maduramicin, monesin, narasin, nicarbazin, oxytetracycline, robenidine, salinomycin, semduramicin, sulfachloropyrazine, sulfamethazine (sulfadimidine),
- the method further comprises an additive that masks the bitter flavour of the berberine alkaloid or acceptable salt.
- composition comprising any two or more of the following: a berberine alkaloid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine,
- the berberine alkaloid and the one or more agents suitable for the prevention or treatment of infectious disease must be stable and compatible with each other and the other components of the formulation and may be formulated for administration. When formulated separately they may be provided in any convenient formulation, conveniently in such a manner as are known for such compounds in the art. It should be understood that the formulations may include other agents as disclosed herein and agents conventional in the art having regard to the type of formulation in question.
- Berberine is an isoquinoline alkaloid extracted from Rhizoma coptidis, Phellodendri chinensis cortex, and other herbs. According to the Chinese Pharmacopoeia, the berberine content of
- Rhizoma coptidis Huanglian in Chinese belongs to family Ranunculaceae and contains three main Coptis species: Coptis chinensis (Weilian in Chinese), Coptis deltoidea (Yalian in Chinese), and Coptis teeta (Yunlian in Chinese). Rhizoma coptidis is harvested in autumn and sliced after the removing the fibrous roots. Those with bright yellow sections and very bitter taste are considered of good quality. The bitter taste of berberine (and other berberine alkaloids as disclosed herein) makes taste-masking/palatability an important issue to consider when formulating berberine alkaloids for administration to animal subjects.
- Berberine is a yellow powder.
- the chloride salt is slightly soluble in cold water, but freely soluble in boiling water. It is practically insoluble in cold ethanol.
- the hemisulfate salt is soluble in about 30 parts water, slightly soluble in ethanol.
- Berberine is a quaternary ammonium cation with molecular formula of C 20 H 18 NO 4 + and molecular weight of 336.36.
- Figure 4 depicts the molecular structure of the berberine ammonium cation, berberine chloride salt, and berberine hemi sulfate salt.
- Berberine may be administered in any form acceptable for enteral administration. Suitable non- limiting forms for enteral administration include tablets, capsules, paste, granules, chewable wafers, gel, oral liquid, injectable liquid, medicated water and medicated feed, and suppositories. However with food producing animals where economic interests are important, the preferred method of administering berberine is via a feed additive in the form of granules, or a medicated feed. It may also be administered via the drinking water of an animal subject by mixing water with a suitable solution or suspension of berberine.
- a palatable berberine alkaloid formulation may comprise berberine and an acceptable excipient which is suitable for forming a granular product.
- the acceptable excipient which is suitable for forming a granular product is, for example, cornstarch or polyvinylpyrollidone (PVP).
- the liquid formulation is a liquid concentrate.
- berberrubine berberrubine, coreximine, tetrahydropalmatine, jatrorrhizine, 13- hydroxyberberine chloride, coralyne chloride, 7,8-dihydro-13-methylberberine, fibrauretin
- Fibrauretin (Palmatine) Fibrauretin or palmatine is a bitter tasting alkaloid extracted from Fibauera recisa Pierre.
- Fibrauera recisa Pierre consists of no less than 2.0% fibrauretin.
- Another source is Coptidis rhizoma, the rhizome of Coptis chinensis Franch, Coptis deltoidea and Coptis teeta Wall.
- Coptidiz rhizoma consists of no less than 1.5% fibrauretin.
- Palmatine chloride is a yellow solid, which is soluble in hot water, sparingly soluble in water, and slightly soluble in ethanol. Its melting point is 196-198 °C. Its molecular formula is C 21 H 22 NO 4 CI with a molecular weight of 387.86.
- the molecular structure of the palmatine quaternary ammonium cation and the structure of the chloride salt are set out in Figure 13.
- the present disclosure contemplates formulations of a berberine alkaloid in combination with other agents suitable for the prevention and/or treatment of infectious disease. Examples of such agents are described below. It will be understood that the invention encompasses isomeric forms, racemates, amorphous or crystalline forms, solvates, acceptable salts, solvates of said salts, or prodrugs or combinations thereof of these agents.
- Arecoline is a major alkaloid constituent extracted from the traditional Chinese medicine Semen arecae, semen of Areca catechu L. According to Chinese Pharmacopoeia the content of
- Arecoline in S Semen arecae is not less than 0.2%. Semen arecae is manufactured primarily in Hainan province China, however Areca catechu L. is produced in southern Yunnan, Fujian, Guangxi, and southern Taiwan and other areas.
- Baicalin and baicalein Baicalin is a major flavonoid constituent found in the traditional Chinese medicine, Scutellaria Root, the root of Scutellaria baicalensis Georgi. According to Chinese Pharmacopoeia the content of Baicalin found in Scutellaria Root is 9.0%. Scutellaria Root is manufactured mainly in Northeast China; Hebei, Shanxi, Henan, Shanxi, Neimeng province et al., although Scutellaria Root can be grown in most provinces of northern China. Baicalin is a pale yellow powder, and is bitter in taste. It is diffluent in N,N-
- Dimethylformamide and soluble in alkaline solution, such as sodium bicarbonate, sodium carbonate, sodium hydroxide (however baicalin is unstable in alkaline environment). It is almost insoluble in water. Its molecular formula is C 21 H 18 0n, molecular weight is 446.36.
- Anemonin Amemonin is the dry root of the Ranunculaceae plant Clematis chinensis osbeck, Clematis ssp Hexapetala Pall and Clematis manshurica rupr.
- the dry root and rhizome is called Clematidis radix et rhizome and according to the Chinese Pharmacopoeia contains 4.6% B l Pulsatilla glycosides.
- Clematidis radix et rhizoma is produced in Jiangsu, Zhejiang, Jiangxi, Anhui and other provinces, with Clematis Hexapetala production mainly in the northeast and Shandong while Manshurica Rupr lotus production is primarily in the northeast.
- Other sources include the dry root of Ranunculus japnicus thunb, R Sceleratus L, Anemone hupehensis lem and Pulsatilla chinesis.
- Anemonin is a white powder of bitter taste. Its melting point is around 158 °C and it is slightly soluble in cold water, soluble in hot water, and soluble in hot ethanol. Its molecular formula is CioH 8 04 with a molecular weight of 192.16. The molecular structure of anemonin is depicted in Figure 14.
- Matrine and its N-oxide derivative, oxymatrine are alkaloids extracted from the legumes (Fabaeceae) and the dry root and fruit of the plant Sophora (Sophora flavescens var.
- Matrine is a white odourless powder of bitter taste.
- the molecular formula is C15H25N2), with a molecular weight of 249.
- oxymatrine is a white powder of bitter taste.
- the molecular formula is C15H24N2O2, with a molecular weight of 264.
- the molecular structures of matrine and oxymatrine are set out in Figure 13.
- Andrographolide is a labdane diterpenoid that is produced by the Andrographis paniculata plant. According to the Chinese Pharmacopoeia, Andrographis paniculata consists of no less than 0.8%) of the active substance.
- Andrographolide is a colourless, powder with a crystalline appearance and is bitter in taste. It is soluble in boiling ethanol, poorly soluble in ethanol at ambient temperature, and almost insoluble in water. It has a melting point of 224-230°C and decomposes upon melting. Its molecular formula is C20H30O5, and has a molecular weight of 350.44. The molecular structure for andrographolide is shown in Figure 14. Piceid
- Piceid is a stilbenoid glucoside and is a major resveratrol derivative in grape juices. It can be found in the bark of Picea sitchensis. It can also be isolated from Fallopia japonica, the
- the composition comprises a berberine alkaloid in combination with at least one other antimicrobial compound selected from arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, piceid, honokiol and thymol.
- at least one other antimicrobial compound selected from arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, piceid, honokiol and thymol.
- composition preferably comprises the following: i. berberine and any one of arecoline, baicalin, baicalein, anemonin, matrine,
- oxymatrine or andrographolide ii. berberine and arecoline as well as any one of baicalin, anemonin, matrine,
- oxymatrine or andrographolide iii. berberine and baicalin as well as any one of anemonin, matrine, oxymatrine or
- preparation of combination feeds can be accomplished using conventional procedures.
- the amounts of antimicrobial compound in the prepared feed can be increased or decreased and the ratio of the individual antimicrobial compounds may be varied.
- the total effective amount or dose of the antimicrobial compound in the prepared feed may range from 0.001 g/kg to 2 g/kg.
- Example amounts of the total amount of antimicrobial compound in the prepared feed are: 0.001 g/kg (0.0001 %), 0.003 g/kg (0.0003 %), 0.01 g/kg (0.001 %), 0.03 g/kg (0.003 %), 0.1 g/kg (0.01 %), 0.3 g/kg (0.03 %), 1.0 g/kg (0.1 %) and 2 g/kg (0.2 %).
- a combination feed which is an example of a combination feed containing the three
- antimicrobial compounds berberine and piceid and baicalin may comprise: " a high amount of berberine, a low amount of piceid, and a low amount of baicalin;
- a combination feed may comprise:
- ⁇ a medium amount of berberine, a medium amount of piceid, and a medium amount of baicalin, such that berberine, piceid and baicalin are in equal amounts;
- a combination feed may comprise:
- ⁇ a low amount of berberine, a medium amount of piceid, and a high amount of baicalin; " a low amount of berberine, a high amount of piceid, and a medium amount of baicalin;
- example combination feeds may comprise beberine:piceid:baicalin in ratios ranging from: 99.0:0.5:0.5; 90:9: 1, 80: 19: 1; 70:29: 1; 60:39: 1; 50:49: 1; 50:40: 10, 40:40:20, 90: 1 :9, 80: 1 : 19; 70: 1 :29; 60: 1 :39; 50: 1 :49; 50: 10:40; 40:20:40 to 33.3 :33.3 :33.3, where berberine ranges from a high to medium amount.
- example combination feeds may have beberine:piceid:baicalin in ratios ranging from 33.3 :33.3 :33.3 to 20:40:40, 10:40:50, 10:50:40, 1 :49:50, 1 :50:49, 1 :39:60; 1 :60:39; 1 :29:70, 1 :70:29; 1 : 19:80; 1 :80: 19; 1 :9:90; 1 :90:9 to 0.5:0.5:90, where berberine ranges from a medium to low amount.
- a combination feed comprising three antimicrobial compounds used for treating a Necrotic Enteritis or an infectious disease caused by Eimeria (Eimeria disease) in chickens is prepared by mixing 0.03 g berberine; 0.0015 g piceid; and 0.0003 g baicalin with commercial feed to give 1 kg of prepared feed.
- berberine represents 0.03 % of the prepared feed
- piceid represents 0.0015 % of the prepared feed
- baicalin represents 0.00003% of the prepared feed.
- the berberine:piceid:baicalin are in a ratio of 0.03 :0.0015:0.0003 or 94.34:4.72:0.94, that is berberine is present in the prepared feed in a high amount; piceid is present in the feed in a medium amount; and baicalin is present in the feed in a low amount.
- the exact total effective amount of antimicrobial, and the ratio of the individual antimicrobial agents depends on the purpose of the treatment and other factors including the animal subject (e.g. chicken versus pig), route of administration, body weight and severity of the disease.
- an example of a combination feed used for treating Spotty Liver Disease (as opposed to Necrotic Enteritis or Eimeria disease) in chickens contains more berberine.
- This combination chicken feed is prepared by mixing 0.1 g berberine; 0.05 g piceid; and 0.0003 g baicalin with commercial feed to give 1 kg of prepared feed comprising three antimicrobial compounds.
- berberine represents 0.1 % of the prepared feed; piceid represents 0.005 % of the prepared feed; and 0.00003%) of the prepared feed.
- piceid:baicalin are in a ratio of 1 :0.5:0.003 or 66.53 :33.27:0.20, that is berberine is present in the prepared feed in a high amount; piceid is present in the feed in a medium amount; and baicalin is present in the feed in a low amount.
- an example of a combination feed used for treating pig disease contains more berberine.
- This combination pig feed is prepared by mixing 0.1 g berberine; 0.05 g piceid; and 0.0003 g baicalin with commercial feed to give 1 kg of prepared feed comprising three antimicrobial compounds.
- berberine represents 0.1 % of the prepared feed
- piceid represents 0.005 % of the prepared feed
- 0.00003%) of the prepared feed is 0.1 % of the prepared feed.
- the berberine:piceid:baicalin are in a ratio of 1 :0.5:0.003 or 66.53 :33.27:0.20, that is berberine is present in the prepared feed in a high amount; piceid is present in the feed in a medium amount; and baicalin is present in the feed in a low amount.
- the disclosure also relates to a combination feed containing two antimicrobial compounds.
- An example of a combination feed containing the two antimicrobial compounds berberine and baicalin for treating Necrotic Enteritis or Eimeria in chickens is prepared by mixing 0.03g berberine and 0.001 g baicalin with commercial feed to give 1 kg of prepared feed.
- berberine represents 0.003 % of the prepared feed
- baicalin represents 0.0001 % of the prepared feed.
- the berberine:baicalin are in a ratio of 0.03 :0.001 i.e., 96.77:3.23 i.e., there is 30 times the amount of berberine in the prepared feed than baicalin.
- an example of a combination feed used for treating Spotty Liver Disease in chickens (as opposed to Necrotic Enteritis or Eimeria disease) or treating pig diseases contains more berberine and baicalin respectively.
- An example combination chicken feed for treating Spotty Liver Disease is prepared by mixing 0.1 g berberine; and 0.01 g baicalin with commercial feed to give 1 kg of prepared feed.
- berberine represents 0.01 % of the prepared feed
- bacalin represents 0.001 % of the prepared feed.
- the berberine :baicalin are in a ratio of 0.1 :0.01 i.e., 91 :9 i.e., there is ten times the amount of berberine in the prepared feed than baicalin.
- the disclosure also relates to a composition
- a composition comprising a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in an animal.
- Agents for treating infectious disease are described above and in, for example, Goodman and Gilman.
- the one or more agents are one or more anti-coccidial agents.
- Anti-coccidial agents are discussed above.
- the compositions disclosed herein further comprise one or more acceptable excipients.
- the one or more acceptable excipients are preferably one or more vehicles or one or more acceptable additives.
- the one or more additives are selected from the group consisting of: buffers, solubilisers, gelling agents, viscosity enhancers, preservatives, oils, antioxidants, emulsifiers, foam forming agents, isotonic agents, a propellant gas, thickeners and combinations thereof.
- the composition preferably further comprises an additive that masks the bitter flavour of the berberine alkaloid.
- compositions of the present disclosure may be formulated for administration by any appropriate route depending on the animal subject.
- oral including buccal or sublingual
- rectal nasal
- topical including buccal, sublingual or transdermal
- vaginal or parenteral including subcutaneous, intramuscular, intravenous or intradermal
- the compositions of the invention may be formulated, for example, as tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions.
- Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association an active ingredient, or combination of active ingredients, of with acceptable excipient(s).
- Such formulations may be prepared as enterically coated granules, tablets or capsules suitable for oral administration and delayed release formulations. The combinations of active ingredients are proposed for both liquid delivery as well as in granules for mixing through animal feeds.
- the dose of each compound may differ from that when the compound is used alone.
- the present disclosure also relates to an animal feed comprising a berberine alkaloid.
- the present disclosure also relates to an animal feed comprising a berberine alkaloid and an animal foodstuff, wherein the berberine alkaloid is in an amount of about 0.001 % to 1% w/w of the animal foodstuff.
- the amount of the berberine alkaloid in the foodstuff may range from 0.001 g/kg to 2 g/kg i.e., 0.001 % to 0.2 % w/w.
- Example amounts of the berberine alkaloid in the foodstuff are: 0.001 g/kg (0.0001 %), 0.003 g/kg (0.0003 %), 0.01 g/kg (0.001 %), 0.03 g/kg (0.003 %), 0.1 g/kg (0.01 %), 0.3 g/kg (0.03 %), 1.0 g/kg (0.1 %) and 2.0 g/kg (0.2%).
- the feed is preferably in the form of a crumble; pellet; or in an aqueous form.
- the present disclosure also relates to an animal feed comprising a composition described herein.
- the present disclosure also relates to an animal feed comprising a composition described herein and an animal foodstuff, wherein the composition is in an amount of about 0.001 % w/w to 1 % w/w of the animal foodstuff.
- the amount of the composition in the foodstuff may range from 0.01 g/kg to 10 g/kg i.e., 0.001 % to 1 % w/w; 0.003 % to 1 % w/w; 0.01 % to 1% w/w; 0.03 % to 1 % w/w; 0.1 % to 1% w/w; 0.3 % to 1 % w/w.
- Example amounts of the composition may be: 0.01 g/kg (0.001%); 0.03 g/kg (0.003%); 0.1 g/kg (0.01%); 0.3 g/kg (0.03 %); 1 g/kg (0.1 %); 3 g/kg (0.3 %); 10 g/kg (1 %).
- Further example amounts of the composition in the foodstuff may be: 0.031 g/kg (0.0031%); 0.06 g/kg (0.006%); 0.11 g/kg (0.011%); 0.1503 g/kg (0.015%).
- the feed is preferably in the form of a crumble; pellet; or in an aqueous form.
- the present disclosure also relates to a dosing regimen comprising administering a berberine alkaloid, or a composition or an animal feed as disclosed herein for 1 to 6 weeks to an animal, wherein the berberine alkaloid, or the composition or animal feed is administered for 1 to 6 weeks and in an amount effective to prevent and/or treat an infectious disease in an animal.
- the berberine or the composition or animal feed is administered for 1, 2, 3, 4, 5 or 6 weeks.
- the berberine or acceptable salt thereof, or the composition or animal feed is administered for 1 to 6; 2 to 5; or between 3 to 4 weeks.
- the berberine alkaloid is administered at a concentration of about 0.6 g/L in-water or about 1.2 g/kg in-feed.
- the amount of the berberine alkaloid in the feed may range from 0.001 g/kg to 2 g/kg i.e., 0.0001 % to 0.2 % w/w.
- Example amounts of the berberine alkaloid or acceptable salt in the foodstuff are: 0.001 g/kg (0.0001%), 0.003 g/kg (0.0003%), 0.01 g/kg (0.0001 %), 0.03 g/kg (0.0003 %), 0.1 g/kg (0.01%), 0.3 g/kg (0.03%), 1.0 g/kg (0.1%), and 2 g/kg (0.2%).
- the disclosure also relates to a method for the reduction of feed conversion ratio in a food- producing animal, wherein the method comprises the step of administering a berberine alkaloid to said food-producing animal.
- the food-producing animal is free of disease.
- the food-producing animal is preferably diseased.
- the food-producing animal is selected from a chicken or a pig.
- the food-producing animal is preferably a chicken.
- the disclosure also relates to a method for preventing or treating an infectious disease in an animal comprising administering a composition or an animal feed described herein.
- the disclosure also relates to a method for preventing or treating an infectious liver disease in an animal comprising administering a composition or an animal feed described herein.
- the disclosure also relates to a method for preventing or treating an infectious intestinal disease in an animal comprising administering a composition or an animal feed described herein.
- the disclosure also relates to a method for preventing or treating Spotty Liver Disease in a chicken comprising administering a composition or an animal feed described herein.
- the chicken is an egg-laying chicken.
- the Spotty Liver Disease is preferably caused by bacteria from the genus Campylobacter.
- the Campylobacter is antibiotic resistant.
- the disclosure also relates to a method for preventing or treating an infectious disease caused by E. coli in an animal comprising administering a composition or an animal feed described herein.
- the infectious disease is selected from: diarrhoea, scour or dysentery and the animal is a pig.
- the infectious disease is preferably caused by an antibiotic-resistant bacterial strain of E. coli.
- the disclosure also relates to a method for preventing or treating an infectious disease caused by Brachyspira in an animal comprising administering a composition or an animal feed described herein.
- the infectious disease is selected from dysentery and the animal is a pig.
- the infectious disease is preferably caused by an antibiotic-resistant bacterial strain from the genus Brachyspira.
- the disclosure also relates to a method for preventing or treating an infectious disease caused by Lawsonia in an animal comprising administering a composition or an animal feed described herein.
- the infectious disease is represented by a group of conditions selected from: porcine intestinal adenopathy, necrotic enteritis, regional ileitis and proliferative haemorrhagic enteropathy and the animal is a pig.
- the infectious disease is preferably caused by an antibiotic- resistant bacterial strain from the genus Lawsonia.
- the disclosure also relates to a method for preventing or treating an infectious disease caused by Eimeria in an animal comprising administering a composition or an animal feed described herein.
- the infectious disease is caused by an antibiotic-resistant bacterial strain from the genus Eimeria.
- the infectious disease is preferably Coccidiosis and the animal is a chicken.
- the disclosure also relates to a method for preventing or treating an infectious disease caused by bacteria from the genus Clostridium in an animal comprising administering a composition or an animal feed described herein.
- the infectious disease is caused by an antibiotic-resistant bacterial strain from the genus Clostridium.
- the bacteria are preferably C. difficile.
- the infectious disease is preferably diarrhoea and the animal is human.
- the infectious disease is colitis and the animal is human.
- the bacteria are preferably C. perfringens.
- the infectious disease is Necrotic enteritis and the animal is a chicken.
- the present disclosure also relates to use of a berberine alkaloid in the preparation of a medicament for the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of any two or more of the following: a berberine alkaloid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, fibrauretin (palmatine), and piceid in the preparation of a medicament for the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in an animal in the preparation of a medicament for the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of a berberine alkaloid in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of any two or more of the following: a berberine alkaloid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatrin, andrographolides, fibrauretin (palmatine), and piceid in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to use of a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in an animal in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to a berberine alkaloid for use in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to any two or more of the following: a berberine alkaloid, arecoline, baicalin, baicalein, anemonin, matrine, oxymatnn, andrographolides, fibrauretin (palmatine), and piceid for use in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal;
- the present disclosure also relates to a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in an animal for use in the prevention and/or treatment of: an infectious disease in an animal; an infectious liver disease in an animal; an infectious intestinal disease in an animal; Spotty Liver Disease in a chicken; an infectious disease caused by E. coli in an animal; an infectious disease caused by Brachyspira in an animal; an infectious disease caused by Lawsonia in an animal; an infectious disease caused by Eimeria in an animal; or treating an infectious disease caused by bacteria from the genus Clostridium.
- the present disclosure also relates to an animal feed preservative comprising a berberine alkaloid.
- the present disclosure also relates to an animal feed preservative comprising a berberine alkaloid, wherein the feed preservative is safe.
- the animal is non-human.
- the non-human animal is preferably a food producing animal.
- the food producing animal is selected from a pig or chicken.
- the food producing animal is preferably a pig.
- the food producing animal is a chicken.
- the residue level is preferably at least below about 13 ng of the berberine alkaloid per g of muscle tissue.
- the residue level is about 10 ng of the berberine alkaloid per g of muscle tissue.
- the residue level is preferably about 5 ng of the berberine alkaloid per g of muscle tissue.
- the berberine alkaloid has been administered in the feed of the chicken in an amount of about 0.3 g/kg.
- the residue levels of the berberine alkaloid in the muscle tissue of the chicken are preferably as follows: about 6.1 ng/g in the muscle tissue in the breast of the chicken; about 5.5 ng/g in the muscle tissue in the lower leg of the chicken; and about 11.6 ng/g in the muscle tissue in the upper leg of the chicken.
- the berberine alkaloid has been administered in the feed of the chicken in an amount of about less than 0.1 g/kg.
- the berberine alkaloid has preferably been administered in the feed of the chicken in an amount of about 0.03 g/kg.
- the residue levels of the berberine alkaloid in the muscle tissue of the chicken are as follows: below 2 ng/g in the muscle tissue in the breast of the chicken; below 2 ng/g in the muscle tissue in the lower leg of the chicken; and below 2 ng/g in the muscle tissue in the upper leg of the chicken.
- the washout period is preferably a period between 1 week and 2 weeks.
- the washout period is selected from a period between
- the washout period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.
- the residue levels of the berberine alkaloid in the muscle tissue of the chicken are as follows: about 5.7 ng/g in the muscle tissue in the breast of the chicken; about 3.2 ng/g in the muscle tissue in the lower leg of the chicken; and about 6.0 ng/g in the muscle tissue in the upper leg of the chicken.
- the residue levels of the berberine alkaloid in the muscle tissue of the chicken are as follows: about 3.6 ng/g in the muscle tissue in the breast of the chicken; about 3.1 ng/g in the muscle tissue in the lower leg of the chicken; and about 4.5 ng/g in the muscle tissue in the upper leg of the chicken.
- the residue levels of the berberine alkaloid in the muscle tissue of the chicken are below 2 ng/g.
- the berberine alkaloid has preferably been administered in the feed of the chicken at a dose of about 0.3 g/kg.
- the level of residue is at least below 13 ng of the berberine alkaloid per g of muscle tissue.
- the level of residue is preferably about 5 ng/g.
- the berberine alkaloid has been administered in the feed of the chicken at a dose of about greater than 0.1 g/kg.
- the berberine alkaloid there is a safe residue level of the berberine alkaloid in the liver and muscle tissue of the chicken after administration.
- the residue levels of the berberine alkaloid in the liver and muscle tissue of the chicken are preferably below 2 ng/g.
- the berberine alkaloid has been administered in the feed of the chicken at a dose of about 0.03 g/kg.
- the washout period is preferably a period between 1 week and 2 weeks.
- the washout period is a period selected from between 1 day and 14 days; between 1 day and 7 days; 1 day and 4 days; and between 1 day and 2 days.
- the washout period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.
- the residue levels of the berberine alkaloid in the muscle tissue of the chicken are as follows: about 5.7 ng/g in the muscle tissue in the breast of the chicken; about 3.2 ng/g in the muscle tissue in the lower leg of the chicken; and about 6.0 ng/g in the muscle tissue in the upper leg of the chicken, and a residue level of the berberine alkaloid in the liver tissue of the chicken of about 8.0 ng/g.
- the residue levels of the berberine alkaloid in the muscle tissue in the breast, lower leg and upper leg of the chicken are below 2 ng/g and the residue level of the berberine alkaloid in the liver tissue of the chicken is about 6.5 ng/g.
- the residue levels of the berberine alkaloid in the muscle tissue in the breast, lower leg and upper leg of the chicken are below 2 ng/g and the residue level of the berberine alkaloid in the liver tissue of the chicken is about 3.0 ng/g.
- the berberine alkaloid has been administered in the feed of the chicken at a dose of about 0.3 g/kg.
- the residue levels of the berberine alkaloid in the liver tissue and muscle tissue in the breast, lower leg and upper leg of the chicken are preferably below 2 ng/g.
- the berberine alkaloid has been administered in the feed of the chicken at a dose of about 0.03 g/kg.
- the washout period is preferably a period selected from between 1 week and 2 weeks.
- the washout period is a period selected from between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days.
- the washout period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.
- the residue level of the berberine alkaloid in the liver tissue of the chicken is about 8.0 ng/g.
- the residue level of the berberine alkaloid in the liver tissue of the chicken is preferably about 6.5 ng/g.
- the residue level of the berberine alkaloid in the liver tissue of the chicken is about 3.0 ng/g.
- the berberine alkaloid has preferably been administered in the feed of the chicken at a dose of about 0.3 g/kg.
- the administration is over 35 days.
- the berberine alkaloid is preferably berberine hemisulfate.
- the berberine alkaloid is berberine chloride.
- the feed preservative preferably further comprises an additive that masks a bitter flavour of the berberine alkaloid.
- the feed preservative further comprises one or more additional preserving agents.
- additional preserving agents are discussed in, for example, Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams and Wilkins, 2005 at pages 1058 to 1060 and the noted references therein. These pages and references are incorporated herein.
- the one or more additional preserving agents are preferably selected from antimicrobial compounds or anti -oxidants.
- the one or more additional preserving agents are one or more antimicrobial compounds selected from arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, piceid, honokiol and thymol.
- the present disclosure also relates to an animal feed comprising a feed preservative disclosed herein.
- the feed is preferably in the form of a crumble or a pellet.
- the berberine alkaloid is present in the feed in an amount of about 0.001 g/kg to about 2 g/kg of feed.
- the berberine alkaloid is present in the feed in an amount of about 0.03 g/kg, wherein the animal is a chicken.
- the berberine alkaloid is present in the feed in an amount of about 1.5 g/kg, wherein the animal is a pig.
- the feed is preferably in an aqueous form.
- the berberine alkaloid is present in the feed in an amount of 0.001 g/L to 0.1 g/L.
- the present disclosure also relates to a composition comprising a berberine alkaloid and any one or more of the following: arecoline, baicalin, baicalein, anemonin, matrine, oxymatrine, andrographolide, piceid, honokiol and thymol.
- the present disclosure also relates to a composition comprising a berberine alkaloid and one or more agents suitable for the prevention and/or treatment of an infectious disease in an animal.
- the compositions further comprise one or more acceptable excipients.
- the composition preferably further comprises an additive that masks the bitter flavour of the berberine alkaloid or acceptable salt.
- the present disclosure also relates to an animal feed comprising a composition disclosed herein.
- the animal feed further comprises an animal foodstuff.
- the present disclosure also relates to an animal feed comprising a composition disclosed herein wherein the composition is in an amount of 0.001 to 1 % w/w of the animal foodstuff.
- the feed is preferably in the form of a crumble; pellet; or in an aqueous form.
- the present disclosure also relates to a method for the prevention and/or treatment of an infectious disease in an animal, wherein the method comprises administering a berberine alkaloid or a composition or an animal feed disclosed herein to said animal, wherein the infectious disease is caused by bacteria from the genus Lawsonia.
- the animal is human.
- the animal is preferably non-human.
- the non- human animal is a food producing animal.
- the food producing animal is preferably selected from a chicken or a pig.
- the infectious disease is diarrhoea and the animal is a pig.
- the infectious disease is preferably caused by an antibiotic-resistant bacterial strain from the genus Lawsonia.
- the infectious disease is caused by Lawsonia intracellularis.
- the infectious disease is preferably represented by a group of conditions selected from: porcine intestinal adenopathy, necrotic enteritis, regional ileitis and proliferative haemorrhagic enteropathy and the animal is a pig.
- the present disclosure also relates to a dosing regimen comprising administering a berberine alkaloid, or a composition or an animal feed according disclosed herein wherein the berberine alkaloid, or the composition or animal feed is administered for 1 to 6 weeks and in an amount effective to prevent or treat an infectious disease in an animal, wherein the infectious disease is caused by bacteria from the genus Lawsonia.
- the present disclosure also relates to use of a berberine alkaloid in the preparation of a medicament for the prevention and/or treatment of an infectious disease caused by bacteria from the genus Lawsonia.
- the present disclosure also relates to use of a berberine alkaloid in the prevention and/or treatment of an infectious disease caused by bacteria from the genus Lawsonia.
- the present disclosure also relates to a method for the reduction of feed conversion ratio in a food-producing animal, wherein the method comprises administering a berberine alkaloid or a composition or an animal feed disclosed herein to the food-producing animal.
- the food-producing animal is free of disease.
- the food-producing animal is preferably diseased.
- the food-producing animal is selected from a chicken or a pig.
- the food-producing animal is preferably a chicken.
- the food-producing animal is a pig.
- Study animals are dosed according to the treatment regime detailed in Table 2 below.
- Medicated feed is provided to chickens in the relevant treatments ad lib as their sole source of feed with potable water also provided ad lib.
- the average weight gain, average daily weight gain over the treatment period can be calculated as well as feed conversion ratio (FCR). Performance of animals can be evaluated by these parameters. Also, food and water intake parameters can provide an indication of medication palatability whereas weight gain and feed conversion ratio (FCR) parameters can provide the antibiotic effect of the IVP i.e. the extent the IVP is promoting growth.
- FCR feed conversion ratio
- the residue level of IRP0001 after observing a wash-out period of 1 week is determined by experiment as follows:
- Samples of muscle from breast, leg and thigh, and liver and kidney are excised from each bird after euthanasia.
- a known weight of tissue (approximately lg) is homogenized in 2 mL water. Samples are centrifuged and a known volume of the supernatant is removed for analysis of
- IRP001 by LC-MS/MS to provide the residue level of berberine in muscle tissue (ng of berberine per g of muscle tissue).
- IRP001 Determination of the efficacy in prevention or treatment of Necrotic Enteritis by administration of IRP001 including investigation of dose response, feed conversion rate, tissue residues and safety.
- IRP001 is administered via feed to broiler chickens artificially challenged with pathogenic strains of Eimeria spp, and Clostridium Perfringens utilizing a proven experimental model. Current industry standard treatment, Zinc Bacitracin, is used for efficacy and FCR comparison.
- Study Design Necrotic Enteritis challenge
- broiler chickens housed in isolators are infected orally at 9 days of age with 5,000 attenuated vaccine strain sporulated oocysts each of E. maxima and E.
- Feed intake, weight gain, mortality and NE lesion scores at autopsy are used as outcome parameters.
- the residue level of IRP0001 can be determined by experiment as follows:
- Samples of muscle from breast, leg and thigh, and liver and kidney are excised from each bird after euthanasia.
- a known weight of tissue (approximately lg) is homogenized in 2 mL water. Samples are centrifuged and a known volume of the supernatant is removed for analysis of IRP001 by LC-MS/MS to provide the residue level of berberine in muscle tissue (ng of berberine per g of muscle tissue).
- the study objective is to evaluate the efficacy of three dose rates of IRP001 in-feed against a mixed moderate coccidiosis challenge (Eimeria spp.) in commercial meat chickens and to assess any occurrence of Necrotic Enteritis or non-specific enteritis. Safety data along with tissue residue data is to be obtained.
- Study Design (Eimeria challenge) - Commercial broiler chickens housed in pens, are infected 14 days of age (Day 14) with wild-type Eimeria oocysts; approximately 12,000 E. tenella, 40,000 E. acervuline and as many E. maxima oocysts as possible per bird. Seven days following oocyst challenge (Days 21), four birds per group are randomly selected from each trial pen and humanely euthanized.
- the residue level of IRP0001 can be determined by experiment as follows:
- Samples of muscle from breast, leg and thigh, and liver and kidney are excised from each bird after euthanasia.
- a known weight of tissue (approximately lg) is homogenized in 2 mL water. Samples are centrifuged and a known volume of the supernatant is removed for analysis of IRP001 by LC-MS/MS to provide the residue level of berberine in muscle tissue (ng of berberine per g of muscle tissue).
- This study and protocol aim to determine the residue depletion profile for a naturally occurring IVP administered at the maximum label dose rate through quantification of the marker tissue residue in broiler chickens treated via feed administration over a full production cycle.
- Antimicrobials are used extensively for animal husbandry purposes for the control and prevention of potentially lethal outbreaks of diseases in the intensive livestock industry. Some see this as a cause for the development of resistant microbes, with government regulators now implementing directives in controlling the use of these antimicrobial agents.
- the Inventors have identified several naturally occurring compounds which can be used as natural antibiotics to replace the current antibiotics used in food producing animals, such as poultry and pig.
- Candidate formulations undergo testing to meet the regulatory standards as required, for example, by the Australian Pesticides & Veterinary Medicines Authority (APVMA) and US Food and Drug Administration (FDA).
- APIMA Australian Pesticides & Veterinary Medicines Authority
- FDA US Food and Drug Administration
- IRP001 has been selected as a candidate IVP as it is well established to be safe and non-toxic. Poultry have been selected as the target animal species due to widespread reliance on antimicrobials in the chicken industry to prevent or treat a number of diseases caused by enteric pathogens. These clinically significant enteric pathogens may potentially respond to IRP001.
- Doses are based on fixed concentrations of IRPOOl CI in feed (0.03 or 0.1 g/kg IRPOOl CI).
- Dose Preparation Powdered IRPOOl CI are incorporated with raw commercial feed ingredients then thoroughly mixed in, for example a "concrete mixer" type apparatus, to provide the final concentrations in feed as outlined.
- Method of Dose Administration Study animals are dosed according to the treatment regime detailed in Table 1 below. Medicated feed will be provided to chickens in the relevant treatments ad libitum as their sole source of feed.
- ANIMAL MANAGEMENT a. Animal Welfare: Study animals are managed similarly and with due regard for their welfare. Study animals are observed according to Animal Ethics Committee (AEC) requirements and a "Record of Animal Care" is completed. b. Health Management: Any routine prophylactic treatments are given as soon as possible, if necessary, and recorded (product name, batch number, expiry date, dose, route and date(s) of administration).
- the study animals are observed twice daily according to the standard operating protocol (SOP) in place commencing on Day 0. Any health problem that requires further examination are recorded. All health problems and adverse events must be reported to the Investigator within one working day. Any adverse event characterised by the Investigator as product related, results in death, is life-threatening, involves a large number of animals, or is a human adverse event, must be recorded and reported to the Sponsor and AEC within one working day. Normal veterinary care and procedures may be performed and are described in the raw data. Concurrent medications may be administered for standard management practice and humane reasons, with prior approval from the Investigator, and Sponsor (if relevant). No treatments similar to the IVP are administered. All concurrent medications are recorded giving identity of materials used (product name, batch number and expiry date), animal ID(s), the reason for use, route of administration, dose and the date(s) administered, and are included in the raw data (Trial Log) and the Study Report.
- SOP standard operating protocol
- Observations Birds are inspected twice daily for general well-being, typically prior to 8am of a morning, and after 4pm of an afternoon. Thus a typical interval between observations would be 9 hours during the day, and 15 hours overnight. Birds showing abnormal clinical signs are recorded, observed closely and euthanized if deemed to be suffering significantly (e.g. marked depression with low likelihood of recovery) by the Investigator.
- Necropsy Examinations All birds are euthanized and necropsied between Days 35 and 49 as per schedule - Table 14.
- Gross Pathology All chickens from all Group 1 through 18 are necropsied and examined for gross visual pathological changes which are described and scored as appropriate by individual bird. f.
- Tissue Residue Analysis Duplicate representative samples of liver, kidney, breast muscle (1), leg muscle (2) [upper and lower thigh] and entire skin with fat intact will be collected and stored frozen ( ⁇ 10 degrees Celsius) from the six (6) heaviest birds in each group (Groups 1 to 18 inclusive) as per schedule, table 1 for subsequent marker residue analysis. Groups 13 to 18 birds shall be sacrificed at Day 35 as untreated control birds with tissues collected for tissue assay requirements.
- Samples will be labelled with adhesive labels listing the study number, animal ID, time point, date, sample type and replicate.
- samples are thawed and a known weight of tissue (approximately lg) homogenized in 2ml water. Samples are centrifuged and a known volume of the supernatant removed for analysis by LC-MS/MS.
- Sample Storage, Transfer & Disposal Sample storage, transfer and disposal are recorded.
- Replicate 1 tissue samples are shipped frozen on wet ice to the Analytical Laboratory at times outlined in Section 10. Samples are transferred according to the standard operating protocol (SOP) with an accompanying temperature data logger and frozen water vial.
- Replicate 2 tissue samples are retained frozen for a period of 6 months after the last sample collection time-point. Beyond that point they may be discarded at the study site's discretion unless specifically requested not to by the Sponsor's Representative.
- SOP standard operating protocol
- Protocol specifications are to supersede facility SOPs. Study forms may be added or amended as required during the study without the need for a Protocol Amendment or Deviation.
- Protocol Approval The Protocol is approved and signed by all relevant personnel (see page 1) prior to study start.
- Amendments/Deviations An amendment is a change or modification of the Protocol made prior to execution of the changed or modified task. Amendments must state the reason for the change and have documented authorisation from the Sponsor. The amendment must be signed by the Investigator, and the Sponsor.
- Deviations from this Protocol or applicable SOPs are to be documented, signed and dated by the Investigator at the time the deviation(s) are identified. An assessment on the impact on the overall outcome or integrity of the study will be made. Deviations must be communicated to the Sponsor as soon as practically possible.
- Salmonella and Campylobacter studies also contemplates the prevention or treatment of infectious disease caused by Salmonella or Campylobacter. Studies for investigating the effectiveness of berberine alkaloids or berberine alkaloid compositions in preventing or treating disease caused by Salmonella or Campylobacter.
- Salmonella or Campylobacter infection are described below. The studies are modelled on published protocols: Alali, W. Q et al. "Effect of essential oil compound on shedding and colonization of Salmonella enteric serovar heidelberg in broilers", Poultry Science, 2013, 92: 836-841; Berghaus, R. et al. "Enumeration of Salmonella and Campylobacter in environmental farm samples and processing plant carcass rinses from commercial broiler chicken flocks", Appl.
- the objective of this study is to evaluate the effectiveness of IVPs as a means to control Salmonella heidelberg in broiler birds.
- Treatment groups are assigned to pens using randomized complete block design (Cochran and Cox, 1992). Treatment groups are as follows: 1. No Treatment - Salmonella heidelberg Challenge Control
- the study begins when birds are placed (day-of-hatch; DOT 0), at which time birds are allocated to experimental pens. Only healthy appearing birds are allocated for study use and final number and disposition of all birds not allocated are documented. No birds are replaced during the course of the study. Bird weights (kg) by pen are recorded at study initiation (DOT 0), DOT 35, and termination (DOT 42).
- Supplemental heat lamps (one [1] lamp per pen) provide heat (when needed). Birds are raised under ambient humidity and are provided a lighting program as per the primary breeder recommendations. At placement, each pen contains approximately four (4) inches of fresh pine shavings. Litter is not replaced during the study course. Each pen contains one (1) tube feeder and one (1) bell drinker resulting in a fifty (50) bird/feeder and drinker ratio.
- DIETS DIETS. Rations are fed as follows: starter DOT 0 through DOT 14, grower DOT 14 through DOT 35, and finisher DOT 35 to DOT 42. Diets are fed as crumbles (starter feed) or pellets (grower and finisher). Feed formulations for this study consist of unmedicated commercial- type broiler starter, grower, and finisher diets compounded with appropriate feedstuffs, calculated analyses to meet or exceed NRC standards, and no antibiotics are added to any feed unless specifically stated as a treatment protocol component. Experimental treatment feeds are prepared from a basal starter feed with quantities of all basal feed and test articles used to prepare treatment batches documented.
- test articles are mixed and pelleted in a California Pellet Mill at 80°C (with pellet temperature recorded). Aftermixing is completed feed is distributed among pens of designated treatment groups. Test article(s) are stored in a SPRG climate controlled storage area. All diets, formulations, and other feed information are documented. FEED CHANGES. Birds receive treatment-appropriate feed from DOT 0 to DOT 42.
- Rations are changed from starter to grower on DOT 14 and from grower to finisher on DOT 35. At that time all previous feed is removed from each pen, individually weighed, and replaced with finisher feed. On DOT 42 all non-consumed finisher feed is removed from pens, individually weighed, and discarded. SALMONELLA INOCULATION. On DOT 0 twenty-five (25) chicks per pen (50% seeders) are tagged, color-coded (for identification), and orally dosed (gavaged) with a 10 ⁇ CFU nalidixic acid-resistant Salmonella heidelberg.
- Bootsocks swab samples are collected for Salmonella environmental contamination determination from all pens DOT 14 and DOT 42. Gloves are changed between completion of each swab to reduce potential sample cross contamination.
- a pre-moistened bootsock swab (Solar Biologicals, Inc., Cat# BT SW-001) is removed from sterile bag, placed onto foot covered with a clean new plastic boot, the perimeter and interior of pen walked, boot sock removed, and placed into sterile bag labeled with pen number. After repeating Iheprocedure for each pen, samples are appropriately stored and then submitted for Salmonella analysis.
- CECAL SALMONELLA CULTURES Cecal sampling is completed on DOT 42. On DOT 42 ten (10) horizontal-exposed (non-tagged) birds are taken from each individual pen, euthanized (by cervical dislocation), and the ceca of each bird is aseptically removed. After removal the cecal sample is placed in one (1) sterile plastic sample bag (Fisher Scientific), labeled, stored on ice, and submitted for Salmonella analysis.
- DOT 42 ten (10) horizontal-exposed (non-tagged) birds are taken from each individual pen, euthanized (by cervical dislocation), and the ceca of each bird is aseptically removed. After removal the cecal sample is placed in one (1) sterile plastic sample bag (Fisher Scientific), labeled, stored on ice, and submitted for Salmonella analysis.
- SALMONELLA ENUMERATION PROCEDURE (MPN METHOD). For all ten (10) horizontal-exposed (non- tagged) and five (5) direct challenged (tagged) samples, a one (1) ml sample of stomachered peptone broth is transferred to three (3) adjacent wells in the first row of a 96-well two (2) ml deep block. A 0.1 ml aliquot of sample is transferred to 0.9 ml of tetrothionate broth in the second row, repeat process for remaining rows (to produce five (5) ten-fold dilutions), and incubate blocks (24 hours at 42 °C) (Table 16).
- Salmonella Specific Antiserum (Mira Vista, Indianapolis, IN). (Berghaus et al., 2013; Alali et al, 2013). Table 16 Salmonella enumeration
- the pen is the unit of measure. Pen security will prevent bird migration.
- MONITORING All birds are monitored for general flock condition, temperature, lighting, water, feed, litter condition, and unanticipated house conditions/events. Findings are documented twice daily during the regular working hours (one [1] observation recorded on final study day). One (1) observation is recorded Saturday, Sunday, and observed holidays.
- MORTALITY Pens are checked daily for mortality. Birds are culled only to relieve suffering. The date and removal weight (kg) are recorded for any bird culled (or found dead), gross necropsy is performed on all culled (or dead) birds, and the following information recorded: gender and probable cause of death.
- SOURCE DATA CONTROL AND HANDLING Data is recorded in indelible ink with legible entries, each source data sheet signed (or initialed), and dated by individual recording entry. All source data errors (and/or changes) are initialed, dated, and a brief explanation statement or error code written directly on the form.
- the study is to determine the efficacy of Investigational Veterinary Products (IVPs) to reduce Campylobacter jejuni shed (horizontal transmission) and colonization in broiler ceca.
- IVPs Investigational Veterinary Products
- Non-SPF One hundred twenty (120) day of age (non-SPF) commercial broilers are received. Five (5) birds are euthanized by cervical dislocation and their ceca are cultured for C. jejuni. The remaining selected one hundred five (105) birds are randomized into three (3) groups in one isolation room subdivided into one-thirds, with thirty-five birds per group. Experimental variables are shown below. All birds are fed a broiler starter crumble diet with treatment as specified below. NUMBER OF ROOMS - 1 Subdivided into 3 bird spaces
- HOUSING AND ENVIRONMENTAL CONTROL At study initiation, one hundred five (105) day-of-hatch Ross 708 male broiler chicks are allocated to one (1) isolation room. The room is subdivided into three (3) equal bird spaces. Thirty -five (35) chicks per space are placed in each room. Each room measures 13.4' x 15.7' (approximately 2.0 foot ⁇ stocking density). The isolation room environment is controlled by an independent HEPA filtration system and heat pump unit with one (1) heat lamp providing supplemental heat during brooding. Birds are reared under ambient humidity. At placement, each pen contains approximately four (4) inches of kiln-dried bagged fresh pine shavings. Litter is not replaced during the course ofthis study. Each space contains one (1) tube feeder and one (1) bell drinker (35 bird/feeder and drinker ratio). Birds are provided lighting twenty -four (24) hours per day.
- DIETS DIETS. Birds are fed a broiler starter diet throughout the study. An unmedicated commercial- type broiler starter diet compounded with appropriate feedstuffs with calculated analyses to meet or exceed NRC standards, and the addition of no antibiotics any feed unless specifically stated as a treatment protocol component is formulated. Feed is prepared from a basal starter feed. After mixing is completed, feed is distributed among pens of designated treatment groups. Test article(s) are stored in a climate controlled area. All diets and formulations and feeds are documented.
- CAMPYLOBACTER COLONIZATION EVALUATION On DOT 0 five (5) birds are cultured for Campylobacter jejuni prevalence; DOT 35, thirty-three (33) birds per treatment are euthanized by cervical dislocation. The ceca of each bird is aseptically removed and placed into sterile plastic sampling bags (Fisher Scientific) for Campylobacter isolation analysis. All samples are stored on ice prior to Campylobacter analysis.
- ENUMERATION PROCEDURE (DIRECT COUNT). For each sample a one (1) ml sample of stomachered Bolton broth willbe transferred to three (3) adjacent wells in the first row of a 96-well two (2) ml deep block. A 0.1 ml aliquot of sample is transferred to 0.9 ml of Bolton broth in the second row, process is repeated for remaining rows (producing twelve (12) ten-fold dilutions), and then 0.1 ml from each well will be spread-plated onto Campy Cefex Agar ⁇ Table 18). Plates are incubated (42 °C for 24 hours) in the presence of Campylobacter gas, final dilution of each sample recorded. Suspect Campylobacter isolates are confirmed by gram stain.
- BIRD IDENTIFICATION The room is the unit of measure. Room security prevents bird migration. MONITORING. All birds are monitored for general flock condition, temperature, lighting, water, feed, litter condition, and unanticipated house conditions/events. Findings are documented twice daily during the regular working hours (one [1] observation recorded Day 35). One (1) observation will be recorded Saturday, Sunday, and observed holidays.
- MORTALITY Rooms are checked daily for mortality. Birds are culled only to relieve suffering. The date and removal weight (kg) is recorded for any bird culled (orfound dead), gross necropsy is performed on all culled (or dead) birds, and the following information is recorded: gender, and probable cause of death.
- SOURCE DATA CONTROL AND HANDLING Data is recorded in indelible ink with legible entries, each source data sheet signed (or initialed), and dated by individual recording entry. All source data errors (and/or changes) are initialed, dated, and a brief explanation statement or error code written directly on the form.
- Necrotic Enteritis is an intestinal gut infection found in food-producing animals such as poultry. First described by Parish in 1961, it is caused in poultry by the bacteria, Clostridium perfringens and may present as acute clinical disease or subclinical disease. Although Clostridium perfringens is recognized as the etiological agent of Necrotic Enteritis, other contributing factors are usually required to predispose the animals to disease. It is accepted that Necrotic Enteritis is a multi-factorial disease process, with numerous risk factors including Eimeria infection, removal of antibiotic-growth promoters, environmental and management conditions, physiological stress and immunosuppression, and nature and form of diet.
- Necrotic Enteritis can cause flock mortality rates up to 1% per day for several consecutive days during the last weeks of the rearing period, with total cumulative mortalities rising to 30-50%.
- damage to the intestinal mucosa leads to decreased digestion and absorption, reduced weight gain and increased feed conversion ratio, resulting in reduction of commercial performance. It is this manifestation of the disease that reportedly causes the greatest economic losses in the poultry production industry.
- Clostridium perfringens in poultry constitutes a risk for transmission to humans through the food chain, with Clostridium perfringens being one of the frequently isolated bacterial pathogens in foodborne disease outbreaks in humans.
- Necrotic Enteritis was previously controlled by well-known antibacterial drugs such as virginiamycin, bacitracin, and so on.
- the banning of antibiotic use in food-producing animals in more and more countries has resulted in Necrotic Enteritis emerging as a serious threat to animal and public health.
- Clostridium perfringens is a gram positive, anaerobic bacteria found in soil, dust, faeces, feed, poultry litter and intestinal contents. It is extremely prolific and is able to produce various toxins and enzymes. Clostridium perfringens strains are classified into five toxinotypes (A, B, C, D and E), based on the production of four toxins ( ⁇ , ⁇ , ⁇ and ⁇ ). It has been proposed that Necrotic Enteritis is caused by type A and to a lesser extent type C, with type A strains producing chromosomal-encoded alpha toxin, while type C strains produce alpha toxins along with beta toxins.
- Alpha toxin is a phospholipase C sphingomyelinase that hydrolyzes phospholipids and promotes membrane disorganization, inducing synthesis of mediators such as leukotrienes, thromboxane, platelet-agglutinating factor and prostacyclin. These mediators cause blood vessel contraction, platelet aggregation and myocardial dysfunction, leading to acute death.
- the beta toxin induces hemorrhagic necrosis of the intestinal mucosa although the exact mechanism is not yet known. The pathology of Necrotic Enteritis is being re-evaluated along with a search for other virulence factors.
- alpha toxin may not have the major role in the pathogenesis of Necrotic Enteritis that has been proposed, with studies reporting an impaired ability to cause the disease using non wild-type alpha toxin.
- the evidence suggests that the molecules in Clostridium perfringens culture supernatant, when infused into the gut, reproduced disease-like pathology.
- Recent evidence also suggests that the NetB toxin from Clostridium perfringens may play a key role in Necrotic Enteritis pathogenesis.
- Clostridium perfringens is found naturally at low levels in the gut, but disturbances to normal intestinal microflora may cause rapid proliferation of the bacteria, resulting in the development of Necrotic Enteritis. Chickens are most commonly affected at 2 to 6 weeks old, however Necrotic Enteritis may occur in birds 7 to 16 weeks old or even up to 6 months.
- the disease is characterized clinically by a sudden increase in flock mortality, often without premonitory signs, although wet litter is sometimes an early indicator.
- Clinical signs can include depression, dehydration, somnolence, ruffled feathers, diarrhoea and decreased feed
- Macroscopical lesions can be found in the small intestine; the duodenum, jejenum and ileum become thin-walled, friable, dilated and filled with gas. In addition, mucosal surfaces are covered with a grey -brown to yellow-green diphteric membrane or pseudomembrane. Lesions may also be found in other organs, as well as atrophy of erythrocytes and bursa. The subclinical form of Necrotic Enteritis is considerably less recognizable and sick birds that respond to treatment with an antibiotic analogue are often deemed to have had the disease.
- Example 1 describes the use of berberine sulfate (IRP001 sulfate) in the prevention or treatment of Necrotic Enteritis.
- NE lesion scores and mortality at autopsy are used as outcome parameters and are shown in Table 22 and Table 23 below. Feed and water intake and weight gain are also measured.
- IRP001 sulfate at either 1.0 g/L in-water or 2.0 g/kg in-feed resulted in a significant reduction in mortalities in the NE challenged broilers, relative to both the nil-treatment groups and the groups treated with either 0.1 g/L in-water or 0.2 g/kg in-feed (See Figure 5).
- Phase 1 On receipt, two hundred and seventy (270) day-old commercial broiler chickens were allocated sequentially as they are received into sixteen (16) individual floor pens, each of 16 or 17 birds, on Day 0.
- Phase 2 On receipt, the ninety (90) day-old commercial broiler chickens were allocated sequentially as they are received into four (4) individual floor pens, each of 22 or 23 birds, on Day 22.
- Feed intake 'Treatment' was significant, 'Day' was highly significant, 'Pen' was not significant. However, no significant differences (at p ⁇ 0.05) were observed on individual pair-wise comparisons of treatments.
- Bodyweight 'Treatment' was significant, 'Day' was highly significant, 'Pen' was not significant. However, no significant differences (at p ⁇ 0.05) were observed on individual pair- wise comparisons of treatments.
- Phase 2 Phase 2 summary data
- Feed intake 'Treatment and 'Pen' were not significant although 'Day'was highly significant. However, no significant differences (at p ⁇ 0.05) were observed on pair-wise comparison of the 2 treatments. Water intake: 'Treatment' was highly significant, 'Day'was highly significant. A significant difference was observed on pair- wise comparison of the 2 treatments, with treated birds (who received treatment in-feed) drinking more water (see Figure 14).
- Bodyweight 'Treatment' and 'Pen' were not significant (although as expected 'Day' was). No significant differences (at p ⁇ 0.05) were observed on pair-wise comparisons of the 2 treatments. Within Phase 2 treatments (in-feed) did not appear to affect either feed intake or bodyweight, while treated birds tended to drink more water (in contrast to Phase 1 where they tended to drink less water when the unmasked treatment was applied in the drinking water).
- Phase 1 + 2 (Group-Pen 2 Untreated vs 15/16, Treated in-water Day 6-42):
- Bodyweight While 'Treatment' was significant in the model (and, as expected 'Day'was highly significant) no significant difference (at p ⁇ 0.05) was observed on pair-wise comparisons of the 2 treatments.
- the chicks were transported to the trial facility and randomized into each of 30 floor pens, placed at 36 chicks per pen (Figure 18). Pens were reduced to half normal size by a divider providing 3.5 m 2 floor space per pen. This was intended to provide a final bird density of approximately 30 kg/m 2 . A further two full size pens had 50 birds placed per pen (these acted as seeders for the coccidiosis challenge).
- Feeds were based on a suitable, balanced basal ration formulation (Starter, Grower and Finisher). Products were added to each of the basal rations as follows ⁇ Table 30).
- Pens were allocated a feed on a randomized complete block basis. Feeds were providedto each pen at 0.7 kg per bird Starter (approximately days 0-14), 1.2 kg per bird Grower (approximately days 15-28) and Finisher feed thereafter until termination (day 42). Seeder bird pens received ration#l (unmedicated). On day 6, the birds in the seeder pens were given the oocyst inoculum by individual gavage (approximately 0.5 mL per bird) using a stepper pipette. Three separate samples of sporulated oocysts from various chicken farm sources were used - given to approximately one third of the birds in each seeder pen.
- the litter in the seeder pens was lightly raked on days 12, 13 and 14. On day 14 the top 2-3 cm of the litter in the seeder pens was collected and mixed well together and weighed (Figure 19). The total weight of the litter was divided by 30 and that amount of litter distributed into each of the trial pens (each pen received 400 gm of mixed seeded litter - Figure 20)
- oocysts counts were performed by suspending 7 gm of litter in 75 mL of saturated sucrose and counting the total number of oocysts visible in a Whitlock Universal counting chamber under 100 x magnification.
- Table 31 shows the identity of Eimeria species included in the inoclula given to the seeder birds, as determined by PCR at Birling Avian laboratories. This PCR is qualitative only but relative abundance of each species can be estimated (shown with increasing numbers of "+” signs if more abundant). Table 31 Eimeria species detected in challenge inoculafor seeder birds
- Table 32 outlines the counts of oocysts per gram of mixed litter samples (samples counted in quadruplicate) derived from the seeder pens 7 days post inoculation. Visible size of the oocysts can be assessed but species cannot be accurately determined. The level of sporulation can be judged in this technique.
- each pen received approximately 6.3 million oocysts in the distributed seeded litter on day 14.
- Oocysts of sizes typical of several species of Eimeria were seen during counting of the challenge seeded litter (percentages estimated in Table 32). However, only the small oocysts seemed to be sporulated, with very few of the other sizes showing signs of sporulation at the time of litter spreading.
- Table 33 shows mean weights at each weighing time and Table 34 shows the mean weight gain in each period.
- Figure 21 shows average daily gain in weight by treatment.
- Table 33 Mean live weights (gm) at each age
- Weights at 14 days had shown significant divergence with treatments with birds receiving 0.3g/kg IRP having significantly lower weights than the negative control and both of the lower IRP dose rates. Both feeds containing salinomycin were intermediate at 14 days. This trend was becoming obvious at 7 days but not significantly at that age. This was also reflected in weight gain over these periods. By 21 days the mean weight of birds in the 0.3g/kg IVP treatment group was significantly lower than any other treatment. IVP at 0.03g/kg at 21 days had the highest numerical mean weight and was significantly greater than the salinomycin + bacitracin group and 0.3g/kg IRP group.
- IVP 0.3g/kg dose can be seen in Figure 21. Birds always appeared healthy.
- the coccidiosis challenge did not depress the growth rate of the negative control
- Table 37 shows the results of coccidial lesions cores at day 21 (7 days post exposure to contaminated litter).
- the coccidial lesions were mainly of those typical of E. acervulina.
- PCR on the challenge litter showed the presence of E. maxima, E. tenella and E. mitis as well. This is consistent with oocyst data prior to challenge insomuch as looking at the oocysts when they were counted prior to challenge, only the smaller oocysts (E. acervulina and E. mitis) appeared to have a good level of sporulation.
- the negative control and the lowest level of the IVP showed the highest lesion scores in duodenum, jejunum and total gut. Location of the lesions and their appearance were typical of E. acervulina (see Figure 24 to Figure 26). In the duodenum, only Salinomycin plus bacitracin reduced the lesions significantly compared to the control and the two lower dose rates of IVP. Jejunal lesions were generally low but there were some significant differences. Overall, the highest level of IRP and Salinomycin containing feeds (#5 & #6) significantly reduced total gut lesion scores.
- Table 39 shows the intestinal lesion scores based on Tierlynck et al. Avian Pathology, 2011, 40: 139-144 (Tierlynck et al, 2011). This is a scoring system aimed at quantifying the level of dysbacteriosis present in a group of birds, attributing scores for certain grossly visible abnormalities. Higher total scores (maximum 10) reflect a higher level of dysbacteriosis, although this may be compromised if coccidiosis is present. For our purposes, the intestinal scores simply reflect gross gut pathology. Examples of some observed intestinal
- E. acervulina Although the coccidial challenge applied contained several species of Eimeria, only the E. acervulina type showed good sporulation at the time of challenge. Sporulation conditions are generally considered to be the same for all species so this observation is unusual and the reason for it unknown. The observation was certainly accurate as only E. acervulina-type lesions were seen at examination on day 21. E. acervulina is a lower pathogenicity species and is not likely to lead to mortality and has less effect on growth rate. It may produce diarrhoea and affect feed conversion efficiency however.
- the challenge applied produced moderate coccidial lesions in the negative control group which were significantly reduced by the feds containing salinomycin and by the feed containing 0.30 g/kg IVP; but not by the lower dose rates. Only the salinomycin containing feeds were able to significantly reduce oocyst levels in faeces at day 21 although all groups receiving IVP levels were numerically lower than the controls. So there would appear to be some effect of IVP against E. acervulina.
- intestinal scores were also moderately correlated to coccidial lesion scores at day 21 we may assume an effect of the coccidial infection continuing on in the gut after their lesions had resolved (no coccidial lesions were observed at day 28).
- the intestinal scoring system is aimed at quantifying the presence and level of the condition known as dysbacteriosis in broiler chickens, and this condition is thought to be provoked by coccidial infection.
- the intestinal integrity scores were higher (i.e. more severe) at day 28 than at day 21 and would suggest a level of dysbacteriosis to be present.
- the IVP provided a similar improvement to salinomycin and salinomycin plus zinc bacitracin. This would be reasonable evidence that the IVP may have some protective effect against dysbacteriosis. Campylobacteriosis
- Campylobacteriosis is a gastrointestinal disease caused by bacteria called Campylobacter (CB).
- CB Campylobacter
- CB chronic myeloma
- CB is mainly spread to humans by eating or drinking contaminated food (mainly poultry), water or unpasteurised milk. CB can also be spread through contact with infected people, or from contact with cats, dogs and farm animals that carry the bacteria (Figure 3 shows the
- Example 4 discloses the antimicrobial activity of certain natural compounds against
- CCSI Clinical and Laboratory Standards Institute
- diarrhoea is the most common and probably the most important. In some outbreaks it is responsible for high morbidity and mortality. In a well-run herd there should be less than 3% of litters at any one time requiring treatment and piglet mortality from diarrhoea should be less than 0.5%. In severe outbreaks levels of mortality can rise to 7% or more and in individual untreated litters up to 100%. The main bacterial cause is E. coli. Scour in the piglet can occur at any age during sucking but there are often two peak periods, before 5 days and between 7 and 14 days.
- Piglet scour is estimated to cost the Australian pig industry more than $7 million each year. The incidence and type of scours, health costs and recovery rate determine the extent of this loss in individual piggeries. Antidiarrhoeal agents such as Bentonite or Kaolin clay are used to protect the gut wall. Addition of electrolytes to drinking is also oftentimes used. Antibiotics are used to reduce the population of bacteria in the gut although drug abuse needs to be avoided as resistance will develop. Current antibiotic medicines are listed in Table 43 below.
- Example 5 discloses the antimicrobial activity of certain natural compounds against pig disease.
- CCSI Clinical and Laboratory Standards Institute
- Clostridium difficile is a bacterium that can cause conditions ranging from diarrhoea to life-threatening inflammation of the colon. Illness from CD most commonly affects older adults or in long-term care facilities and typically occurs after use of antibiotic medications. However, studies show increasing rates of CD infection among people traditionally not considered high risk, such as younger and healthy individuals without a history of antibiotic use or exposure to health care facilities. Each year in the United States, about a half million people get sick from CD, and in recent years, CD infections have become more frequent, severe and difficult to treat with the rise of antimicrobial resistance.
- the antibiotics that most often lead to CD infections include Fluoroquinolones, Cephalosporins, Penicillins and Clindamycin.
- the standard treatment for CD is another antibiotic.
- Metronidazole taken orally is often prescribed despite not FDA approved.
- Vancomycin taken orally is prescribed.
- Fidaxomicin is another approved option to treat CD but costs considerably more. Up to 20 percent of people with CD get sick again. After two or more recurrences, rates of further recurrence increase up to 65 percent.
- Treatment for CD recurrence typically involves Vancomycin.
- Fecal microbiota transplant or stool transplant may be considered but is not yet FDA approved.
- the present disclosure relates to a method for preventing or treating an infectious disease caused by bacteria from the genus Clostridium in humans comprising administering a berberine alkaloid or composition as described herein.
- the present disclosure also contemplates that a berberine alkaloid or composition or animal feed described herein may inhibit spore formation.
- the overgrowth of spores after antibiotic treatment is acknowledged to be a problem in humans.
- the present disclosure relates to preventing C. difficile spores overgrowing after antibiotic treatment by administration of a berberine alkaloid or composition as described herein.
- Example 6 discloses the antimicrobial activity of certain natural compounds against Clostridium. EXAMPLE 6 Laboratory work
- MIC Minimum inhibitory concentration assays were conducted for a Necrotic Enteritis strain of C. perfringens and a clinical isolate of C. difficile using Berberine sulfate as the test agent and Vancomycin as an established control. Berberine sulfate with a purity of 98.0% was obtained as a natural extract from Sichuan BioFarm Inc. The MIC of Berberine for C. perfringens was 125 ⁇ g/ml, however partial inhibition of growth could be seen at a concentration of 62.5 ⁇ g/ml, indicating the true MIC is in between these two values.
- the Minimum Bacterial Concentration (MBC) of Berberine for C. perfringens was equal to the MIC (125 ug/ml), with 100%) killing of viable cells observed at this concentration.
- the MIC of Berberine for C. difficile was found to be 500-1000 ug/ml (variation between the replicates).
- the MBC of Berberine for C. difficile was 1000 ug/ml.
- the MIC and MBC values for Berberine for C. difficile were equal to or within a 2-fold dilution of values from a previous study.
- Vancomycin MICs were within the expected range for both C. perfringens and C. difficile.
- EXAMPLE 7 This study aims to determine tissue residues of the naturally occurring plant compound IRP001 chloride (berberine chloride) when administered orally via feed to commercial broiler chickens.
- Broiler chickens received either 0.3g/kg or 0.03g/kg IRP001 chloride mixed into their feed, or received regular feed without additive (i.e. control groups). Treatment began immediately after the birds were housed in pens (in groups of 10) and treatment continued for 35 days. Birds were either euthanized on day 35 for tissue collection or were fed beyond day 35 on regular feed for up to 7 days to examine residues after a washout period. Two other groups received IRP001 chloride feed additive for 28 days at either 0.3g/kg or 0.03g/kg mixed into their feed (i.e., 0.3 g IRP001 chloride in 1 kg of feed or 0.03 g IRP001 chloride in 1 kg of feed) and were
- IRP001 chloride was extracted from lg samples of three muscle tissues taken from each bird (in each case from breast, upper leg and lower leg). The residual mass of IRP001 chloride was determined using LC-MS/MS. The method allowed IRP001 to be detected with a lower limit of 2 ng IRPOOl/g tissue. The assay was fully validated during each assay run and proved to be quantitative to be better than ⁇ 20 % accuracy at 5 ng/g tissue. Levels lower than 2 ng IRPOOl/g were found to be within the baseline noise of the assay and were below the lower limit of detection (LLOD), i.e. IRP001 was not detectable.
- LLOD lower limit of detection
- the method was optimized so that IRP001 chloride could be detected with certainty at 2ng/g tissue.
- the assay was fully validated during each assay run and proved to be quantitative to better than +20% accuracy at 4ng/g or 5ng/g tissue. Levels of lng/g tissue or below were found to be within the baseline noise of the assay and were below the lower limit of quantitation (LLOQ).
- Residues of berberine were detectable and quantifiable after feeding for 35 days at the high IRP001 chloride concentration.
- a washout effect was evident at the high feed additive concentration in all three muscle tissues, reaching levels of approximately lng/g, below the LLOQ after 4 days washout.
- the mean residue levels were less than lng/g, below the LLOQ, in all cases, with or without washout.
- the residue levels in the liver after the high feed additive concentration were above 13 ng/g without washout but below 13 ng/g after one day of washout. Given the average consumption of chicken liver is limited, the levels of IRPOOl in liver do not represent cause for concern.
- the data taken as a whole indicate that the risk of cancer resulting from consumption of chicken meat from IRPOOl chloride-fed chickens is less than one in a million at feed additive levels equal to or less than 0.3g berberine/kg feed.
- Berberine levels in chicken muscle were below the LLOD after dosing at 0.03 IRPOOl/kg feed, or after 4 days of washout after dosing at 0.3 g IRPOOl/kg feed.
- Berberine alkaloids including berberine
- Berberine has been used as a dietary supplement by humans for many years and is available from several manufacturers in capsule form. It is sold for use once or twice daily at doses as high as 400mg berberine chloride per capsule. Further, in experiments leading to the present invention, no adverse reaction or unanticipated event has been observed in broilers treated with berberine at a dose of 1 g berberine in 1 kg of commercial feed over 42 days (see EXAMPLE 8).
- FDA Food and Drug Administration
- the FD&C Act requires that compounds intended for use in food-producing animals are shown to be safe and that food produced from animals exposed to these compounds is shown to be safe for consumption by people.
- the use in food-producing animals of any compound found to induce cancer when ingested by people or animal is prohibited by statute (21 CFR Part 500, Subpart E - Regulation of carcinogenic compounds used in food-producing animals) unless certain conditions are met (the so-called "Diethylstilbestrol (DES) Proviso").
- DES Diethylstilbestrol
- no residue refers to any residue remaining in the edible tissues that is so low that it presents an insignificant risk of cancer to consumers. More specifically, an insignificant risk of cancer is defined as a 1 in 1 million increase in risk.
- berberine peaks from tissue samples could be detected at concentrations as low as lng/g tissue, but intereference due to tissue matrix effects and analyte carryover at lng/g tissue made quantitation of IRP001 difficult at this or lower concentrations.
- the assay could be validated as accurate at ⁇ _20% true analyte
- concentration Realistically a concentration of less than 2ng/g can be considered to be below the lower limit of quantitation (LLOQ).
- LLOQ lower limit of quantitation
- Tissue samples from 3 birds from each feed additive group were received by the Monash analytical team and analysed by LC-MS/MS. A single sample from each control group was assayed.
- Table 47 shows mean concentration of berberine and standard deviation determined for each muscle tissue excised from 3 birds in each group. One representative from each control group was assayed and these values were found to be effectively zero, expressed in the results table as below the LLOD " ⁇ LLOD", i.e. not detectable.
- Table 48 shows mean concentration of berberine and standard deviation determined for liver tissue excised from 3 birds in each group. One representative from each control group was assayed and these values were found to be effectively zero, expressed in the results table as below the LLOD " ⁇ LLOD", i.e. not detectable.
- Residue levels in liver were above the limits of quantitation after birds were fed with 0.3g IRPOOl/kg feed, were reduced by washout period over 7 days, and reduced to below the limit of quantitation after a 14-day washout. Residue levels in liver after birds were fed with 0.03 g IRPOOl/kg feed were below the limit of detection before and after washout.
- tissue homogenates were distributed into Eppendorf tubes in 200 ⁇ L aliquots.
- the method was validated for selectivity, linearity, LLOQ, accuracy, precision, recovery, stability and matrix effect.
- matrix recovery samples were prepared by extracting blank tissue and then spiking with the analyte solutions to give various concentration levels up to 500 ng/g (5 replicates each). The recovery was defined by the ratio of the mean peak area of extracted samples to the mean peak area of matrix recovery samples. 6. To evaluate bench-top stability, 4 concentration levels of 20, 50, 100 and 500 ng/g were prepared at 5 replicates each, where they were kept at room temperature for 30 minutes prior to extraction. The mean peak area was compared to that of freshly-prepared standards.
- ME matrix effect
- ANIMAL MANAGEMENT a. Animal Welfare: Study animals were managed similarly and with due regard for their welfare. Study animals were observed according to Animal Ethics Committee (AEC) requirements and a "Record of Animal Care" was completed. b. Health Management: Any routine prophylactic treatments were given as soon as possible, if necessary, and recorded (product name, batch number, expiry date, dose, route and date(s) of administration).
- Body Weights Chickens were weighed on Days 0 (Group weight) and 7, 14, 21, 28 and 35 days - individual animal weights were recorded. Weigh scales were checked pre- and post-weighing with calibrated test weights and recorded. Body weights at study termination were compared between groups to determine treatment effects (if any).
- Examinations Individual clinical examinations were performed on euthanasia at the time of gross pathology and tissue collection. Clinical examinations were recorded. Digital still images may be recorded as appropriate.
- Observations Birds were inspected twice daily for general well-being, typically prior to 8am of a morning, and after 4pm of an afternoon. Thus a typical interval between observations would be 9 hours during the day, and 15 hours overnight. Birds showing abnormal clinical signs were recorded, observed closely and euthanized if deemed to be suffering significantly (e.g.
- Samples were labelled with adhesive labels listing the study number, animal ID, time point, date, sample type and replicate.
- samples were thawed and a known weight of tissue (approximately lg) homogenized in 2ml water. Samples were centrifuged and a known volume of the supernatant removed for analysis by LC-MS/MS.
- Sample Storage, Transfer & Disposal Sample storage, transfer and disposal were recorded. Replicate 1 tissue samples were shipped frozen on wet ice to the Analytical
- Protocol specifications are to supersede facility SOPs. Study forms may be added or amended as required during the study without the need for a Protocol Amendment or Deviation.
- Protocol Approval The Protocol is to be approved and signed by all relevant personnel (see page 1) prior to study start.
- Amendments/Deviations An amendment is a change or modification of the Protocol made prior to execution of the changed or modified task. Amendments must state the reason for the change and have documented authorisation from the Sponsor. The amendment must be signed by the Investigator, and the Sponsor.
- Deviations from this Protocol or applicable SOPs are to be documented, signed and dated by the Investigator at the time the deviation(s) are identified. An assessment on the impact on the overall outcome or integrity of the study is to be made. Deviations must be communicated to the Sponsor as soon as practically possible.
- a Study Report was prepared by the Investigator, or designee. Data listings of each variable measured was included. The study Investigator's Compliance Statement was included in the Study Report. The original signed Study report with raw data and Statistical Report appended was submitted to the Sponsor and archived.
- the objective of this study was to test the general safety of IRPOOl chloride in broilers reared to market weight by examination of histology.
- the experiment consisted of the following treatments (1 pen per treatment, Table 54).
- the pen had 5 feet high side walls with a bottom 1 1/2 feet being of solid wood to prevent bird migration.
- the temperature of the building was monitored. Environmental conditions during the trial (temperature) were appropriate (optimum) to the age of the animals. Illumination was provided by fluorescent bulbs placed above the pens. The diets were provided ad libitum in one tube-type feeder per pen. From DO until D7, feed was also supplied on a tray placed on the litter of each pen. Water was provided ad libitum from one Plasson drinker per pen.
- Standard floor pen management practices were used throughout the experiment. Animals and housing facilities were inspected twice daily, observing and recording the general health status, constant feed and water supply as well as temperature, removing all dead birds, and recognizing unexpected events. Birds found dead during the study were noted on the Daily Mortality Record, and were not replaced. Pen number, the date of mortality, sex, weight, and diagnosis were recorded.
- BIRDS Day of hatch male Cobb chicks were obtained and ten male broiler chicks were placed in each pen. Accountabilities of all test animals and any extra birds were recorded on animal disposition form. The birds were sexed at the hatchery. The breeder flock history and vaccination record at the hatchery were recorded. Bird weights by pen were recorded on DO and 42.
- Treatment feed samples ( ⁇ 150g each) were collected and blended: one each from the beginning, middle, and end of each batch of treatment diet. Samples are retained until directed to ship or discarded 2 months post submission of report.
- the main ingredients used were corn, soybean meal and animal by-product.
- the basal feed did not contain any probiotic/ prebiotic feed additives, NSPases, coccidiostats or antibiotic growth promoter. All diets contained phytase.
- Standard floor pen management practices were used throughout the experiment. The temperature of the building was monitored. Environmental conditions during the trial (temperature) were appropriate (optimum) to the age of the animals. Illumination was provided by fluorescent bulbs placed above the pens. The lighting scheme was 24 hours of light from DO to D14, then 18 hours of light to D42.
- the diets were provided ad libitum in one tube-type feeder per pen. From day 1 until day 7 feed was also supplied on a tray placed on the litter on each pen.
- Source data were entered with indelible ink. Entries were legible, signed or initialed, and dated by the person making the observation entry. Each sheet of source data was signed by the person(s) attributed to the data. Any mistakes or changes to the source data were initialed and dated and a correction code or statement added as to why the changes were made.
- This study measures the anticoccidial efficacy/sensitivity of IRPOOl against a mixture of Eimeria acervulina, E. maxima, and E. tenella.
- the experiment consisted of 72 cages starting with 8 male chicks. The treatments were replicated in 6 blocks, randomized within blocks of 12 cages each. A randomization procedure for pen assignment for treatments and blocks was provided by Southern Poultry Research, Inc. (SPR, Athens, GA 30607) who conducted the study for the Sponsor.
- Treatment groups are set out in Table 57.
- Experimental Ration An unmedicated commercial starter ration compounded with feedstuffs commonly used in North Georgia was formulated. This ration (in mash form) was fed ad libitum from the date of chick arrival until completion of the study. Experimental diets were prepared from a uniform basal diet. Quantities of all basal feed and test articles used to prepare treatment batches were documented. Treatment diets were mixed to assure a uniform distribution of test article. The mixer was flushed between medicated treatment diets. The feed was transferred to building #2 and distributed among cages of the same treatment.
- Chicks (Cobb 500) were obtained for the study. The strain, source, and vaccination record were recorded. Upon arrival, chicks were assigned to treatment battery cages. Chicks (DOT 0) was grouped into sets of 8, weighed, and placed into assigned cage. The total number of birds entering the test was 576. Accountabilities of all birds were recorded in the source data.
- Coccidial oocyst inoculation procedures are described in SPR SOP.
- Tl birds received 1 ml of distilled water by oral pipette (p.o.).
- All other birds received the coccidial inoculum diluted to a 1 ml volume (p.o.).
- the inoculum was a mixture of Eimeria acervulina (100,000 oocysts/ bird), E. maxima (50,000 oocysts/ bird), and E. tenella (75,000 oocysts/ bird).
- Results for Feed Intake adjusted feed conversion ratio (Adj. FCR), weight gain (Wt. Gain), Mortality (% Cocci Mort.); lesion scores; and fecal oocyst counts (for Eimeria acervulina (E. acer), E. maxima, and E. tenella) are shown in Table 58.
- the objective of this study is to demonstrate the efficacy of IRPOOl in the control of Swine Ileitis using a challenge model.
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- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
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- Birds (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Communicable Diseases (AREA)
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AU2018241245A AU2018241245B2 (en) | 2017-03-28 | 2018-03-28 | Berberine alkaloid formulations in the prevention and/or treatment of infectious disease |
BR112019020228-5A BR112019020228A2 (en) | 2017-03-28 | 2018-03-28 | BERBERINE ALKALOID FORMULATIONS FOR THE PREVENTION AND / OR TREATMENT OF INFECTIOUS DISEASES |
MX2019011714A MX2019011714A (en) | 2017-03-28 | 2018-03-28 | Berberine alkaloid formulations in the prevention and/or treatment of infectious disease. |
CA3057996A CA3057996A1 (en) | 2017-03-28 | 2018-03-28 | Berberine alkaloid formulations in the prevention and/or treatment of infectious disease |
EP18777835.2A EP3600305A4 (en) | 2017-03-28 | 2018-03-28 | Berberine alkaloid formulations in the prevention and/or treatment of infectious disease |
CN201880033270.9A CN110709080A (en) | 2017-03-28 | 2018-03-28 | Use of berberine alkaloid preparation in preventing and/or treating infectious diseases |
US16/499,163 US20210329946A1 (en) | 2017-03-28 | 2018-03-28 | Berberine alkaloid formulations in the prevention and/or treatment of infectious disease |
KR1020197031821A KR20190139908A (en) | 2017-03-28 | 2018-03-28 | Berberine Alkaloid Formulations in the Prevention and / or Treatment of Infectious Diseases |
PH12019502240A PH12019502240A1 (en) | 2017-03-28 | 2019-09-27 | Berberine alkaloid formulations in the prevention and/or treatment of infectious disease |
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AU2017901105A AU2017901105A0 (en) | 2017-03-28 | Berberine alkaloids in the prevention and/or treatment of intestinal disease | |
AU2017901105 | 2017-03-28 | ||
AU2017903261A AU2017903261A0 (en) | 2017-08-15 | Berberine alkaloids in the prevention and/or treatment of intestinal disease | |
AU2017903261 | 2017-08-15 |
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PCT/AU2018/050288 WO2018176093A1 (en) | 2017-03-28 | 2018-03-28 | Berbine alkaloid formulations in the prevention and/or treatment of infectious disease |
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US (2) | US20230404107A1 (en) |
EP (2) | EP3600304A4 (en) |
KR (2) | KR20190139236A (en) |
CN (2) | CN110650739A (en) |
AU (2) | AU2018241231A1 (en) |
BR (2) | BR112019020225A2 (en) |
CA (2) | CA3057858A1 (en) |
MX (2) | MX2019011678A (en) |
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WO2021072473A1 (en) * | 2019-10-14 | 2021-04-22 | IRP Health Pty Ltd | Antimicrobials as feed preservatives |
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EP3810137A4 (en) | 2018-05-29 | 2022-03-09 | IRP Health Pty Ltd | Nitroimidazole formulations |
WO2022192535A1 (en) * | 2021-03-10 | 2022-09-15 | Cornell University | Methods for improving poultry health |
CN113817842B (en) * | 2021-09-26 | 2024-05-03 | 中国农业大学 | SNP molecular marker related to chicken eimeria tenella diclazuril drug resistance and application thereof |
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EP3600304A4 (en) | 2021-01-06 |
BR112019020225A2 (en) | 2020-04-22 |
CN110709080A (en) | 2020-01-17 |
WO2018176079A8 (en) | 2020-02-13 |
KR20190139236A (en) | 2019-12-17 |
CA3057858A1 (en) | 2018-10-04 |
WO2018176079A1 (en) | 2018-10-04 |
CN110650739A (en) | 2020-01-03 |
EP3600304A1 (en) | 2020-02-05 |
WO2018176093A8 (en) | 2018-11-15 |
US20210329946A1 (en) | 2021-10-28 |
KR20190139908A (en) | 2019-12-18 |
EP3600305A4 (en) | 2021-06-16 |
PH12019502241A1 (en) | 2020-07-06 |
BR112019020228A2 (en) | 2020-06-16 |
PH12019502240A1 (en) | 2020-07-06 |
AU2018241231A1 (en) | 2019-11-07 |
EP3600305A1 (en) | 2020-02-05 |
MX2019011678A (en) | 2020-02-12 |
CA3057996A1 (en) | 2018-10-04 |
AU2018241245A1 (en) | 2019-11-07 |
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