WO2007095830A1 - Powder perfusion agent used in the treatment of bovine mastitis and its preparing process - Google Patents

Abstract

A perfusion agent and preparation method thereof, the perfusion agent is in a sterilized-powder form, and comprises Nisin and a physiologically acceptable acid, used to manufacture of a medicament for the treatment of bovine mastitis.

Description

 Powdered breast perfusion agent for treating cow mastitis and preparation method thereof

 The invention relates to a pharmaceutical preparation for treating cow mastitis and a preparation method thereof. Background technique

 Mastitis is the most serious type of disease that jeopardizes dairy farming. About 50% of cows worldwide suffer from various types of mastitis. Cows with mastitis have reduced milk production and quality. The annual direct economic loss caused by cow mastitis in the United States amounts to $2 billion. About 70% of the damage caused by mastitis in dairy cows is caused by the decline in milk production caused by recessive mastitis. The annual loss of only hidden mastitis in China is at least 135 million yuan. The main cause of mastitis in cows is pathogenic microbial infection. Therefore, the main drug currently used to treat mastitis is antibiotics. Long-term, extensive, high-dose antibiotics for the treatment of dairy disease have led to a number of adverse consequences. The first is the public health problem of food: After treatment with antibiotics, the risk of drug residues in milk may have an adverse effect on human health. Secondly, the increase of drug-resistant strains: the resistance of pathogenic bacteria, the more the mastitis becomes The more difficult it is to cure, the veterinarians of the cattle farm have to increase the dose of antibiotics, which increases the risk of drug residues; other hazards of drug residues include inhibition of the fermentation process in dairy production, resulting in reduced product yield and quality. Therefore, people have been looking for a more effective and effective treatment for dairy cow mastitis than antibiotics.

 In this regard, there are many preparations such as Chinese herbal medicine and lysozyme.

 In the long-term practice, Chinese veterinary medicine has accumulated rich clinical experience and a large number of Chinese herbal medicines for the prevention and treatment of mastitis in dairy cows. However, the effective scores of these prescriptions and their contents are uncertain, and the quality is difficult to be effectively controlled. Traditional Chinese herbal medicines are directly used. The original medicinal material has a large dosage and the drug effect is not significant; and the herbal composition is complex and is applied to the intramammary perfusion, which is stimulating.

 Lysozyme can prevent and cure diseases by lysing cell walls, dissolving and killing various pathogenic bacteria. However, the osmotic effect of the enzyme is not strong. When the bacteria are in the deep part of the breast tissue, the lysozyme has no effect. In addition, the stability of the enzyme preparation is poor, and the quality is difficult to control.

According to reports, in 1970 Gross and MoreU clarified the structure of the nisin (Nisin) molecule. Nisin is a new type of oligopeptide, which exists in six types, namely A, B, C, D, and EZ. Among them, Nisin A and Nisin Z are the most active types of research. The two differ only in the 27 amino acid residues, and their antibacterial properties are almost the same. The Nisin-class structure consists of 34 amino acids with amino and carboxyl groups at the end of the molecule, and five sulfur bridges form the inner ring. The molecular formula is C ₁₄₃ H ₂₂₈ N ₄₂ 0 ₃₇ S ₇ and the average molecular weight is 3510. Nisin active molecules often Dimers and tetramers have molecular weights of 7000 and 14,000, respectively.

 Nisin has a lot of research on the mechanism of action of microorganisms. Nisin can adsorb the cell pancreas of G+ bacteria, and then form a cavity in the cell membrane, so that the intracellular substances are discharged, leading to cell disintegration and death. Nisin mainly inhibits the growth of most G+ bacteria. Common cow mastitis pathogens are mainly G+ bacteria, such as Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis. In the study table, Nisin is highly active against these pathogens, and Penicillin-resistant pathogens also have a strong killing effect.

 Nisin is a natural product contained in dairy products. For several years, it has been ingested with food and no toxicity problems have been found. A systematic study of the safety of Nisin has now been conducted. Rats have an oral LD50 of approximately 7000 mg / kg body weight. Studies have shown that Nisin is particularly sensitive to proteolytic enzymes (such as trypsin), so it can be quickly hydrolyzed into amino acids by proteolytic enzymes in the digestive tract after consumption. After ingesting lOmin, people can not detect the activity of Nisin in saliva, so it will not remain in the body, nor will it change the normal intestinal flora. It is a safe preservative. In 1969, FAO I WHO Joint Expert Committee on Food Additives approved Nisin as a food additive; on March 29, 1990, the Ministry of Health of China listed it in the 1990 supplement of GB2760-86, which can be used for canned food, vegetable protein food, Dairy and meat products. Nisin has been approved as a pure natural food preservative in more than 50 countries and regions.

 U.S. Patent No. 5,762,948 and Chinese Patent No. 1,118,413 disclose a wet wipe for the prevention and treatment of mastitis in cows, the composition of which is nisin. However, the preparation can only be used for disinfection outside the dairy cow's milk area, and can not be used for intramammary injection. Chinese Patent No. N03115388 discloses a bactericidal preparation for preventing mastitis of dairy cows, the main component of which is a synthetic antimicrobial peptide and Lysococcal enzyme, but no country has yet approved the use of this antimicrobial peptide for the treatment of dairy cow mastitis. In the previous application, the applicant filed a patent application for the treatment of cow mastitis with lactic acid streptomycin (Patent Publication No. CN1840173), but our later studies found that the aqueous solution of nisin was stored for several months, antibacterial The potency will drop significantly. Summary of the invention

 SUMMARY OF THE INVENTION An object of the present invention is to provide a pharmaceutical preparation containing nisin as a main component for the treatment of cow mastitis which is stable and convenient for veterinary clinical use and which is harmless to the human body.

 Another object of the present invention is to provide a process for the preparation of the above pharmaceutical preparation.

 The present invention provides a powder injection for treating cow mastitis comprising nisin and a physiologically acceptable acid.

In the powder injection of the present invention, each administered dose of the powder injection contains a potency of 200,000-12,500,000 units of nisin, preferably from 1,000,000 to 5,000,000 units.

 In the powder injection of the present invention, when the unit injection of the powder is completely dissolved in 20 to 50 ml of water or physiological saline, the titer of nisin is a titer of nisin of 10,000 to 250,000 units/ml, the most Good is 50,000-100,000 units / ml.

 In the powder injection of the present invention, the physiologically acceptable acid is selected from the group consisting of vitamins. And citric acid, malic acid, citric acid, tartaric acid, oxalic acid, oxalic acid, succinic acid, glutaric acid, boric acid, acetic acid, and the like, and mixtures thereof. In the powder injection of the present invention, the acid is used in an amount ranging from 20 to 80% by weight, preferably in the range of from 40 to 60%.

 The powder injection of the present invention has a pH of 1 to 4, preferably 2-3 after dissolution.

 The powder injection of the present invention is suitable for use as a medicament for treating cow mastitis.

 The present invention also relates to a method of preparing the above powder injection, comprising the steps of: mixing a powder of nisin powder and a physiologically acceptable acid under aseptic conditions.

 The advantages and positive effects of the Nisin powder of the present invention are as follows: (1) The Nisin powder of the present invention is stored at room temperature for one year, and the potency is not significantly lowered; (2) Since the powder contains an acidic substance such as vitamin C, in water or physiological In saline, Nisin is easy to dissolve and is convenient for clinical application. (3) The main component of the preparation is nisin, which is used to treat milk mastitis. It does not form harmful residues in milk, and there is no withdrawal period after treatment. , no need to abandon milk.

 Description of "potency of nisin": "The titer of nisin" is used to measure the antibacterial activity of Nisin, expressed in international units. The World Health Organization Expert Committee in Biological Standardization defines a standard substance containing 25 mg of pure Nisin per gram, equivalent to 1 million international units. This standard substance was used as a control when detecting Nisin activity. Example 1: A method for preparing a nisin powder having a titer of 3 million units/vial includes the following steps:

(1) Mix 10 g of vitamin C and 10 g of potency at 15,000 units/mg nisin by aseptic method;

(2) The mixed powder of (1) is dispensed in 50 sterilized glass ampoules of 5 to 20 ml in an aseptic manner at 400 mg per serving. Example 2: A method for preparing a nisin powder having a titer of 2 million units/vial comprises the following steps: (1) 14 g of vitamin C and 10 g of a lactic acid having a titer of 15,000 units/mg aseptically. Chain Fully mixed with colistin;

 (2) The mixed powder of (1) is dispensed into 75 sterilized glass ampoules of 5 to 20 ml in an aseptic manner in an amount of 320 mg per serving. 'Example 3: A method for preparing a nisin powder having a titer of 4 million units/vial includes the following steps:

(1) 100 g of vitamin C and 100 g of a titer of 20,000 units/mg of nisin are thoroughly mixed by aseptic method;

 (2) The mixed powder of (1) is dispensed into 500 sterilized glass ampoules of 5 to 20 ml in an aseptic manner at 400 mg per serving. Example 4: A method for preparing a nisin powder having a titer of 4 million units/vial includes the following steps:

(1) 100 g of citric acid and 100 g of a titer of 20,000 units/mg of nisin are thoroughly mixed by aseptic method;

 (2) Dispense the mixed powder in (1) in 400 sterilized glass ampoules with a capacity of 5 to 20 ml in an aseptic manner at 400 mg per serving. Example 5: A method for preparing a nisin powder having a titer of 4 million units/vial includes the following steps -

(1) 100 g of malic acid and 100 g of 20,000 gram/mg nisin are thoroughly mixed by aseptic method;

 (2) The mixed powder of (1) is dispensed into 500 sterilized glass ampoules of 5 to 20 ml in an aseptic manner at 400 mg per serving. Observation Example 1 Stability of different Nisin mastitis infusions

 (1) Purpose of the experiment

The stability of storage of different nisin breast perfusions No. 1, No. 2, No. 3 and No. 4 at 4 ° C, 25 ° C and room temperature was observed. Table 1. Different breast perfusion formulations

 (2) Storage conditions

 The samples of the above four formulations were placed under the conditions of temperature 25 ± 2 ° C, 4 Ό and room temperature (ie placed indoors). After the monthly inspection, the inspection was carried out once a month, and every 3 months after 3 months, A total of 2 years of investigation.

 (3) Test items and methods

 Test items: nisin titer, pH.

 Detection method: According to the national standard GB2760-86 nisin potency test method to detect nisin titer, using a common pH meter to detect the PH value of the solution.

 (4) Results of the investigation

 Table 2. PH values of each formula

 Type 1 No. 2 No. 3 No. 4

 ι, ί\ 4°C 25 °C Room yi3⁄4 4°C 4°C 4°C 25 °C Room Temperature

0 2.21 2.21 2.21 2.64 2.50 2.78 2.78 2.78

January 2.36 2.42 2.35 2.52 2.86 2.65 2.82 2.69

February 2.78 2.24 2.23 3.06 3.59 2.71 2.72 2.72

March 2.24 2.35 2.30 3.64 3.94 2.71 2.74 2.74

June 3.65 4.36 2.46 3.42 4.42 2.68 2.71 2.78 Table 3. Potency of each formula (unit: IU/ml)

 No. 1 No. 2 No. 3 No. 4

 Time Ι, ί\ 4°C 25 V Room 4°C 4°C 4. C 25. Room C y

 0933 99771 99771 99771 88348 90082 72125 72125 72125

January 85688 73983 51828 58239 68377 70392 68392 69586

February 82097 70984 47904 66444 57313 71657 68631 69595

March 61005 59963 46714 62303 55284 71447 70756 70950

June 59103 59063 46285 47793 46152 69960 66158 69534 (v) Conclusion

 Powders are stable in pH and potency under the three storage conditions, and liquid formulations have unstable effects such as reduced nisin titer and increased pH, regardless of the solvent used. The medium nature is the most stable. Observation Example 2 Therapeutic effect of Nisin mastitis infusion on clinical mastitis in dairy cows

1 Materials and methods

1.1 Test drug

 Breast infusion No. 2 (powder needle) was the product prepared in the above Example 2. Test Ranch conventional antibiotic preparations: All are commercially available products.

1.2 Test animals and their grouping

 There were 54 natural cases of clinical mastitis in dairy farms in the suburbs of Hangzhou, which were divided into experimental group and control group.

1.3 Test plan

 1.3.1 Before treatment Aseptically, collect the milk sample from the diseased milk area and isolate and identify the bacteria. The specific operating procedures are as follows: ' 1 milk sample inoculated blood agar plate, 37 ° C constant temperature culture 241! ~ 48h, observe bacterial growth and perform colony counting.

 2 Pick a single colony suspected of pathogenic bacteria and inoculate ordinary broth. Incubate at 37 °C for 18~24h to observe the growth of bacteria.

 3 smear the broth culture in step 2, Gram stain microscopy.

 4 According to the results of the above 3 steps, the species of the bacteria can be initially determined: Staphylococcus, Streptococcus, Enterobacter.

5 broth cultures were inoculated on a nutrient agar plates, and cultured for 24 hours at 37 °C for biochemical identification. The bacterial species were determined according to the relevant literature.

 1.3.2 Treatment experiment Intramaural perfusion of breast infusion in the experimental group No. 2, 1 bottle / time, 2 times / day; dissolve with 20mL sterile saline before injection, shake well. The control group was treated with conventional antibiotics in the pasture, mainly penicillin, streptomycin, gentamicin, erythromycin and sulfadiazine.

1.3.3 After the treatment, the first and second weeks after treatment, the milk samples from the milk group of the test group and the control group were aseptically processed, and the HMT (Hangzhou dairy cow mastitis diagnostic reagent) test, milk cell count and bacteriological examination were performed respectively. . 1.3.4 Susceptibility test For the Staphylococcus aureus isolated from the milk sample, the drug sensitivity test was carried out with penicillin G susceptibility paper (batch number 20040218, product of Hangzhou Tianhe Microbial Reagent Co., Ltd.). 1.4 Judging criteria

 1.4.1 Determination criteria for treatment effect

 1 Complete cure: The clinical symptoms such as redness, swelling and heat pain in the diseased milk area completely disappeared, the lactation basically returned to normal, and no pathogenic bacteria were detected in the milk sample;

 2 Clinical cure: The clinical symptoms such as redness, swelling and heat pain in the diseased milk area disappear completely, the lactation is basically restored to normal, and the pathogenic bacteria can be detected in the milk sample;

 3 Invalid: The clinical symptoms of the diseased dairy area did not significantly improve or aggravate, or re-occurred within two weeks after treatment, both of which were considered ineffective.

 1.4.2 Milk sample HMT test results judgment standard

 1 Negative (1) : Evenly mixed, no precipitation occurs;

 2 Suspicious (Shi): A small amount of precipitate appears, disappears after shaking;

3 weak positive (+) _: a small amount of precipitate appeared, disappeared after shaking;

 4 positive ( + + ) : There is a lot of gelatinous appearance, agglutination to the center after agitation;

 5 Strong positive ( + + + ) : Rapid formation of a gelatinous precipitate, which bulges when it shakes and sticks to the bottom of the disk.

2 Results and discussion

 2.1 The clinical cure rate of Nisin in the treatment of mastitis is higher than that of conventional antibiotics. 39 breasts (43 breast areas) were treated with breast perfusion No. 2, and the average number of treatments per breast area was 3.8. No adverse reactions were found after treatment. 23 breast areas were completely cured, the cure rate was 53.5%, and 38 breasts were cured clinically. District, the clinical cure rate was 88.4%; the control group treated 15 (19 breast areas), the average number of treatments per breast area was 5.1, completely cured 10 breast areas, the cure rate was 52.6%, clinically cured 12 breast areas, clinical The cure rate was 63.2%. After statistical analysis, there was no significant difference in the cure rate between the two groups (P>0.05), but the clinical cure rate of the test group was significantly higher than that of the control group (P<0.05).

 Table 1 Clinical mastitis treatment results

2.2 Bacterological changes before and after treatment in the infected area. For the 43 infected areas, the bacteriological examination of the milk samples was performed before and 1 and 2 weeks after treatment, including 32 milk areas in the test group and 11 milk in the control group. District, see Table 2. The bacterial conversion rate of the experimental group was lower than that of the control group, which may be due to the fact that the clinical symptoms disappeared quickly in cases treated with nisin. The medication was stopped very quickly, and the number of treatments was small (average 3.8), but the pathogens were not cleared from the affected area.

 Number of milk areas detected before and after treatment

 * Check for pre-treatment.

 2.3 After treatment, the milk sample Hangzhou mastitis diagnostic reagent test results are shown in Table 3. There was no significant difference in the proportion of positive (++) and strong positive (+++) areas between the two groups.

 Table 3 Results of milky recessive mastitis in the milk area after treatment, Unit: Number of tested milk areas (%)

* There are 2 breast areas with clinical mastitis. 2.4 The results of the detection of the number of somatic cells (SCC) after treatment are shown in Table 4. There was no significant difference in the proportion of milk cells with a somatic cell content of more than 500,000/ml.

 Results of the number of somatic cells in the milk area after treatment, unit: number of detected milk areas (%)

 * Two of the breast areas have clinical mastitis; the percentages in parentheses indicate the total treated area.

 2.5 Penicillin G Sensitivity Test The penicillin G susceptibility test was performed on the isolated Staphylococcus aureus. The results showed that all the isolated Staphylococcus aureus were resistant to penicillin.

 3 Conclusion

 Using nisin to treat clinical mastitis in dairy cows, the clinical cure rate is lower than that of conventional antibiotics, but there is little difference between the removal of pathogenic bacteria and the reduction of the number of milk somatic cells.

Claims

A powdered breast infusion for treating cow mastitis comprising nisin and a physiologically acceptable acid.  2. The powdered breast-filling agent according to claim 1, wherein each dose of the powdered breast-filling agent comprises nisin having a titer of 200,000 to 12,500,0 00 units.  3. The powdered breast infusion of claim 2, wherein each dose of powdered breast infusion comprises a potency of from 1,000,000 to 5,000,000 units.  4. The powdered breast infusion according to claim 1, wherein the titer of nisin is 10,000-250,000 when each dose of the powdered breast infusion is completely dissolved in 20 to 50 ml of water or physiological saline. Unit / ml.  5. The powdered breast infusion according to claim 4, wherein the titer of nisin is 50,000 when each dose of the powdered breast infusion is completely dissolved in 20 to 50 ml of water or physiological saline. 100,000 units / ml.  The powdered breast infusion agent according to claim 1, wherein the powdered breast infusion agent has a pH of 1 to 4 after dissolution.  7. The powdered breast-filling agent according to claim 6, wherein the powdered breast infusion agent has a pH of 2-3 after dissolution.  8. The powdered breast infusion of claim 1, wherein the physiologically acceptable acid is selected from the group consisting of vitamin C, citric acid, malic acid, citric acid, tartaric acid, oxalic acid, oxalic acid, succinic acid, Glutaric acid, boric acid, acetic acid, and mixtures thereof.  9. The powdered breast infusion of claim 1 for use as a medicament for the treatment of cow mastitis. 10. A method of preparing a powdered breast-filling agent according to claim 1, comprising the step of: mixing the nisin powder with a physiologically acceptable acid powder under sterile conditions.