Use of berberine alkaloid in preventing and/or treating intestinal diseases
阅读说明：本技术 小檗碱生物碱在预防和/或治疗肠疾病中的作用 (Use of berberine alkaloid in preventing and/or treating intestinal diseases ) 是由 D·X·于 Z·肖 C·波顿 Z·何 于 2018-01-02 设计创作，主要内容包括：本发明涉及小檗碱生物碱类,其制剂及它们在预防和/或治疗动物中的传染病,特别是产气荚膜梭菌和艾美球虫属引起的疾病中的用途。本发明还涉及通过施用小檗碱组合物来改善食物生产动物中的饲料转化率。(The present invention relates to berberine alkaloids, formulations thereof and their use in the prevention and/or treatment of infectious diseases in animals, in particular diseases caused by clostridium perfringens and eimeria. The invention also relates to improving feed conversion ratio in food producing animals by administering the berberine composition.)
1. A method for preventing and/or treating an infectious disease in an animal, wherein the method comprises administering berberine alkaloids to the animal.
2. The method of claim 1, wherein the animal is a human.
3. The method of claim 1, wherein the animal is a non-human animal.
4. The method of claim 1, wherein the non-human animal is a food-producing animal.
5. The method of claim 4 wherein the food producing animal is selected from a chicken or a pig.
6. The method of any one of claims 1-5, wherein the infectious disease is an intestinal disease.
7. The method of any one of claims 1-6, wherein the infectious disease is caused by a parasite of the genus Eimeria.
8. The method of claim 7, wherein the parasite is selected from the group consisting of Eimeria maxima (E.maxima), Eimeria acervulina (E.acervulina), and Eimeria brunetti (E.brunette).
9. The method of any one of claims 1-6, wherein the infectious disease is caused by an antibiotic-resistant parasite of the genus Eimeria (genus Eimeria).
10. The method of claim 9, wherein the antibiotic-resistant bacterial parasite is selected from the group consisting of eimeria maxima, eimeria acervulina, and eimeria brunetti antibiotic-resistant parasites.
11. The method of any one of claims 7-10, wherein the infectious disease is coccidiosis and the animal is a chicken.
12. The method of any one of claims 1-6, wherein the infectious disease is caused by a bacterium of the genus Clostridium (genus Clostridium), wherein the bacterium is Clostridium perfringens (C.
13. The method of any one of claims 1-6, wherein the infectious disease is caused by an antibiotic resistant bacterium of the genus Clostridium, wherein the antibiotic resistant bacterium is antibiotic resistant Clostridium perfringens.
14. The method of claim 12 or claim 13, wherein the infectious disease is necrotic enteritis and the animal is a chicken.
15. The method of claim 14, wherein the necrotic enteritis is caused by a clostridium perfringens type a strain.
16. The method according to claim 15, wherein the clostridium perfringens type a strain is clostridium perfringens type a strain EHE-NE 36.
17. The method according to claim 15, wherein the clostridium perfringens type a strain is clostridium perfringens type a strain EHE-NE 18.
18. The method of claim 14, wherein the necrotic enteritis is caused by a clostridium perfringens type C strain.
19. The method of any one of claims 14 to 19, wherein the administering is via the feed or water of the chicken.
20. The method of claim 20, wherein the feed is in the form of a crumbled or pelletized material.
21. The method according to claim 20 or claim 21, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of 0.001 to 2g/kg feed.
22. The method according to claim 20, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of 0.001 to 0.1g/L water.
23. The method of claim 11 or any one of claims 14 to 23, wherein the lesion score is decreased and/or the fecal oocyst count is decreased.
24. The method of any one of claims 14 to 24, wherein morbidity is reduced.
25. The method according to claim 11 or any one of claims 14 to 25, wherein mortality is reduced.
26. The method according to any one of claims 14 to 26, wherein safe residual levels of berberine alkaloids are present in the chicken's muscle tissue after the treatment period.
27. The method of claim 27, wherein the residual level is at least less than about 13ng berberine alkaloid per g muscle tissue.
28. The method of claim 27, wherein the residual level is about 10ng berberine alkaloid per g muscle tissue.
29. The method of claim 27, wherein the residual level is about 5ng berberine alkaloid per g muscle tissue.
30. The method according to claim 27 or claim 28, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about 0.3 g/kg.
31. The method of claim 31, wherein the residual levels of berberine alkaloids in the chicken muscle tissue are as follows:
about 6.1ng/g in the muscle tissue of the chicken breast;
about 5.5ng/g in the muscle tissue of the chicken calf; and
about 11.6ng/g in the muscle tissue of chicken thigh.
32. The method according to any one of claims 27 to 30, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about less than 0.1 g/kg.
33. The method according to any one of claims 27 to 30, wherein the berberine alkaloid is administered at a dose of about 0.03g/kg in the feed of the chicken.
34. The method of claim 34, wherein the residual levels of berberine alkaloids in the chicken muscle tissue are as follows:
the content of the chicken breast muscle tissue is lower than 2 ng/g;
the muscle tissue of the chicken calf is lower than 2 ng/g; and
less than 2ng/g in the muscle tissue of chicken thigh.
35. The method according to any one of claims 14 to 26, wherein safe residual levels of berberine alkaloids are present in the chicken's muscle tissue after the treatment and clearance periods.
36. The method of claim 36, wherein the washout period is a period between 1 week and 2 weeks.
37. The method of claim 36, wherein the washout period is selected from the following periods: 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.
38. The method of claim 36, wherein the washout period is a period selected from 1 day, 2 days, 4 days, 7 days, and 14 days.
39. The method according to claim 39, wherein after a 1 day washout period, the residual levels of berberine alkaloids in the chicken's muscle tissue are as follows:
about 5.7ng/g in the muscle tissue of chicken breast;
about 3.2ng/g in the muscle tissue of the chicken calf; and
about 6.0ng/g in the muscle tissue of chicken thigh.
40. The method according to claim 39, wherein after a 2 day washout period, the residual levels of berberine alkaloids in the chicken's muscle tissue are as follows:
about 3.6ng/g in the muscle tissue of the chicken breast;
about 3.1ng/g in the muscle tissue of the chicken calf; and
about 4.5ng/g in the muscle tissue of chicken thigh.
41. The method according to claim 39, wherein the residual level of berberine alkaloids in the chicken's muscle tissue is below 2ng/g after 4, 7 and 14 days of washout period.
42. The method according to any one of claims 36 to 42, wherein the berberine alkaloid is administered at a dose of about 0.3g/kg in the feed of the chicken.
43. The method according to claim 36 or claim 37, wherein the residual level is at least below 13ng of the berberine alkaloid per g of muscle tissue.
44. The method of claim 36 or 37, wherein the residual level is about 5 ng/g.
45. The method according to any one of claims 36, 37, 44 or 45, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about more than 0.1 g/kg.
46. The method according to any one of claims 14 to 26, wherein after the treatment period, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken.
47. The method according to claim 47, wherein the residual level of berberine alkaloids in the liver and muscle tissue of the chicken is below 2 ng/g.
48. The method according to claim 48, wherein the berberine alkaloids are administered in the feed of the chicken at a dose of about 0.03 g/kg.
49. The method according to any one of claims 14 to 26, wherein safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken after the treatment and clearance periods.
50. The method of claim 50, wherein the washout period is a period between 1 week and 2 weeks.
51. The method of claim 50, wherein the washout period is a period selected from the group consisting of: 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.
52. The method of claim 50, wherein the clearance period is a period selected from 1 day, 2 days, 4 days, 7 days, and 14 days.
53. The method of claim 53, wherein after a 1 day washout period, the residual levels of berberine alkaloids in the chicken's muscle tissue are as follows:
about 5.7ng/g in the muscle tissue of chicken breast;
about 3.2ng/g in the muscle tissue of the chicken calf; and
about 6.0ng/g in the muscle tissue of chicken thigh,
and berberine alkaloid residue level in liver tissue of chicken is about 8.0 ng/g.
54. The method of claim 53, wherein after a 7 day washout period, the residual level of berberine alkaloids in the muscle tissue of the breast, calf and thigh of the chicken is below 2ng/g and the residual level of berberine alkaloids in the liver tissue of the chicken is about 6.5 ng/g.
55. The method of claim 53, wherein after a 14 day washout period, the residual level of berberine alkaloids in the muscle tissue of the breast, calf and thigh of the chicken is below 2ng/g and the residual level of berberine alkaloids in the liver tissue of the chicken is about 3.0 ng/g.
56. The method according to any one of claims 50 to 56, wherein the berberine alkaloid is administered at a dose of about 0.3g/kg in the feed of the chicken.
57. The method according to any one of claims 14 to 26, wherein safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken after the treatment period.
58. The method of claim 58, wherein the residual level of berberine alkaloids in the liver tissue and muscle tissue of the breast, calf and thigh of the chicken is below 2 ng/g.
59. The method according to claim 58 or claim 59, wherein the berberine alkaloid is administered at a dose of about 0.03g/kg in the feed of the chicken.
60. The method according to any one of claims 14 to 26, wherein safe residual levels of berberine alkaloids are present in the liver tissue of the chicken after the treatment and clearance periods.
61. The method of claim 61, wherein the washout period is selected from a period between 1 week and 2 weeks.
62. The method of claim 61, wherein the washout period is a period selected from the group consisting of: 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.
63. The method of claim 61, wherein the washout period is a period selected from 1 day, 2 days, 4 days, 7 days, and 14 days.
64. The method of claim 64, wherein the residual level of berberine alkaloids in the liver tissue of the chicken is about 8.0ng/g after a 1 day washout period.
65. The method of claim 64, wherein the residual level of berberine alkaloids in the liver tissue of the chicken is about 6.5ng/g after a 7 day washout period.
66. The method of claim 64, wherein the residual level of berberine alkaloids in the liver tissue of the chicken is about 3.0ng/g after a washout period of 14 days.
67. The method according to any one of claims 61 to 67, wherein the berberine alkaloid is administered at a dose of about 0.3g/kg in the feed of the chicken.
68. The method of any one of claims 27-68, wherein the treatment period is 35 days.
69. The method according to any one of claims 1 to 69, wherein the berberine alkaloid is berberine hemisulfate.
70. A method according to any one of claims 1 to 69, wherein the berberine alkaloid is berberine chloride.
71. The method according to any one of the preceding claims, further comprising an additive that masks the bitter taste of the berberine alkaloid.
72. An animal feed comprising berberine alkaloids and an animal food product, wherein the berberine alkaloids are present in an amount of 0.001% w/w to 2% w/w of the animal food product.
73. The animal feed of claim 73, wherein the feed is a crumbled feed; in the form of pellets; or an aqueous form.
74. A dosing regimen comprising administering the berberine alkaloid or animal feed according to claim 73 or claim 74, wherein the berberine alkaloid or animal feed is administered for 1 to 6 weeks in an amount effective to prevent or treat an infectious disease in the animal.
75. A method for reducing feed conversion ratio in a food-producing animal, wherein the method comprises the step of administering to the food-producing animal a berberine alkaloid according to claim 73 or claim 74, or an animal feed.
76. The method of claim 76 wherein the food producing animal is free of disease.
77. The method of claim 77 wherein the food producing animal is diseased.
78. The method of any one of claims 76 to 78 wherein the food producing animal is selected from a chicken or a pig.
79. The method of claim 79 wherein the food producing animal is a chicken.
80. A method for preventing and/or treating infectious disease in an animal comprising administering an animal feed according to claim 73 or claim 74.
81. A method for preventing and/or treating infectious intestinal disease in an animal comprising administering an animal feed according to claim 73 or claim 74.
82. A method for preventing and/or treating infectious disease in an animal caused by Eimeria comprising administering an animal feed according to claim 73 or claim 74.
83. The method of claim 82, wherein the infectious disease is caused by an antibiotic-resistant parasite of the genus Eimeria.
84. The method of claim 82 or claim 83, wherein the infectious disease is coccidiosis and the animal is a chicken.
85. A method of preventing and/or treating infectious disease in an animal caused by Clostridium bacteria comprising administering an animal feed according to claim 73 or claim 74, wherein the bacteria is Clostridium perfringens.
86. The method of claim 86, wherein the infectious disease is caused by antibiotic resistant Clostridium perfringens.
87. The method of claim 86 or claim 87, wherein the infectious disease is necrotic enteritis and the animal is a chicken.
88. Use of berberine alkaloids for the preparation of a medicament for the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
89. Use of berberine alkaloids in the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
90. Berberine alkaloids for the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
Technical Field
The present invention relates to berberine alkaloids, formulations thereof and their use in the prevention and/or treatment of infectious diseases (ifectious diseases) in animals. In particular, the present invention relates to berberine alkaloids, formulations thereof and their use in the prevention and/or treatment of infectious diseases including bacterial, viral, parasitic or fungal infections (infections) in food-producing animals.
Background
The use of antibiotics has been a major problem for animal production worldwide for decades. It is estimated that about 63,000 tons of antibiotics are used worldwide each year to feed cattle, chickens and pigs, which is about twice as much as antibiotics developed by global doctors to combat human infections, and the current trend suggests that the worldwide consumption of antibiotics by animals will increase by two thirds in the next 20 years.
Antibiotics have been supplemented in animal and poultry feed not only to treat and control infections, but also as growth promoters at low doses and are believed to improve product quality, resulting in lower fat percentages and higher protein content in meat. According to the national animal health administration (NOAH, 2001), they are used to "help growing animals digest their food more efficiently, gain the maximum profit therefrom, and grow them into strong and healthy individuals", thus bringing economic advantages to farmers. Therefore, it is important to increase and develop a drug library (armamentarium) that has the potential to combat infectious diseases as an antibiotic and is cost effective.
Antimicrobial resistance (AMR) is a natural process in which microorganisms are evolved to resist the action of drugs, rendering them ineffective. Over time, this causes the antibiotic to become less effective and, in extreme cases, ultimately useless. AMR is increasingly a problem because the speed of finding new antibiotics has been greatly reduced and, therefore, the number of new drugs is very limited. Meanwhile, the use of antibiotics is exponentially increased, and the development of resistance is increased.
Recently, the use of antibiotics in food-producing animals has been reviewed again, and there is an increasing concern that their overuse will promote the spread of antibiotic resistance genes by promoting the selection of antibiotic-resistant bacteria in animals. Furthermore, the waste material produced by animals may contain antibiotic residues, resulting in a more widespread distribution of them in the environment. These are the major problems with intensive farming methods, and the problems caused by their use are mainly problems in developed countries rather than in developing countries.
Antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing spectrum of infections caused by bacteria, parasites, viruses and fungi. AMR poses an increasingly serious threat to global public health, requiring action by all government agencies and society. The widespread and excessive use of antibiotics in food-producing animals has led to the emergence of antibiotic-resistant bacteria that contaminate the food, and then to the appearance of consumers who, in turn, may develop antibiotic-resistant infections. Fig. 1 depicts the transmission of AMR from food producing animals to humans.
There is a concern that the overuse of antibiotics in food-producing animals leads to the spread of resistant bacteria to humans, which in turn leads to "superbacteria" that are resistant to several classes of antibiotics. It is estimated that superbacteria have caused over 320,000 deaths annually in china and the united states, and by 2050, the number of deaths is expected to exceed 1000 million, with losses in the world of over 100 trillion dollars.
The global burden of infection with resistance to existing antimicrobial drugs is growing at an alarming rate. Methicillin-resistant Staphylococcus aureus (MRSA) and klebsiella pneumoniae (klebsiella pneumoniae) are the major causes of hospital-acquired infections. Klebsiella pneumoniae is a common enterobacterium, which in some countries has even developed resistance to last resort to beta-lactam carbapenem antibiotics. In many parts of the world, treatment of urinary tract infections caused by e.coli has been ineffective due to resistance to fluoroquinolone antibiotics.
The use of beta-lactam antibiotics and fluoroquinolones can lead to secondary infections and further complications, such as the overgrowth of Clostridium Difficile (CD). CD is a bacterium that can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. CD disease commonly affects the elderly in long-term care facilities, often following treatment with antibiotic drugs. However, studies have shown that the rate of CD infection is rising in people who are not traditionally considered to have a high risk, such as young and healthy individuals who have no history of antibiotic use or who have not been exposed to healthcare facilities. In the united states, about fifty thousand people become ill each year due to the release of CD toxins, and in recent years, with the rise in antimicrobial resistance, CD infections have become more frequent, severe and difficult to treat. Ironically, the standard treatment for CD is other antibiotics: metronidazole is used for mild to moderate infections; vancomycin is used for more severe infections. However, as many as 20% of people with CD get ill again. After two or more relapses, further relapse rates increased to 65%.
Infected patients caused by resistant bacteria are at worse clinical outcome and risk of death and consume more health care resources than patients infected by non-resistant strains of the same bacteria. Antimicrobial resistance is a complex problem that affects society as a whole and is driven by many interrelated factors. Single, isolated intervention measures 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 address the global resistance issues facing human and animal health.
Major governmental regulatory agencies, including the european union, FDA, australian department of agriculture and health, have now implemented strict new directives and legislation on the control of the use of antibiotics in food production animals to reduce the selection of resistance. Major companies in the food industry, such as mcdonald and walmart, are proposing their own initiatives to reduce the use of antibiotics in food.
Elimination or prohibition of antibiotic use in animals will and has led to a number of consequences. The U.S. animal health institute estimates that if growth-promoting antibiotics are not used, the U.S. will require an additional 4.52 million chickens, 2300 million cattle and 1200 million pigs to reach the production levels achieved by current practice, which results in a greater economic burden on agriculture.
Even more alarming, the reduction or cessation of antibiotics and changes in farming practices have led to certain animal diseases becoming more prevalent and prevalent. Such as Necrotic Enteritis (Necrotic enterititis) in poultry. This is reported in european countries (e.g. france and scandinavia peninsula) that ban the use of antibiotic growth promoters with a dramatic increase in the incidence of necrotic enteritis, suggesting that antibiotic growth promoters have a prophylactic effect in controlling the disease. With the increasing number of countries implementing policies to reduce antibiotic use, the cost of necrotic enteritis in the domestic poultry industry is currently estimated to be about $ 20 billion per year, and is expected to increase further. Other diseases cause significant losses to the poultry industry, such as coccidiosis, macular liver disease have become a major cause of layer death and reduce egg production.
Reducing or stopping the use of antibiotics and changes in animal husbandry practices have also affected the swine industry, where diseases become more prevalent and prevalent. Diarrhea associated with Enterotoxigenic e.coli and swine dysentery outbreaks associated with Brachyspira are responsible for high mortality and morbidity in pigs. Also detrimental to the swine industry is a group of conditions associated with Lawsonia intracellularis (Lawsonia intracellularis) and affecting ileitis of the small intestine. This group of conditions includes porcine intestinal glandular disease, necrotic enteritis, crohn's disease, and proliferative hemorrhagic bowel disease.
Salmonellosis is one of the most common and most widely distributed food toxicities, caused by salmonella. It is estimated that tens of millions of human cases occur worldwide each year, and that this disease causes more than a hundred thousand deaths. Antimicrobial resistance of salmonella serotypes has been a global problem. The monitoring data showed that the overall antimicrobial resistance of salmonella increased significantly from 20% to 30% in the early 1990 s to 70% in some countries in the early century. Salmonella lives in the intestines of farm animals, especially chickens and cattle. It can be found on water, food or surfaces contaminated with feces of infected animals or humans (fig. 2 depicts various aspects of salmonella infection and food poisoning).
Campylobacteriosis (campylobacter) is a gastrointestinal disease caused by bacteria called Campylobacter (CB), and is also a major cause of food-borne diseases. CB is primarily transmitted to humans by eating or drinking contaminated food (mainly poultry), water, or unpasteurized milk. CB can also be transmitted by contact with infected (infested) people or by contact with bacteria-carrying cats, dogs and livestock (farm animals). Epidemiology is shown in figure 3.
Most people infected with CB develop diarrhea, colic, abdominal pain and fever, which lasts for one to two weeks. Symptoms usually appear within 2 to 5 days after infection. Diarrhea may contain blood or mucus. In rare cases, CB can enter the blood and cause more serious disease. Any person can suffer campylobacteriosis, although the risk of infection is greater for very young children, the elderly, immunocompromised persons and persons who have a race with livestock. Treatment usually involves fluid replacement, but in severe or complex cases, antibiotics such as erythromycin should be prescribed to reduce the duration of the disease.
More specifically, CB contamination of poultry carcasses/meat continues to occur. The methods of controlling CB contamination by cleaning and desmutting have been focused on process plants. However, it is believed that contamination of processed birds can be reduced if the colonization of CB in the gut of the birds can be controlled prior to slaughter.
The forced reduction or cessation of antibiotics has led to the entry into the "post-antibiotic age," which has led to a need to consider and develop alternatives to treat, control and protect food-producing animals (and humans) from disease. Currently, there is a need for drugs comprising medicated feeds that can be used to alleviate problems associated with reducing or stopping the use of antibiotics and the consequent outbreaks of disease. To date, no cost-effective prophylactic or therapeutic agent has been found that can replace antibiotics in animal feed.
Disclosure of Invention
The present disclosure relates to methods for preventing and/or treating infectious diseases in an animal, wherein the methods comprise administering berberine alkaloid (berberine alkaloid) to the animal.
The present disclosure also relates to an animal feed comprising berberine alkaloids and an animal food (foodstuff), wherein the amount of berberine alkaloids is about 0.001% w/w to 2% w/w of the animal food.
The present disclosure also relates to a dosing regimen comprising administering the berberine alkaloids or animal feed disclosed herein, wherein the berberine alkaloids or animal feed is administered for 1 to 6 weeks in an amount effective for preventing and/or treating an infectious disease in an animal.
The present disclosure also relates to a method for reducing feed conversion ratio in a food-producing animal, wherein the method comprises the step of administering to the food-producing animal the berberine alkaloids or animal feed disclosed herein.
The present disclosure also relates to methods for preventing or treating infectious disease in an animal comprising administering the animal feed disclosed herein.
The present disclosure also relates to methods of preventing or treating infectious intestinal disease in an animal comprising administering the animal feed disclosed herein.
The present disclosure also relates to methods for preventing or treating infectious disease caused by Eimeria (Eimeria) in an animal comprising administering the animal feed disclosed herein.
The present disclosure also relates to methods of preventing or treating infectious disease in an animal caused by Clostridium bacteria comprising administering an animal feed disclosed herein, wherein the bacteria is Clostridium perfringens (c.
The invention also relates to the use of berberine alkaloids for the preparation of a medicament for the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
The present disclosure also relates to the use of berberine alkaloids for the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
The present disclosure also relates to berberine alkaloids for the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
Definition of
The term "acceptable excipient" as used herein refers to a solid or liquid filler, carrier, diluent or encapsulating substance that may be safely used for administration. Depending on the particular route of administration, a variety of carriers well known in the art may be used. These carriers or excipients may be selected from sugars, starches, cellulose and its derivatives, malt (malt), gelatin, 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.
The term "acceptable salt" as used herein refers to a salt that is toxicologically safe for systemic administration. Acceptable salts, including acceptable acidic/anionic or basic/cationic are described in the following: gould, International Journal of pharmaceuticals, 1986, month 11, 33(1-3), 201-; berge et al, Journal of Pharmaceutical Science, month 1 1977, 66(1), 1; heinrich Stahl, Camile G.Wermuth (eds.), Handbook of Pharmaceutical Salts: Properties, Selectionatend Use, second revision, Wiley, 2011. Acceptable salts of the acidic or basic compounds of the invention can, of course, be prepared by conventional methods (e.g., by reacting the free acid with the base or acid to form the desired salt).
Acceptable salts of acidic compounds include salts with cations and may be selected from salts of alkali or alkaline earth metals, 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 formed with organic bases. Suitable organic bases include N-methyl-D-glucamine, arginine, benzathine, diethanolamine, ethanolamine, procaine and tromethamine.
Acceptable salts of the basic compounds include salts with anions and may be selected from organic or inorganic acids. Suitable anions include acetate, acylsulfate, acylsulfonate, adipate, ascorbate, benzoate, benzenesulfonate, bromide (bromide), camphorsulfonate, decanoate, hexanoate, octanoate, chloride (chloride), citrate, dioctylsulfonate (docusate), ethanedisulfonate, propionate dodecylsulfate (estolate), formate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, heconate, hydrobromide, hydrochloride, iodide (iodide), isethionate, lactate, lactobionate, laurate, malate, maleate, methanesulfonate, methylbromide, methylsulfate, naphthalenesulfonate, nitrate, octanoate, oleate, pamoate, phosphate, polygalacturonate, salicylate, stearate, succinate, sulfate, sulfonate, sulfosalicylate, tannate, tartrate, terephthalate, tosylate, triethyliodide (triethiodide), and the like.
Berberine is a positively charged quaternary ammonium cation. Acceptable salts of berberine include, but are not limited to, chloride, hemisulfate (hemisulfate) and iodide salts.
As used herein, an "acceptable solvent" is a solvent that, for the purposes of this disclosure, may not interfere with the biological activity of the solute. Examples of 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. The term "solvate" refers to a complex of variable stoichiometry formed by a solute (e.g., a berberine alkaloid) and a solvent. In particular, the solvent used is an "acceptable solvent" as defined herein. When water is the solvent, the molecule is referred to as a hydrate.
As used herein, "IRP 001" refers to berberine, which is a quaternary ammonium cation and plant natural product having antimicrobial activity, as described herein. The terms "IRP 001" and "berberine" are used interchangeably herein. As used herein, "IRP 001 chloride" or "IRP 001 Cl" refers to chloride salts of berberine; and "IRP 001 sulfate" is the hemisulfate salt of berberine. Thus, it is to be understood that the terms "IRP 001 sulfate", "berberine sulfate", "IRP 001 hemisulfate, and" berberine hemisulfate "are equivalent herein. The molecular structures of berberine quaternary ammonium cations and chlorides and hemisulfates are shown in figure 4.
As used herein, the term "berberine alkaloid(s)" refers to berberine and compounds having similar structure and characteristics to berberine and suitable for the compositions/methods/uses of the present invention. Such compounds include, but are not limited to protoberberine (protoberberine): berberrubine, bicuculline, tetrahydroafrican tetrandrine, jateorhizine, 13-hydroxyberberine chloride, coralyne chloride, 7, 8-dihydro-13-methyl berberine, fibrauretine and 13-benzyl berberine.
The berberine alkaloids may exist in different isomers or different isomeric forms, such as various tautomeric or tautomeric forms. It will be understood that the term "berberine alkaloid(s)" encompasses different isomeric forms as well as combinations, separated from each other.
Berberine alkaloids may also exist in various amorphous and crystalline forms (i.e., polymorphs). It is also to be understood that the term "berberine alkaloid(s)" encompasses different amorphous and crystalline forms as well as combinations, isolated from each other.
As used herein, the term "berberine alkaloid(s)" includes acceptable salts, solvates of said salts or prodrugs thereof.
As used herein, the term "food-producing animal" refers to an animal that is bred to produce food for consumption by another animal, such as a human. It is to be understood that the term "food-producing animal" includes, for example, chickens or pigs.
It will be understood that the term "isomer" refers to structural or architectural isomers, tautomers, regioisomers, geometric isomers or stereoisomers, including enantiomers or diastereomers. In addition, a racemate is understood to comprise an equimolar mixture of a pair of enantiomers.
It will be understood that the term "prodrug" refers to an inactive form of a compound that is converted in vivo to an active form. Suitable prodrugs include esters, phosphonates, and the like of the active forms of the compounds. Further discussion regarding prodrugs 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.
As used herein, a "safe" residual level of berberine is a level that poses no significant risk of causing disease, particularly cancer.
As used herein, the terms "treat," "treating," or the like, refer to the control, cure, or amelioration of a disease, disorder, or condition, or the reduction in the rate of progression of a disease, disorder, or condition, or the prevention or inhibition of symptoms or side effects, the reduction in the severity of symptoms or side effects development, and/or the reduction in 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. The term "treatment" includes use in palliative setting.
The terms "prevent", "preventing", and the like, as used herein, are intended to encompass treatment for delaying or slowing the development of a disease, disorder, or condition, or a symptom or side effect thereof.
With respect to "prevention" and "treatment," as used herein, the term "effective amount" refers to an amount that achieves a desired effect when administered to an animal. For example, an effective amount of a composition disclosed herein is an amount that prevents or treats necrotic enteritis in chickens. The exact total effective amount of the antimicrobial depends on the purpose of the treatment and other factors, including the animal subject (e.g., chicken and swine), route of administration, weight, and severity of the disease.
Drawings
Fig. 1 depicts the transmission of AMR from food producing animals to humans. The figures are taken fromhttps://www.cdc.gov/ foodsafety/challenges/from-farm-to-table.html。
FIG. 2 depicts various aspects of Salmonella infection and food poisoning. The figures are taken fromhttp://thelancet.com/ journals/lancet/article/PIIS0140-6736(11)61752-2/fulltextAndhttps:// www.epainassist.com/abdominal-pain/stomach/food-poisoning。
FIG. 3 depicts Campylobacter epidemiology. The figures are taken fromhttps://wwwnc.cdc.gov/eid/article/10/ 6/04-0403-f1。
Figure 4 depicts the molecular structures of berberine quaternary ammonium cations, berberine chlorides and berberine hemisulfates.
Fig. 5 to 12 depict the results of a lead study of necrotic enteritis in chickens as described in example 1.
Figure 5 is a graph of mortality of birds prior to necropsy for each group.
Fig. 6 is a graph depicting median small bowel disease scores by treatment/challenge group.
Figure 7 depicts necrotic enteritis lesion scores.
FIG. 8 photograph of duodenum of avian group 9; NE-attacked, IVP/berberine water 1.0g/L
FIG. 9 is a photograph of duodenum of avian group 6; NE-challenged, berberine-free treatments
FIG. 10 is a photograph of duodenum of group 12 avian; NE attacked IVP/berberine feed 2.0g/kg
FIG. 11 photograph of duodenum of group 4 avian; 1.0g/L of non-attack IVP berberine water.
FIG. 12 photograph of duodenum of avian group 6; NE-challenged, without IVP/berberine.
Fig. 13 depicts the molecular structures and names of representative compounds referred to in this disclosure.
Figure 14 depicts the molecular structures and names of other representative compounds of the invention:
figure 15 depicts the total individual water intake (stage 1) of the necrotizing enteritis pilot study described in example 2.
Figure 16 depicts the total individual water intake of the necrotizing enteritis pilot study described in example 2 (stage 2).
Figure 17 depicts the feed conversion ratio (stages 1 and 2) of the necrotic enteritis lead study described in example 2.
Figure 18 depicts a barn arrangement with day-old chicks for the study described in example 3.
Fig. 19 depicts litter collected from a seeder (seeder) barn on day 14 of the study described in example 3.
Fig. 20 depicts 400 grams of dunnage dispensed per barn on 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: mean daily gain (ADG) (g/day) on the y-axis versus the growth phase (days) on the x-axis. Treatment group 1 (control group): 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 + Bacitracin zinc (Zn Bacitracin)50ppm (salino Zn bac)).
Fig. 22 depicts a comparison between IVP used in control fattening period (finisher) and example 3, treatment group 2 (dose of IVP 0.30 g/kg).
Fig. 23 depicts the feces of birds from treatment group 2 of example 3 at day 42 (dose of IVP 0.30 g/kg).
Figure 24 eimeria acervulina (e.acervulina) type lesions from example 3 (from outside and inside the duodenum), score + 1.
Figure 25 eimeria acervulina (e.acervulina) type lesions from example 3, scored +2 and + 3.
Figure 26 eimeria acervulina (e. acervulina) type lesions of example 3, scored + 4.
Fig. 27 body lumen inflation of intestine from example 3 (balloon).
FIG. 28 Upper bowel (upper gut) hyperemia (white arrow; left-side heading) and bowel translucency (black arrow; right-side heading) of example 3.
Fig. 29 aqueous intestinal contents from example 3, including orange mucus.
Figure 30 depicts the correlation between corrected feed conversion at 42 days and total score of intestinal coccidiosis lesions at 21 days. The solid line shows the best fit line; the dashed line shows the 95% confidence interval.
Specific embodiments of the present disclosure are described below. It will be appreciated that these embodiments are illustrative and not restrictive.
Detailed Description
The present disclosure relates to methods of preventing and/or treating infectious diseases in an animal, wherein the methods comprise administering to the animal a berberine alkaloid, or an acceptable salt thereof.
In the methods (and animal feeds; dosing regimens and uses) disclosed herein: the animal is preferably a human. The animal is preferably a non-human. Preferably, the non-human animal is a food producing animal. The food-producing animal is preferably selected from chicken or pig. Preferably, the animal is an aquatic animal. The aquatic animal is preferably finfish. Preferably, the aquatic animal is a shellfish (shellfish). The shellfish (shellfish) is preferably selected from crustaceans or molluscs. Preferably, the crustacean is selected from the group consisting of crab, crayfish (crayfish), lobster, prawn (prawn) and shrimp. The mollusk is preferably selected from the group consisting of clams (clam), mussels, oysters, scallops and viviparies (winkle). Preferably, the animal is a mammal. The mammal is preferably a human, horse, dog, cat, sheep, cow, pig or primate. Preferably, the animal is a bird. The fowl is preferably chicken, goose, turkey or duck.
Spot liver disease
Preferably, the infectious disease is a liver disease or an intestinal disease. Preferably, the infectious disease is an intestinal disease. The liver disease is preferably a macular liver disease and the animal is a chicken. Preferably, the chicken is a laying hen. Plaque liver disease is preferably caused by bacteria of the genus Campylobacter. Preferably, the campylobacter is antibiotic resistant.
Salmonellosis (Salmonella)
Preferably, the infectious disease is associated with food poisoning. Preferably, the food poisoning is salmonellosis. Preferably, the salmonellosis is caused by an antibiotic resistant strain of salmonella.
Campylobacter disease (Campyylobacter)
Preferably, the infectious disease is campylobacteriosis. Campylobacteriosis is preferably caused by antibiotic resistant strains of campylobacter.
Infectious diseases with pathogens of escherichia coli: porcine diarrhea/diarrheal disease (Scour)
Preferably, the infectious disease is caused by escherichia coli.
Among all diseases of sucking piglets (sucking pig), diarrhea is the most common, and probably the most important. In some outbreaks, it is responsible for high morbidity and mortality. In a well-functioning group, less than 3% of the litters should be treated at any time, and the mortality rate of piglets due to diarrhea should be less than 0.5%. However, in severe outbreaks mortality levels may rise to 7% or higher, and in individual untreated piglets mortality levels may rise up to 100%. The main bacterial cause is escherichia coli. Diarrhoea in piglets can occur at any age during suckling, but there are usually two peak periods before 5 days and between 7 and 14 days.
The infectious disease is preferably diarrhea, and the animal is a pig. Preferably, the infectious disease is diarrhoea and the animal is a pig. Preferably, the infectious disease is dysentery and the animal is a pig.
Preferably, the infectious disease is caused by an antibiotic-resistant strain of escherichia coli.
Swine dysentery associated with Brachyspira
Swine Dysentery (SD) is caused by a type of cyclonic bacteria known as Brachyspira, which includes Brachyspira hyodysenteriae (Brachyspira hyodysenteriae), Brachyspira pilosus (Brachyspira pilosicoli) and Brachyspira hanpro (Brachyspira hampsonii). This organism causes severe inflammation of the large intestine, which is accompanied by bloody mucous diarrhea (blody mucous diarrheea). The high costs of the disease are associated with morbidity, mortality, growth inhibition and feed conversion efficiency and constant costs of in-feed drug therapy.
Preferably, the infectious disease is caused by a bacterium of the genus brachyspira. Preferably, the infectious disease is dysentery and the animal is a pig. Preferably, the infectious disease is caused by an antibiotic resistant strain of the genus brachyspira.
The infectious disease is preferably caused by a bacterium of the genus Lawsonia (Lawsonia). Preferably, the infectious disease is caused by an antibiotic-resistant bacterial strain of the genus lawsonia. Infectious diseases are preferably caused by Lawsonia intracellularis (Lawsonia intracellularis).
Lawsonia intracellularis related to porcine ileitis
Ileitis comprises a group of conditions involving pathological changes in the small intestine associated with the bacterium lawsonia intracellularis. The disease has four different forms. The first form is porcine adenopathy (PIA), which is an abnormal proliferation of cells lining the intestine. PIA can develop into three other rare forms: necrotic Enteritis (NE), in which proliferating cells of the small intestine die and shed, with an overall thickening of the small intestine (hosipipe gut). Crohn's disease (RI), inflammatory and proliferative hemorrhagic bowel disease (PHE) or "blood gut" (bloody gut), in which the small intestine bleeds extensively. PHE is the most common form of ileitis in growing pigs. PHE is more common in 60 kg pigs and replacement gilts (gilt).
Preferably, the infectious disease is represented by a group of conditions selected from: porcine intestinal disease, necrotic enteritis, crohn's disease, and proliferative hemorrhagic bowel disease, and the animal is a pig.
Infectious diseases with Eimeria (Eimeria) as the pathogen
Preferably, the infectious disease is caused by a parasite of the genus eimeria. The parasite is preferably selected from the group consisting of Eimeria maxima (E.maxima), Eimeria acervuline (E.acervuline) and Eimeria brunette (E.brunette). Preferably, the infectious disease is caused by an antibiotic-resistant parasite of the genus eimeria. The antibiotic-resistant parasite is preferably selected from the group consisting of E.maxima, E.acervuline and E.brunetti antibiotic-resistant bacterial strains. Preferably, the infectious disease is coccidiosis and the animal is a chicken.
Infectious diseases with clostridium as pathogen
Preferably, the infectious disease is caused by a bacterium of the genus clostridium. The bacteria are preferably selected from: clostridium difficile (Clostridium difficile) and Clostridium perfringens (Clostridium perfringen).
Preferably, the bacterium is clostridium difficile (c. Preferably, the infectious disease is diarrhea and the animal is a human. Preferably, the infectious disease is colitis and the animal is a human.
Clostridium perfringens and necrotic enteritis in chickens
Preferably, the infectious disease is caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens. Infectious diseases are caused by antibiotic-resistant bacteria of the genus clostridium, wherein the antibiotic-resistant bacteria is antibiotic-resistant clostridium perfringens.
The infectious disease is preferably necrotic enteritis and the animal is a chicken. Preferably, the necrotic enteritis is caused by a clostridium perfringens type a strain. The clostridium perfringens type a strain is preferably clostridium perfringens type a strain EHE-NE 36. Preferably, the Clostridium perfringens type A strain is Clostridium perfringens type A strain EHE-NE 18. Necrotic enteritis is preferably caused by clostridium perfringens type C strains.
Preferably, administration is via the feed or water of the chicken. The feed is preferably in the form of crumble (crumble) or pellet (pellet).
Preferably, the berberine alkaloid is administered in the feed of the chicken at a dose of 0.001g/kg to 2.0g/kg of feed. The berberine alkaloid is preferably administered in the feed at a dose of 0.003 to 0.3g/kg of feed. The berberine alkaloid is preferably administered in water of the chicken at a dose of 0.001g/L to 1g/L water.
Preferably, the lesion score is reduced and/or the fecal oocyst count (fecal oocyst count) is reduced. Preferably, the lesion score is reduced. Preferably, fecal oocyst count is decreased. Preferably, the incidence is reduced. Preferably, mortality is reduced. Preferably, the FCR is decreased. Preferably, there is an increase in the average daily weight gain.
Feed safety and residue level
For safety reasons, strict regulations have been placed on human and animal pharmaceuticals and animal feed additives. In australia, the therapeutic product administration (TGA) is responsible for the management of therapeutic products for human use, while the australian pesticide and veterinary drug administration (APMVA) is responsible for the evaluation and registration of pesticides and veterinary drugs. In the united states, the Food and Drug Administration (FDA) is responsible for approving human and animal drugs and feed additives regulated by the federal food, drug and cosmetic Act (FD & C Act).
The FD & C act requires that compounds for food-producing animals be proven safe, and that food produced by animals exposed to these compounds be safe for human consumption. In particular, regulations (part 500 of 21CFR, subsection E-provisions of carcinogenic compounds used in food-producing animals) prohibit the use of any compound in food-producing animals that is found to induce cancer when ingested by a human or animal unless certain conditions are met (so-called "Diethylstilbestrol (DES) restriction provisions (Proviso)"). According to DES restrictive terms, the use of suspected carcinogenic compounds is not prohibited if it can be determined by a prescribed examination method that they are found "residue-free" in food produced by food-producing animals under conditions of use that are reasonably certain to be followed in practice.
Although the safety of berberine alkaloids is demonstrated by the widespread use of e.g. berberine alkaloids as dietary supplements for humans, berberine is suspected to be a carcinogen, despite the anticancer activity of berberine itself. (Ma, W.; Zhu, M.; Zhang, D.; Yang, L.; Yang, T.; Li, X.; and Zhang, Y. "Berberine inhibitors the promotion and migration of Breast cancer ZR-75-30 ls by targeting Ephrin-B2" photomedicine 2017,25: 45-51). Thus, if the FDA decides to administer berberine as a carcinogenic compound, U.S. regulations prohibit the use of berberine in food producing animals unless the "no residue" DES restriction clause is applied.
The term "residue-free" means that any residual content remaining in the edible tissue is so low that it presents no significant carcinogenic risk to the consumer. More specifically, an insignificant risk of cancer is defined as a 1 part per million increase in risk.
As used herein, a "safe" residual level of berberine is a level that poses an insignificant risk of disease, particularly cancer.
Preferably, the residual level of berberine alkaloids in the animal after the treatment period is low. After the treatment period, safe residual levels of berberine alkaloids are preferably present in the animal.
Preferably, safe residual levels of berberine alkaloids are present in the chicken muscle tissue after the treatment period. The residual level per g of muscle tissue is at least less than about 13ng berberine alkaloids.
Preferably, the residual level per g of muscle tissue is about 10ng berberine alkaloid. The residue content is preferably about 5 ng/g.
Preferably, the berberine alkaloids have been administered in the feed of the chicken at a rate of about 0.3 g/kg. The residual levels of berberine alkaloids in the muscle tissue of the chicken are preferably as follows:
about 6.1ng/g in the muscle tissue of chicken breast (breast of chicken);
about 5.5ng/g in the muscle tissue of chicken calf (lower leg of chicken); and
about 11.6ng/g in the muscle tissue of chicken thigh (upper leg of chicken).
Preferably, the berberine alkaloids are administered in the feed of the chicken at a dose of less than about 0.1 g/kg.
Preferably, the berberine alkaloids are administered in the feed of the chicken at a dose of about 0.03 g/kg. 35. The residual levels of berberine alkaloids in the chicken muscle tissue are preferably as follows:
the content of the chicken breast muscle tissue is lower than 2 ng/g;
the muscle tissue of the chicken calf is lower than 2 ng/g; and
less than 2ng/g in the muscle tissue of chicken thigh.
Preferably, low residual levels of berberine alkaloids are present in the muscle tissue of the animal after the treatment period and the washout period. After the treatment and clearance periods, safe residual levels of berberine alkaloids are preferably present in the muscle tissue of the animal.
Preferably, safe residual levels of berberine alkaloids are present in the chicken muscle tissue after the treatment and clearance periods.
Preferably, the washout period is 1-2 weeks. The clearance period is preferably selected from the following periods: 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. Preferably, the clearance period is a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.
Preferably, after a 1 day washout period, the residual levels of berberine alkaloids in the chicken muscle tissue are as follows:
about 5.7ng/g in the muscle tissue of chicken breast;
about 3.2ng/g in the muscle tissue of the chicken calf; and
about 6.0ng/g in the muscle tissue of chicken thigh.
Preferably, after a 2-day washout period, the residual levels of berberine alkaloids in the chicken muscle tissue are as follows:
about 3.6ng/g in the muscle tissue of the chicken breast;
about 3.1ng/g in the muscle tissue of the chicken calf; and
about 4.5ng/g in the muscle tissue of chicken thigh.
Preferably, the residual level of berberine alkaloids in the muscle tissue of the chicken is below 2ng/g after a washout period of 4, 7 and 14 days.
Preferably, the berberine alkaloids are administered in the feed of the chicken at a dose of about 0.3 g/kg.
The residual level is preferably at least below 13ng berberine alkaloid per g muscle tissue. The residual level is preferably about 10ng berberine alkaloid per g muscle tissue. Preferably, the residual level is about 5 ng/g.
Preferably, the berberine alkaloids are administered in the feed of the chicken at a dose of about more than 0.1 g/kg.
Preferably, the residual level of berberine alkaloids present in the liver and muscle tissue of the animal after the treatment period is low. Preferably, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the animal after the treatment period.
Preferably, after the treatment period, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken. The residual level of berberine alkaloids in the liver and muscle tissue of the chicken is preferably below 2 ng/g. Preferably, the berberine alkaloids are administered in the feed of the chicken at a dose of about 0.03 g/kg.
Preferably, low residual levels of berberine alkaloids are present in the liver and muscle tissue of the animal after the treatment period and the clearance period. Preferably, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the animal after the treatment and clearance periods.
Preferably, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken after the treatment and clearance periods. The washout period is preferably a period between 1 and 2 weeks. Preferably, the clearance 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 clearance period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.
Preferably, after a 1 day washout period, the residual levels of berberine alkaloids in the chicken muscle tissue are as follows:
about 5.7ng/g in the muscle tissue of chicken breast;
about 3.2ng/g in the muscle tissue of the chicken calf; and
about 6.0ng/g in the muscle tissue of chicken thigh,
and berberine alkaloid residue level in liver tissue of chicken is about 8.0 ng/g.
Preferably, after a 7 day washout period, the residual level of berberine alkaloids in the muscle tissue of the chicken's breast, calf and thigh is below 2ng/g and the residual level of berberine alkaloids in the chicken's liver tissue is about 6.5 ng/g.
Preferably, after a 14 day washout period, the residual level of berberine alkaloids in the muscle tissue of the breast, calf and thigh of the chicken is below 2ng/g and the residual level of berberine alkaloids in the liver tissue of the chicken is about 3.0 ng/g.
Preferably, the berberine alkaloids are administered in the feed of the chicken at a dose of about 0.3 g/kg.
Preferably, after the treatment period, low residual levels of berberine alkaloids are present in the liver and muscle tissue of the animal. Preferably, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the animal after the treatment period.
Preferably, after the treatment period, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken. The residual level of berberine alkaloids in the liver tissue and muscle tissue of the breast, calf and thigh of the chicken is preferably below 2 ng/g. Preferably, the berberine alkaloids are administered in the feed of the chicken at a dose of about 0.03 g/kg.
Preferably, safe residual levels of berberine alkaloids are present in the liver tissue of the chicken after the treatment and clearance periods. The washout period is preferably selected from a period between 1 week and 2 weeks. Preferably, the clearance 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 clearance period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.
Preferably, after a 1 day washout period, the residual level of berberine alkaloids in the liver tissue of the chicken is about 8.0 ng/g. After a 7 day washout period, the residual level of berberine alkaloids in the liver tissue of the chicken is preferably about 6.5 ng/g. Preferably, after a washout period of 14 days, the residual level of berberine alkaloids in the liver tissue of the chicken is about 3.0 ng/g. The berberine alkaloid is preferably administered in the chicken feed at a dose of about 0.3 g/kg.
Preferably, the treatment period is 35 days.
"residual studies" are described elsewhere. The residual level of berberine alkaloids can be determined experimentally. An exemplary protocol for determining berberine alkaloid residual levels in animal tissue using LC-MS/MS is as follows:
after euthanasia, breast, calf (leg) and thigh (thigh) muscle and liver and kidney samples were removed from each bird. A known weight of tissue (about 1g) was homogenized in 2mL of water. Samples were centrifuged and a known volume of supernatant removed to analyze berberine by LC-MS/MS to provide residual levels of berberine in muscle tissue (ng of berberine per g of muscle tissue).
Administration of berberine formulations
Preferably, the berberine alkaloid is berberine hemisulphate. The berberine alkaloid is preferably berberine chloride.
Preferably, the method further comprises an additive that masks the bitter taste of the berberine alkaloid or acceptable salt.
Berberine
Berberine is an isoquinoline alkaloid extracted from Coptis chinensis (Rhizoma coptidis), phellodendron amurense (Phellodendri chinensis cortex) and other herbs. According to the Chinese pharmacopoeia, berberine contents of coptis chinensis, Phellodendron amurense (Phellodendron chinense), Phellodendron amurense (Phellodendron amurense) and barberry (Berberidis radix) are 5.5%, 3.0%, 0.6% and 0.6%, respectively. Coptis (huang) (huang yin is Huanglian) belongs to Ranunculaceae (Ranunculaceae), and contains three main species of coptis: radix et rhizoma Rhei, rhizoma Coptidis (Coptis chinensis, Pinyin is Weilian), rhizoma Coptidis (Coptis deltoidea, Pinyin is Yalian) and radix et rhizoma Rhei (Coptis teeta, Pinyin is Yunlian). Coptidis rhizoma is harvested in autumn, and cut into slices after removing fibrous root. Those coptis chinensis that have a bright yellow part and a very bitter taste are considered to be excellent. The bitter taste of berberine (and other berberine alkaloids disclosed herein) makes taste masking/palatability an important consideration when formulating berberine alkaloids for administration to an animal subject.
Berberine is yellow powder. Chloride salts are slightly soluble in cold water, but readily soluble in boiling water. It is practically insoluble in cold ethanol. The hemisulfate salt is soluble in about 30 parts water and slightly soluble in ethanol. Berberine is a quaternary ammonium cation with molecular formula of C20H18NO4 +And the molecular weight is 336.36. Figure 4 depicts the molecular structures of berberine ammonium cation, berberine chloride salt and berberine hemisulfate salt.
Berberine may be administered in any form acceptable for enteral administration. Suitable non-limiting forms for enteral administration include tablets, capsules, pastes, granules, chewable wafers, gels, oral liquids, injections, medicated waters and feeds (mediated feeds), and suppositories. However, for food producing animals where economic interest is important, the preferred method of administration of berberine is by feed additives in pellet form or medicated feed. Berberine can also be administered through the drinking water of the animal subject by mixing water with a suitable solution or suspension of berberine.
The present disclosure also contemplates providing granule and liquid formulations that can be added to food and water, which make the formulations disclosed herein more palatable to, for example, food-producing animal subjects. For example, a palatable berberine alkaloid formulation may comprise berberine and acceptable excipients suitable for forming a granulated product. Acceptable excipients suitable for forming granular products are, for example, corn starch or polyvinylpyrrolidone (PVP). In one example, the liquid formulation is a liquid concentrate.
There are also many compounds that have similar structures and properties to berberine, including protoberberine: berrubine, allopiceide peoniflorine, tetrahydroafrican tetrandrine, jateorhizine, 13-hydroxyberberine chloride, phellodendrine, 7, 8-dihydro-13-methyl berberine, fibrauretine (african tetrandrine) and 13-benzyl berberine. Protoberberine together with berberine are suitable for use in the composition/method/use of the invention and are referred to in the specification as "berberine alkaloids".
Fibrauretine (African tetrandrine)
Fibrauretine or african tetrandrine is bitter alkaloid extracted from caulis Fibauera recisa Pierre. According to the regulations of Chinese pharmacopoeia, the fibraurea recisa contains fibrauretine not less than 2.0%. Other sources are the rhizome of Coptis chinensis Franch, Coptis deltoidea and Coptis spruce Wall. The Coptidis rhizoma contains fibrauretine not less than 1.5%.
The chlorinated african tetrandrine is yellow solid, soluble in hot water, slightly soluble in ethanol. The melting point is 196 ℃ and 198 ℃. The molecular formula is C21H22NO4Cl, molecular weight 387.86. Figure 13 lists the molecular structure of the quaternary ammonium cation of african stephanine and the structure of the chloride salt.
The total effective amount or dose of antimicrobial compound in the prepared feed may be 0.001g/kg to 2 g/kg. Exemplary amounts of the total amount of antimicrobial compounds in the prepared feed are: 0.001g/kg (0.0001%), 0.003g/kg (0.0003%), 0.01g/kg (0.001%), 0.03g/kg (0.003%), 0.1g/kg (0.01%), 0.3g/kg (0.03%), 1.0g/kg (0.1%) and 2g/kg (0.2%).
The present disclosure also relates to an animal feed comprising berberine alkaloids and an animal food, wherein the amount of berberine alkaloids is between about 0.001% and 1% w/w of the animal food.
The amount of berberine alkaloid in the food product may be in the range of 0.001g/kg to 2g/kg, i.e. 0.001% to 0.2% w/w. Exemplary contents of berberine alkaloids in the food product are: 0.001g/kg (0.0001%), 0.003g/kg (0.0003%), 0.01g/kg (0.001%), 0.03g/kg (0.003%), 0.1g/kg (0.01%), 0.3g/kg (0.03%), 1.0g/kg (0.1%) and 2.0g/kg (0.2%).
The feed is preferably a crumble (crumble); in the form of pellets; or an aqueous form.
The present disclosure also relates to a dosing regimen comprising administering to an animal the berberine alkaloids or animal feed disclosed herein, wherein the berberine alkaloids or the composition or animal feed is administered in an amount effective for preventing and/or treating an infectious disease in the animal for 1 to 6 weeks.
Preferably, the berberine alkaloid or animal feed is fed for 1, 2, 3, 4, 5 or 6 weeks. Preferably, the berberine alkaloid or animal feed is fed for 1 to 6 weeks; 2 to 5 weeks; or between 3 and 4 weeks.
Preferably, the berberine alkaloids are administered at a concentration of about 0.6g/L in water or about 1.2g/kg in feed. The amount of berberine alkaloid in the feed may be in the range of 0.001g/kg to 2g/kg, i.e. 0.0001% to 0.2% w/w. Exemplary amounts of berberine alkaloids or acceptable salts in the food product are: 0.001g/kg (0.0001%), 0.003g/kg (0.0003%), 0.01g/kg (0.0001%), 0.03g/kg (0.0003%), 0.1g/kg (0.01%), 0.3g/kg (0.03%), 1.0g/kg (0.1%) and 2g/kg (0.2%).
The present disclosure also relates to a method for reducing feed conversion ratio in a food-producing animal, wherein the method comprises the step of administering berberine alkaloids to the food-producing animal.
Preferably, the food producing animal is disease free. The food-producing animal is preferably diseased. Preferably, the food-producing animal is selected from a chicken or a pig. The food producing animal is preferably a chicken.
The present disclosure also relates to a method for preventing or treating infectious disease in an animal comprising administering the animal feed disclosed herein.
The present disclosure also relates to a method for preventing or treating infectious intestinal disease in an animal comprising administering an animal feed disclosed herein.
The present disclosure also relates to a method for preventing or treating infectious disease caused by eimeria in an animal comprising administering the animal feed disclosed herein.
Preferably, the infectious disease is caused by an antibiotic-resistant parasite of the genus eimeria. Preferably, the infectious disease is coccidiosis and the animal is a chicken.
Preferably, the infectious disease is necrotic enteritis and the animal is a chicken.
The present disclosure also relates to the use of berberine alkaloids for the preparation of a medicament for the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
The present disclosure also relates to the use of berberine alkaloids in the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
The present disclosure also relates to berberine alkaloids for the prevention and/or treatment of:
infectious diseases in animals;
infectious bowel disease in animals;
infectious disease in animals caused by eimeria; or
Infectious disease caused by a bacterium of the genus clostridium, wherein the bacterium is clostridium perfringens.
The development of formulations, dosages and protocols for the prevention or treatment of infectious diseases in animals is described in the following studies.
Formulation and palatability Studies
Studies to determine feed palatability and poultry productivity after administration of four IRP001 formulations to broiler (broilers) chicks.
Study design-upon receipt, two hundred and forty (240) day-old commercial broiler chicks were evenly distributed on separate ground animal houses and acclimatized for 7 days. On day 7, birds were weighed and assigned to sixteen (16) groups of 15 birds in order. Feed intake, water intake, weight gain and mortality were used as outcome parameters.
Veterinary products of investigative research (IVP)
Table 1 IVP for formulation and palatability studies
Name (R)
Components
Dose level (g/kg)
Masked IRP001 chloride
30％IRP001
2.7, 5.3 and 10.7
Masked IRP001 sulfate
10％IRP001
8.0, 16.0 and 32.0
Unmasked IRP001 chloride
100％IRP001
0.4, 0.8 and 1.6
Unmasked IRP001 sulfate salt
100％IRP001
0.4, 0.8 and 1.6
The dosage is based on a fixed concentration of IRP001 in the feed
Study animals were dosed according to the treatment regimen detailed in table 2 below. Medicated feed is provided to chickens without limitation in the relevant treatment because the sole feed source for the chickens, drinking water, is also provided without limitation.
TABLE 2 treatment protocol
TABLE 3 event Schedule
From the above, the food and water intake and body weight of the animals (and organs after euthanasia) can be recorded. The average body weight gain, average daily body weight gain and Feed Conversion Ratio (FCR) can be calculated over the treatment period. Animal performance can be assessed by these parameters. Likewise, food and water intake parameters may indicate palatability of the drug, while weight gain and Feed Conversion Ratio (FCR) parameters may provide the antibiotic effect of IVP, i.e., the degree to which IVP promotes growth.
Study on feed conversion efficiency
Study to determine the feed conversion efficiency and tissue residues of IRP001 when administered to commercial broiler chicks by feed. 1 week washout residues were also studied.
Veterinary products of investigative research (IVP)
TABLE 4 IVP for study of feed conversion efficiency
Name (R)
Components
Dosage level (g/kg)
IRP001 chloride
100% IRP001 chloride
1.0, 0.1, 0.03 and 0.01
TABLE 5 treatment protocol
TABLE 6 event Schedule
After observing a 1-week washout period, the residual level of IRP0001 was determined by the following experiment:
after euthanasia, muscle samples of the breast, calf and thigh, and liver and kidney were removed from each bird. A known weight of tissue (about 1g) was homogenized in 2mL of water. Samples were centrifuged and a known volume of supernatant was removed to analyze IRP001 by LC-MS/MS to provide residual levels of berberine in muscle tissue (ng berberine per g muscle tissue).
Efficacy study of IRP001 against the Industrial Standard Bacitracin Zinc (Zinc Bacitracin)
Efficacy in preventing or treating necrotic enteritis was determined by administration of IRP001, including study of dose response, feed conversion ratio, tissue retention and safety. Using a validated experimental model, IRP001 was administered via feed to broiler chicks challenged artificially with an Eimeria spp and pathogenic strain of clostridium perfringens. Current industry standard treatments, bacitracin zinc, were used for efficacy and FCR comparisons.
Study design (necrotic enteritis challenge) -commercial broiler chicks raised (used) in an isolator were orally infected at 9 days of age with 5,000 sporulated oocysts of each of the attenuated vaccine strains eimeria maxima and eimeria acervulina (e.acervuline) and 2,500 sporulated oocysts of eimeria brunetti in 1mL of 1% (w/v) sterile saline.
Six days after oocyst challenge (day 15), a known pathogenic clostridium perfringens strain (type a strain NE18), i.t, (c) c
Feed intake at necropsy, weight gain, mortality and NE lesion scores were all used as outcome parameters.
Veterinary product for research (IVP) -IRP001
TABLE 7 IVP and dose levels for efficacy studies of IRP001 on industry Standard bacitracin Zinc
Name (R)
Composition of
Dosage level (g/kg)
IRP001
100％IRP001
1.0、0.3、0.1、0.03
Bacitracin zinc
Industrial markQuasi-drug
Industry standard
TABLE 8 challenge and treatment protocol
TABLE 9 event schedules
Residual levels of IRP0001 can be determined by the following experiment:
after euthanasia, muscle samples of the breast, calf and thigh, and liver and kidney were excised from each bird, and a known weight of tissue (approximately 1g) was homogenized in 2mL of water. Samples were centrifuged and a known volume of supernatant removed to analyze IRP001 by LC-MS/MS to provide residual levels of berberine in muscle tissue (ng of berberine per g of muscle tissue).
Dose rate study
The aim of the study was to evaluate the efficacy of three dose rates of IRP001 in the feed for moderate mixed coccidiosis challenge (eimeria species) in commercial broiler chickens, and to evaluate any occurrence of necrotic enteritis or nonspecific enteritis. Security data and organization residual data will be obtained.
Study design (eimeria challenge) -commercial broiler chicks raised in a barn were infected with wild-type eimeria oocysts at 14 days of age (day 14); approximately 12,000 eimeria tenella (e.tenella), 40,000 eimeria acervulina (e.acervuline) and as many eimeria maxima oocysts per bird as possible.
Four birds per group were randomly selected from each experimental barn and humanely euthanized 7 days after oocyst challenge (day 21).
Overall intestinal health (enteritis) and lesion scores at day 21 and necropsy were evaluated, and feed conversion rates per time period were calculated using feed intake, weight gain, and mortality as outcome parameters.
Veterinary product for research (IVP) -IRP001
TABLE 10 IVP and control for dose Rate study
Name (R)
Composition of
Dosage level (g/kg)
IRP001
100％IRP001
1.0, 0.3 and 0.1
Salinomycin
Industry standard
60ppm
Serithromycin + bacitracin zinc
Industry standard
60ppm+50ppm
TABLE 11 treatment and challenge regimens
Table 12 event schedule
Residual levels of IRP0001 can be determined by the following experiment:
after euthanasia, muscle samples of the breast, calf and thigh, and liver and kidney were excised from each bird, and a known weight of tissue (approximately 1g) was homogenized in 2mL of water. Samples were centrifuged and a known volume of supernatant removed to analyze IRP001 by LC-MS/MS to provide residual levels of berberine in muscle tissue (ng of berberine per g of muscle tissue).
Study of residues
This study and protocol is aimed at determining the residual consumption profile of naturally occurring IVP administered at the maximum tag dose rate by quantifying the marker tissue residues in broiler chicks treated by feed feeding throughout the production cycle.
Background
Antimicrobial drugs are widely used in animal husbandry to control and prevent the potentially fatal disease outbreaks in intensive animal husbandry. This is believed to be the reason for the development of resistant microorganisms and government regulatory agencies are now implementing instructions to control the use of these antimicrobial agents.
The inventors have identified several naturally occurring compounds that can be used as natural antibiotics to replace the antibiotics currently used in food-producing animals such as poultry and swine.
Candidate formulations are tested to meet regulatory standards, such as those required by, for example, the australian pesticide and veterinary administration (APVMA) and the us Food and Drug Administration (FDA). In this regard, determining the residual consumption status of an animal's health care treatment is an essential part of the product development process. This allows government regulators to set appropriate retention periods (WHPs) to protect human health and agricultural product trade.
IRP001 has been selected as a candidate IVP because it has been determined to be safe and non-toxic. Since the chicken industry has extensively relied on antimicrobial drugs to prevent or treat a variety of diseases caused by enteric pathogens, poultry has been selected as the target animal species. These clinically important enteropathogens may potentially respond to IRP 001.
Compliance device
This tissue residual consumption study should be performed according to agreed protocols using SOP and good scientific practices.
Design of research
a. Experimental unit: the experimental and observation units were animals and the statistical unit was the treatment group.
b. Animal model: feed intake, daily water intake, weight change, mortality and marker residues in the tissues were used as outcome parameters.
c. Inclusion criteria were: animals will be selected for study if they meet the criteria outlined in section 10 below.
d. Exclusion and removal criteria: investigators considered animals that were weak, diseased, injured, or otherwise unsuitable for inclusion in the study to be excluded.
After selection, animals that may be considered unsuitable for further study were removed only with written consent from the sponsor or investigator. The reason for any removal will be well documented and documented in the raw data and study reports. The animals removed from the study will receive appropriate veterinary care.
e. Distributing: broiler chicken: upon receipt, one hundred eighty (180) broiler chicks meeting inclusion criteria were assigned to eighteen (18) individual treatment groups of ten (10) birds per group in sequence after being removed from the shipping container. The methods of assignment and randomization will be described in the original data and study reports.
f. Blinding: not applicable.
Veterinary products of investigative research (IVP)
All formulation details, including lot number, expiration date, receipt and use were recorded.
a. Veterinary products of the investigational study: IRP001 Cl as 100% IRP001 Cl.
b. The source is as follows: the IVP will be provided by the sponsor.
c. And (3) storage: the IVP should be stored at ambient temperature in a specified temperature region. The storage location and conditions of the IVP are recorded.
d. Safety: the sponsor provides the SDS or equivalent thereof (if any).
e. And (3) analysis: the IVP is provided with the certificate of analysis (if any).
f. And (3) drug treatment: all remaining IVP treatments were recorded.
Treatment of
a. Dose calculation: the dosage was based on a fixed concentration of IRP001 Cl in the feed (0.03 or 0.1g/kg IRP001 Cl).
b. Preparation of dosage: powdered IRP001 Cl is incorporated into commercial feed ingredient and then thoroughly mixed in, for example, a "concrete mixer" type apparatus to provide the final concentration in the feed outlined.
c. The dose administration method comprises the following steps: study animals were dosed according to the treatment regimen detailed in table 1 below. In the relevant treatment, the chickens should be provided with medicated feed without restriction as their sole feed source.
TABLE 13 treatment protocol-feed conversion ratio
'Duan' euthanasia
Attention is paid to: drug-containing feed was withdrawn from groups 6 and 12 on day 28 to allow for a 14 day washout period for these groups.
Event timetable
TABLE 14 event Schedule
Test system
Animal details were recorded in the raw data. Namely: species, broiler chicks; number, 180; source, commercial (90 in a batch); age, one day of age.
Animal management
a. Animal welfare: study animals were similarly managed with due consideration of their welfare. The study animals were observed according to the requirements of the Animal Ethics Committee (AEC) and "animal care records" were completed.
b. Health management: if necessary, any conventional prophylactic treatment should be performed as soon as possible and recorded (product name, batch number, expiration date, dose, route of administration and date).
From day 0, study animals were observed twice daily at appropriate locations according to Standard Operating Protocol (SOP). Any health issues that require further examination are recorded.
All health issues and adverse events must be reported to the investigator within one working day. Any adverse event deemed by investigators to be product-related, leading to death, life-threatening, involving a large number of animal or human adverse events must be recorded and reported to the initiating person and AEC within one working day.
Routine veterinary care and procedures can be performed and described in the raw data. For standard regulatory practices and humanistic reasons, drug therapy may be administered in combination with prior approval by the investigator and sponsor (if relevant). No treatment similar to IVP was administered. All concomitant medications were recorded to provide identification of the material used (product name, lot number and expiration date), animal ID, reason for use, route of administration, dosage and date of administration, and included in the raw data (test log) and study report.
If the injury or disease results in euthanasia or death of the study animal, this should be recorded and necropsied by the veterinarian, if possible. Included in the raw data are "necropsy reports" including possible causes of death.
All health issues, adverse events and animal mortality, including their relationship to treatment, were included in the study report.
c. Livestock house environment: treatment groups raised chickens in dedicated chicken floor barns in two separate and discrete controlled environment rooms within an approved animal facility. One room may hold all non-dosed birds from groups 13 to 18 (including groups 13 and 18), and a second room may hold all dosed birds from groups 1 to 12 (including groups 1 and 12). The floor space of each animal house is about 1.5m2. According to normal commercial practice, chickens are raised on bedding.
By day 49, there were 18 ground barns, 10 chickens per barn. At the end of the study, the maximum chicken weight per house was well below the maximum broiler weight of 40kg/m2 recommended in australian utility standards.
Note-birds from groups 13 to 18 (including groups 13 and 18) (untreated control animals) were kept in similar but physically separate isolation rooms from drug-treated birds from groups 1 to 12, ensuring no cross-contamination during the study.
d. Experiment diet: throughout the study, a formulated commercial brood-time (starter) ration followed by a brood-time (growth) ration was administered. Copies of the feedbag labels or equivalent showing the feed ingredients are contained in the raw data.
e. Feed and water intake: the daily feed addition was weighed and recorded and the daily feed intake (by treatment group) was calculated. Daily water volume was measured and recorded and daily water intake (by treatment group) was calculated.
f. Animal treatment: the study animals were humanely euthanized according to AEC approval and recorded at intervals listed in the event schedule (table 14).
Study procedure
a. Test logs: all planned and unplanned events during the study were recorded.
Evaluation of effects
a. Weight: chickens were weighed on day 0 (group body weight) and on days 7, 14, 21, 28 and 35-the body weight of each animal was recorded. Before and after weighing, the weighing scale was checked using a calibrated test weight and recorded. Body weights at study termination were compared between groups to determine the effect of treatment (if any).
b. And (4) checking: at the time of gross pathology and tissue collection, euthanasia was subjected to a separate clinical examination. Clinical examinations were recorded. Digital still images can be recorded appropriately.
c. And (4) observing results: birds were checked twice daily for overall health, usually before 8 am and after 4 pm. Thus, a typical interval between two observations is 9 hours a day and 15 hours a night. If the investigator considers that the abnormal clinical symptoms are significant (e.g., significant weakness and low likelihood of recovery), the birds showing the abnormal clinical symptoms are recorded, carefully observed and euthanized.
d. Autopsy examination: between days 35 and 49, all birds were euthanized and necropsied according to schedule-table 14.
e. Gross pathology: all birds from all groups 1 to 18 were necropsied and examined for gross visual pathology changes, which were appropriately described and scored (by individual bird).
f. Analysis of tissue residues: according to the schedule, table 1, duplicate representative samples of liver, kidney, breast muscle (1), leg muscle (2) [ upper and lower thighs ] and total skin with intact fat were collected from the six (6) heaviest birds in each group (groups 1 to 18 (including groups 1 and 18)) and stored frozen (<10 degrees celsius) for subsequent marker residue analysis. Birds from groups 13 to 18 were sacrificed on day 35 as untreated control birds and tissues were collected to meet the tissue assay (tissue assay) requirements.
The samples will be labeled with a sticker label that lists the study number, animal ID, time point, date, sample type and replicate (duplicate).
For residual analysis, the samples were thawed and then a known weight of tissue (about 1g) was homogenized in 2ml of water. The samples were centrifuged and a known volume of supernatant was removed for analysis by LC-MS/MS.
TABLE 15 analysis matrix
g. Storage, transfer and disposal of the sample: the storage, transfer and disposal of the samples were recorded. Replicate sample 1 tissue samples were shipped frozen on wet ice to the analysis lab for the times outlined in section 10. The samples were transferred with an accompanying temperature data recorder and a frozen water bottle according to Standard Operating Protocol (SOP). After the last sample collection time point, repeat sample 2 tissue samples were stored frozen for a period of 6 months. Beyond this point, it may be discarded at the decision of the research site unless explicitly required by the sponsor representative to not do so.
Statistical analysis
The methods are recorded in the study report.
Data recording
The recipe specification will replace the facility SOP. Study forms can be added or modified as needed during the study without recipe revisions or deviations.
a. Protocol approval: the protocol was approved and signed by all relevant personnel before the study began (see page 1).
b. Modification/deviation: a modification is a change or modification made to a schema prior to performing the task of changing or modifying. The modification must account for the reason for the change and be authorized by the originator of the written record. The modification must be signed by the investigator and the initiator.
In determining the deviation, the investigator should record, sign and date the deviation from the present scenario or applicable SOP. The impact on the overall outcome or completeness of the study will be assessed. The departure must be notified to the originator as soon as practicable.
All protocol modifications and deviations are recorded accordingly and numbered sequentially according to the date of occurrence or the identified date.
c. And (3) file annotation: the file annotation is recorded accordingly to clarify events or situations that might not otherwise be apparent from the raw data. The originator must be informed of the file annotation as soon as feasible.
d. Modifications of the investigators: changes by the research investigator or other responsible investigator should be recorded accordingly.
e. Raw data: all initial raw data pages are paginated by the person observing and the person recording the information, identified and signed with the study number and dated.
f. Communication log: the investigator keeps a copy of all correspondence relevant to the study. Any telephone conversations that would result in a change in study documentation, design, progress, or study report are recorded.
g. And (4) permission: the studies detailed in this project should be included in government agency licenses (e.g., APVMA bench licenses).
Research report
The study report is prepared by the investigator or a designated person. Including a data list of each variable measured. The compliance statements of the study investigators are included in the study reports. The original signed study report, with the original data and statistics attached, will be submitted to the sponsor and archived.
Investigation of Salmonella and Campylobacter
The present disclosure also contemplates preventing or treating infectious diseases caused by salmonella or campylobacter. The following describes studies for studying the effectiveness of berberine alkaloids or berberine alkaloid compositions in the prevention or treatment of a disease caused by salmonella or campylobacter infection. These studies were modeled on published protocols: alali, W.Q et al, "efficiency of infection oil compound on sharing and organization of Salmonella enterica seroverberg in bridges", Poultry Science,2013,92: 836-841; berghaus, R. et al, "energy of Salmonella and Campybacter in environmental fast and processing plants from commercial boiler chips tanks", appl.Environ.Microbiol.2013, 1-37; cochran, W.G., and G.M.Cox, Experimental design, second edition John Wiley & Sons, New York, NY, pp.582-.
Salmonella study
The objective of this study was to evaluate the effectiveness of IVP as a means of controlling Salmonella medibergiensis (Salmonella heidelberg) in broiler chicks.
Design of experiments
In this study of twelve (12) barns, six hundred (600) chicks were assigned to three (3) treatment groups of four (4) replicate blocks (replicate blocks) and divided into fifty (50) birds per barn.
Treatment groups were assigned to the barn using a randomized complete block design (Cochran and Cox, 1992). The treatment groups were as follows:
1. no treatment-Haidelberg Salmonella challenge control
2. Treatment of 1-Heidelberg Salmonella challenge
3. Treatment of salmonella 2-Heidelberg challenge
The study began with the laying of birds (day of hatch; DOT0) at which time the birds were assigned to the experimental barn. Only birds that appeared healthy were assigned to study use and the final number and disposition of all unassigned birds were recorded. During the study, no birds were changed. Poultry weight (kg) was recorded at the house at the beginning of the study (DOT0), at DOT 35 and at the end (DOT 42).
Materials and methods
A poultry feed. Six hundred (600) hatching day Ross x Ross straight-hatched (straight-run) broiler chicks were obtained. Birds received conventional vaccination (HVTSB1) and herd number information was recorded. All birds were vaccinated with the commercial coccidiosis vaccine at the recommended dose.
Animal houses and environmental control. At the beginning of the study, fifty (50) broiler chicks were distributed to twelve (12) ground stalls in a modified conventional poultry house with firm sides and a muddy ground, the size of the barn being 5x 10(1.00 ft)2Poultry stocking density). The facility is cooled by a fan. Thermostatically controlled gas heaters are the primary source of heat. Auxiliary heating lamp (one for each animal house 1]Individual lamps) provide heat (when needed). According to the recommendations of the main breeder, birds are raised under ambient humidity and provided with a lighting program. When placed, each barn contained approximately four (4) inches of fresh pine wood chips. No dunnage was changed during the study. Each barn contained one (1) tube feeder and one (1) bell-shaped drinker, resulting in a ratio of fifty (50) birds/feeder and drinkers.
Application and drinking methods are not limited.
And (4) eating. The rations administered were as follows: DOT0 to DOT 14 at brooding stage, DOT 14 to DOT 35 at brooding stage, and DOT 35 to DOT42 at fattening stage. The diet is administered in the form of crumbled feed (feed for brood) or pellets (feed for brood and fattening). The feed formulations of this study included non-dosed commercial broiler brood, finishing and fattening diets, mixed with appropriate feeds, that were calculated to meet or exceed the NRC standard, and that were not supplemented with antibiotics unless explicitly specified as part of a treatment regimen. Experimental treatment feeds were prepared from basal brood time feeds, all basal feeds and the number of test items used to prepare treatment batches being recorded. To ensure uniform distribution of all tested items, the treated feed was mixed and pelleted in a california pellet mill at 80 ℃ (pellet temperature was recorded). After mixing was complete, the feed was dispensed into the barn for the indicated treatment group. The test items are stored in the SPRG climate-controlled storage area. All diet, formulation and other feed information was recorded.
And (5) changing the feed. Birds received appropriate treatment feed from DOT0 to DOT 42. The ration is changed from brood to fertile at DOT 14 and from fertile to fattening at DOT 35. At that time, all previous feed was removed from each barn, weighed separately and replaced with fattening feed. At DOT42, all the unconsumed fattening feed was removed from the barn, weighed separately and discarded.
And (4) inoculating salmonella. At DOT0, twenty-five (25) chicks (50% of the breeders) per barn were marked, color-coded (for identification), and administered orally (by gavage) 107CFU against nalidixic acid salmonella heidelberg.
And (4) sampling salmonella. Bootsocks swab (bootsock swab) samples were collected from all barns DOT 14 and DOT42 to determine the environmental contamination of Salmonella. Gloves were changed between completion of each swab to reduce potential sample cross-contamination. Pre-wetted bootock swabs (Solar Biologicals, inc., catalog number BT SW-001) were removed from the sterile bag, placed on feet covered with clean new plastic boots, walked around and inside the barn, removed, and placed in a sterile bag labeled with the barn number. After repeating this procedure for each barn, the samples were suitably stored and then submitted for salmonella analysis.
And (5) culturing the salmonella cececal. Cecal sampling is completed at DOT 42. Ten (10) horizontally exposed (unlabeled) birds were removed from each barn on DOT42, euthanized (by cervical dislocation) and the cecum of each bird removed aseptically. After removal of the cecal sample, it was placed into one (1) sterile plastic sample bag (Fisher Scientific), labeled, stored on ice, and submitted for salmonella analysis.
And (4) separating and identifying salmonella. All samples submitted for salmonella isolation and identification (bootsock swabs and/or cecum) were stored on ice in sterile Whirl Pack bags prior to analysis. Upon arrival, tetrathiosulphate (tetrathionate) broth was added to the Bootsock swab samples while weighing the ceca, sterile saline was added, and the samples were homogenized (stomacher). One (1) mL aliquot was taken for MPN analysis, a 10X tetrathiosulphate broth (Difco) solution was added, and the sample was incubated overnight at 41.5 ℃. A loop of the sample was knocked into a xylose lysine tergitol-4 agar (XLT-4, Difco) plate and incubated overnight at 37 ℃. A maximum of 3 (three) black colonies were selected and confirmed to be positive for salmonella using Poly-O salmonella specific antiserum (MiraVista, Indianapolis, indiana, IN). (Berghaus et al, 2013; Alali et al, 2013)
Salmonella enumeration procedure (MPN method). For all ten (10) horizontal exposures (unlabeled) and five (5) direct challenge (labeled) samples, one (1) ml of homogenized (stomacher) peptone broth sample was transferred to three (3) adjacent wells in the first row of a 96-well two (2) ml deep module (block). A 0.1ml aliquot of the sample was transferred to a second row of 0.9ml of tetrathiosulphate broth, the procedure described above was repeated for the remaining rows (to produce five (5) ten-fold dilutions), and the module was incubated (at 42 ℃ for 24 hours) (table 16). One (1) μ l of each well was transferred to XLT-4 agar (containing nalidixic acid) using a needle tool replicator, the plates were incubated (at 37 ℃ for 24 hours), and the final dilution of each sample was recorded and input to the MPN calculator (determine sample MPN). The suspected salmonella isolates have been confirmed by Poly-O salmonella specific antiserum (MiraVista, Indianapolis, IN). (Berghaus et al, 2013; Alali et al, 2013).
TABLE 16 Salmonella count
Disease and coccidiosis control. All birds were vaccinated with the american agricultural sector (USDA) approved coccidial vaccine at one (1) day of age via a spray cabinet. No concomitant medication was used during the study. To prevent cross-contamination, plastic disposable boots were worn into the stall and replaced between each stall.
Identification of birds. Barns are units of measure. The safety of the animal house will prevent the birds from migrating.
And (5) monitoring. Overall flock (cock) status, temperature, light, water, feed, bedding status and accidental henhouse status/events were monitored for all birds. The findings were recorded twice daily during normal working hours (one [1] observation was recorded on the last study day). One (1) observation was recorded on Saturday, Sunday and observed holidays.
The mortality rate. The mortality rate of the barn was checked daily. The birds are eliminated only to relieve the pain. The date and removed weight (kg) of any rejected (or found dead) birds were recorded, necropsies were performed on all rejected (or dead) birds, and the following information was recorded: gender and possible cause of death.
Poultry and feed handling. All birds, mortality and residual feed, including mixer flushes (mixer flushes), were disposed of by appropriate ethical methods.
And controlling and processing source data. The data is recorded in unwashed ink with clear entries, each source data table is signed (or initialized) and dated with a separate record entry. All source data errors (and/or changes) have been initialized, dated, and a short explanatory statement or error code written directly on the form.
And (5) managing data. Data management and statistical analysis of weight gain, feed consumption, feed conversion and salmonella results were performed.
Event calendar
TABLE 17 Salmonella study event calendar
Campylobacter study
This study was to determine the efficacy of the Investigational Veterinary Product (IVP) in reducing shedding (shed) (horizontal spread) and colonization of campylobacter jejuni in the caecum of broiler chickens.
Design of experiments
One hundred twenty (120) day-old (non-SPF) commercial broiler chickens were received. Five (5) birds were euthanized by cervical dislocation and their cecum was cultured for campylobacter jejuni. The remaining selected one hundred and five (105) birds were randomly divided into three (3) groups in an isolation chamber, which was again divided into three portions, each group having 35 birds. The experimental variables are shown below. Broiler chicks were given the initial broke feed to all birds and treated as specified below.
The number of rooms is-1, and the rooms are divided into 3 bird spaces
Total number of chicks-120
Number of chicks euthanized immediately-05
The number of birds which can be subdivided into treatment groups-105 treatment groups-3
Repeat Block-N/A
Birds subdivided per house-35
Treatment group
1. No treatment-Campylobacter jejuni challenge
2. Treatment of 1-Campylobacter jejuni challenge
3. Treatment of 2-Campylobacter jejuni challenge
Materials and methods
A poultry feed. One hundred and ten (110) hatchery Ross 708 male broiler chicks were obtained. Birds were sexed, received a conventional vaccination (HVTSB1) and herd number information was recorded. According to the manufacturer's recommendations, birds receive one (1) dose of a commercially approved coccidial vaccine at one (1) day of age.
Animal houses and environmental control. At the beginning of the study, one hundred and five (105) hatchery day Ross 708 male broiler chicks were assigned to one (1) isolation room. The room is subdivided into three (3) equal bird spaces. Thirty-five (35) chickens per room are placed in each room. The dimensions of each room were 13.4 'x 15.7' (approximately 2.0 feet)2Stocking density). The environment of the isolated room was controlled by a separate HEPA filtration system and a heat pump unit with one (1) heat lamp that provided supplemental heat during the brooding process. The birds are raised under ambient humidity. When placed, each barn contained about four (4) inches of kiln dried bags of fresh pine wood chips. No dunnage was changed during this study. Each space contained one (1) tube feeder and one (1) bell-shaped drinker (35 birds/feeder to drinker ratio). Birds were provided with light for twenty-four (24) hours per day.
Application and drinking methods. Without limitation.
And (4) eating. Throughout the study, the birds were fed broiler chicks with a brood time diet. The unfed commercial broiler brooding diet was mixed with appropriate feed and calculated to meet or exceed the NRC standard without the addition of antibiotics to any of the feeds unless explicitly specified as part of the treatment regimen. The feed is prepared from basic brood time feed. After mixing was complete, the feed was dispensed into the barn for the indicated treatment group. The test items are stored in a climate controlled area. All diets, preparations and feeds were documented.
And (5) changing the feed. Birds received feed from DOT0 to DOT 35 during brooding.
Method of administration of campylobacter jejuni: in DOT 14, 35 birds per treatment were orally gavaged with 0.1ml Campylobacter jejuni JB strain broth containing about 106CFU/ml (chick dose about 10)5CFU/ml)。
Campylobacter colonization evaluation: at DOT0, five (5) birds were cultured for prevalence of campylobacter jejuni; DOT 35, thirty-three (33) birds were euthanized by cervical dislocation for each treatment. The cecum of each bird was removed aseptically and placed into sterile plastic sampling bags (Fisher Scientific) for campylobacter isolation analysis. All samples were stored on ice prior to campylobacter analysis.
Campylobacter counting procedure: campylobacter count program (direct count). For each sample, one (1) ml of homogenized Bolton broth sample was transferred to three (3) adjacent wells in the first row of a 96-well two (2) ml deep module. 0.1ml aliquots were transferred to the second row of 0.9ml Bolton broth, the procedure was repeated for the remaining rows (yielding twelve (12) ten-fold dilutions), and then 0.1ml of each well was spread onto Campy Cefex agar (Table 18). The plates were incubated (42 ℃ for 24 hours) in the presence of campylobacter gas and the final dilution of each sample was recorded. The isolated strain of Campylobacter suspected was confirmed by gram staining.
TABLE 18 Campylobacter count
And (4) controlling diseases. No concomitant drug therapy will be used during the study. To prevent cross-contamination, the rooms were entered with disposable plastic boots and replaced between each room.
Identification of birds. Barns are units of measure. The safety of the animal house will prevent the birds from migrating.
And (5) monitoring. Overall flock condition, temperature, light, water, feed, bedding condition and accidental henhouse condition/event were monitored for all birds. The results of the findings were recorded twice daily during normal working hours (one [1] observation was recorded on day 35). One (1) observation was recorded on Saturday, Sunday and observed holidays.
The mortality rate. The rooms were checked daily for mortality. The birds are eliminated only to relieve the pain. The date and removed weight (kg) of any rejected (or found dead) birds were recorded, necropsies were performed on all rejected (or dead) birds, and the following information was recorded: gender and possible cause of death.
Poultry and feed handling. All birds, mortality and residual feed, including mixer flushes (mixer flushes), were disposed of by appropriate ethical methods.
And controlling and processing source data. The data is recorded in unwashed ink with clear entries, each source data table is signed (or initialized) and dated with a separate record entry. All source data errors (and/or changes) have been initialized, dated, and a short explanatory statement or error code written directly on the form.
And (5) managing data. Data management and statistical analysis of weight gain, feed consumption, feed conversion and campylobacter results were performed.
Event calendar
TABLE 19 Campylobacter event calendar
Examples
Necrotic enteritis
Necrotic enteritis is an intestinal infection found in food producing animals such as poultry. It was first described by Parish in 1961 and is caused by the bacterium Clostridium perfringens (Clostridium perfringens) in poultry and may manifest as an acute clinical disease or a subclinical disease. Although clostridium perfringens is considered to be the causative agent of necrotic enteritis, other contributing factors are often required to predispose an animal to disease. It is well recognized that necrotic enteritis is a multifactorial disease process with many risk factors, including eimeria infection, removal of antibiotic growth promoters, environmental and regulatory conditions, physiological stress and immunosuppression, and the nature and form of the diet.
Necrotic enteritis is a potentially fatal disease that can lead to up to 1% of group mortality per day for several consecutive days during the last few weeks of the feeding period, with cumulative total mortality rising to 30-50%. In the subclinical form, damage to the intestinal mucosa leads to reduced digestion and absorption, reduced weight gain and increased feed conversion rates, resulting in a decrease in commercial performance. It is the manifestation of this disease that reportedly causes the greatest economic loss in the poultry industry. In addition, clostridium perfringens in poultry is at risk of transmission to humans through the food chain, where clostridium perfringens is one of the frequently isolated bacterial pathogens in human food-borne disease outbreaks.
Necrotic enteritis has previously been controlled by well-known antibacterial agents such as virginiamycin (virginiamycin), bacitracin, and the like. More and more countries prohibit the use of antibiotics in food-producing animals, leading to the occurrence of necrotic enteritis, which poses a serious threat to animals and public health.
Clostridium perfringens is a gram-positive anaerobic bacterium present in soil, dust, manure, feed, poultry bedding and intestinal contents. It is extremely prolific, being able to produce a variety of toxins and enzymes. Clostridium perfringens strains are classified into five toxin types (a, B, C, D and E) based on the production of four toxins (α, β, epsilon and iota). Necrotic enteritis has been suggested to be caused by type a, and to a lesser extent by type C, where type a strains produce chromosomally encoded alpha toxin, while type C strains produce alpha toxin as well as beta toxin.
Alpha toxin is a phospholipase C sphingomyelinase which hydrolyzes phospholipids and promotes tissue disassembly of membranes, inducing synthesis of mediators such as leukotrienes, thromboxanes, platelet aggregation factor and prostacyclin. These mediators cause vasoconstriction, platelet aggregation and myocardial dysfunction, leading to acute death. Beta toxin induces hemorrhagic necrosis of the intestinal mucosa, although the exact mechanism is not clear. The pathology of necrotic enteritis is being reevaluated, while other virulence factors are being sought. Recently, there is evidence that alpha toxin may not have a major role in the pathogenesis of necrotic enteritis that has been proposed, and studies report that the use of non-wild type alpha toxin impairs the ability to cause the disease. Evidence suggests that molecules in clostridium perfringens culture supernatants recapitulate disease-like pathology upon injection into the intestine. Recent evidence also suggests that NetB toxin from clostridium perfringens may play a key role in the pathogenesis of necrotic enteritis.
Clostridium perfringens is naturally present at low levels in the intestinal tract, but disturbances in the normal intestinal microflora may lead to rapid proliferation of the bacterium, leading to the development of necrotic enteritis. Chickens are most commonly infected at 2 to 6 weeks of age, but necrotic enteritis may occur in birds of 7 to 16 weeks of age, or even up to 6 months of age.
The clinical feature of the disease is a sudden increase in flock mortality, usually without prior notice, although wet litter is sometimes an early indicator. Clinical symptoms may include weakness, dehydration, lethargy, feathering, diarrhea and reduced feed consumption, although the duration of clinical disease prior to death is short, so the reduction in weight gain is not significant. Macroscopic lesions were visible in the small intestine. The duodenum, jejunum and ileum become thin-walled, fragile, distending and filled with gas. In addition, the mucosal surface is covered with a gray-brown to yellowish-green amphoteric or pseudomembrane. Lesions may also be found in atrophy of other organs as well as red blood cells and bursa. The subclinical form of necrotic enteritis is rather difficult to identify and sick birds that respond to antibiotic analogue treatment are generally considered to suffer from the disease. Although wet litter does not always originate from clostridia, wet litter will often prompt poultry farms to take immediate antibiotic treatment. In addition, mild necrosis of the intestinal mucosa was reported in subclinical necrotic enteritis. Example 1 describes the use of berberine sulphate (IRP001 sulphate) in the prevention or treatment of necrotic enteritis.