复合蛋白酶乳房注入剂在奶牛的药动学、药效学及安全性试验研究

发布时间：2018-06-05 23:23

本文选题：胰蛋白酶 + 糜蛋白酶　；参考：《南京农业大学》2015年硕士论文

【摘要】：胰蛋白酶、糜蛋白酶作为治疗炎症的酶类药物,在国外临床上已得到广泛应用。鉴于胰蛋白酶和糜蛋白酶在人医临床上的广泛应用,我们拟尝试使用以胰蛋白酶和糜蛋白酶为主要组份的复合蛋白酶乳房注入剂治疗奶牛乳房炎,本研究依据农业部《兽药研究技术指导原则编汇》进行了该制剂在奶牛体内的药动学、药效学和安全性试验研究,为该制剂在兽医临床上的使用积累试验和理论依据。为了研究复合蛋白酶乳房注入剂在奶牛体内药代动力学特征,获取该药的试验药代参数,为该药在兽医临床上的使用提供剂量和方法保证,本试验进行了复合蛋白酶乳房注入剂在奶牛(牛奶)的药代动力学研究。试验结果显示,在牛奶中胰蛋白酶最低检测限浓度为72 mu/mL,平均回收率为71.9%;糜蛋白酶最低检测限浓度142 mu/mL,平均回收率为70.1%。同时依据本试验建立的检测方法,选取受试奶牛,于每个乳区注入1支复合蛋白酶乳房注入剂,采集给药乳区牛奶。结果显示,受试奶牛乳区中胰蛋白酶活性在乳房注入12 h后趋于0或者降低到检测限以下,连续检测24 h、36 h胰蛋白酶活性仍然低于检测限;受试奶牛乳区中糜蛋白酶活性在乳房注入后8 h降低到检测限以下,连续检测12 h、24 h糜蛋白酶活性仍低于检测限。胰蛋白酶在受试奶牛乳区中消除速度相对较慢、药效时间长,给药间隔可以以胰蛋白酶消除时间作为依据,建议给药间隔为12 h。为了评价复合蛋白酶乳房注入剂对临床型奶牛乳房炎的治疗效果、确定其临床使用剂量等问题,本试验通过对乳房炎患牛体细胞、奶牛乳房细菌分离鉴定以及其它临床疗效指标的检测和观察,评估了复合蛋白酶注入剂对临床型奶牛乳房炎的治疗效果。试验结果显示,按照1支/乳区·次,连续用药3次,每次间隔12 h给药的临床使用方案,复合蛋白酶乳房注入剂(糜蛋白酶8 mg+胰蛋白酶8 mg组)剂量组制剂对临床型奶牛乳房炎的有效率达到88.2%,治愈率高达70.6%;对患病乳区病原菌菌株数清除作用减少50%以上;奶牛乳房炎评分均值从2.80提升到4.30;体细胞均值从209.80万个/mL降低到125.94万个/mL。建议使用主药含量为糜蛋白酶8 mg+胰蛋白酶8 mg剂量的复合蛋白酶乳房注入剂作为临床推荐剂量。作为一种酶制剂用做奶牛乳房炎的治疗药物,复合蛋白酶乳房注入剂为避免细菌耐药性和药物残留等问题,为复合蛋白酶制剂的开发和应用于治疗奶牛乳房炎提供了新思路。为了解泌乳期奶牛对复合蛋白酶乳房注入剂的耐受程度(最大耐受剂量),明确超过推荐剂量使用复合蛋白酶乳房注入剂后可能对受试奶牛造成的包括临床症状、乳房局部症状以及对血液、体细胞数的影响和危害,本试验利用不同剂量的复合蛋白酶乳房注入剂对奶牛进行了靶动物安全性试验评估。试验结果显示,分别按照推荐剂量的1、3、5倍剂量为受试奶牛乳区给药,所有受试奶牛均未观察到明显的不良临床表现。受试奶牛血液常规和血液生化指标在给药前后均处在奶牛正常指标范围之内;分别在受试奶牛给药的0d、3d、5 d、10 d采集奶样进行体细胞检测,发现3×推荐剂量组、5×推荐剂量组药物对奶牛乳房有一定程度的刺激作用,且5×推荐剂量组药物比3×推荐剂量组药物对奶牛乳房的刺激作用更大,建议临床用药时不要一次性使用超过3倍剂量的药物;尽管3×推荐剂量和5×推荐剂量组药物在给药3 d和5 d后受试奶牛乳汁中的体细胞数高于50万个/mL,但给药10 d后乳汁中体细胞数降至20万个/mL左右,说明该药物会在短时间内引起奶牛乳中体细胞数量的上升,药效结束后,奶牛体细胞数量会降至给药前水平,刺激强度可为奶牛所承受,该药物在临床上使用时安全性较好。
[Abstract]:Trypsin, chymotrypsin, as an enzyme drug for the treatment of inflammation, has been widely used in clinic abroad. In view of the widespread application of trypsin and chymotrypsin in human medical clinic, we intend to try to treat dairy cow mastitis with trypsin and chymotrypsin as the main component of compound protease breast injecting agent. In order to study the pharmacokinetic characteristics of the compound proteinase breast injecting agent in the cow body, the experiment of pharmacokinetics, pharmacodynamics and safety tests of the preparation in dairy cows was carried out according to the compilation of the guiding principles of veterinary medicine research technology in the Ministry of agriculture. The pharmacokinetics of the compound proteinase breast injecting agent in dairy cows (milk) was studied in this experiment. The test results showed that the lowest detection limit of trypsin in milk was 72 mu/mL, the average recovery rate was 71.9%, and the minimum detection limit of chymotrypsin was concentrated. The average recovery rate was 142 mu/mL, and the average recovery rate was 70.1%. and the test method established in this experiment was selected. The tested dairy cows were selected and 1 compound proteinase breast injections were injected into each dairy area to collect milk. The results showed that the trypsin activity in the dairy area of the dairy cows tended to 0 or lower to the detection limit after the breast injection 12 h. The activity of trypsin was still lower than the detection limit of 24 h and 36 h, and the activity of chymotrypsin decreased to below the detection limit after the breast injection of 8 h, 12 h was continuously detected, and 24 h chymotrypsin was still lower than the detection limit. It is suggested that the interval of the protease elimination is 12 h. In order to evaluate the therapeutic effect of the compound proteinase breast injecting agent on the clinical dairy cow mastitis, to determine the clinical dosage and so on. The therapeutic effect of compound proteinase injecting agent on the clinical Dairy Mastitis was evaluated. The results showed that the clinical use of the compound protease breast injecting agent (chymotrypsin 8 mg+ trypsin 8 mg group) was used for the clinical dairy cow mastitis according to the clinical use of 1 consecutive doses of 1 branches / milky areas and 12 consecutive intervals. The effective rate was 88.2%, the cure rate was up to 70.6%, the scavenging effect of the pathogenic bacteria number of the infected dairy region was reduced by more than 50%, the mean value of the cow mastitis score increased from 2.80 to 4.30, the mean of the body cell mean from 2 million 98 thousand /mL to 1 million 259 thousand and 400 /mL. suggested that the compound proteinase breasts of the main drug content was the chyloase 8 mg+ trypsin 8 mg dose. Injecting agent as a clinical recommended dose. As a kind of enzyme preparation for dairy cow mastitis, compound proteinase breast injecting agent is to avoid bacterial resistance and drug residue. It provides a new idea for the development and application of compound protease preparation for the treatment of dairy cow mastitis. The tolerance level of the breast injecting agent (maximum tolerable dose), clearly beyond the recommended dosage of the compound proteinase breast injecting agent, may include the clinical symptoms, the local symptoms of the breast, the influence and harm to the blood, the number of somatic cells, and the use of different doses of compound proteinase breast injecting agent in this experiment. The test results of the target animal safety test were conducted. The results showed that all the dairy cows were not observed the obvious adverse clinical manifestations according to the 1,3,5 times of the recommended dose, and the blood routine and blood biochemical indexes of the tested cows were within the normal range of the dairy cows before and after the Administration. The 0d, 3D, 5 d, 10 d collected milk samples were tested by the tested cows. The 3 x recommended dose group was found. The 5 x recommended dose group had a certain degree of stimulation to the cow breasts, and the 5 * recommended dose group was more stimulative to the cow breasts than the 3 x recommended dose group. It is suggested that the drug should not be used once in the clinical medication. More than 3 times the dosage of the drug; although the number of somatic cells in the milk of the dairy cows was higher than 500 thousand /mL after the 3 * recommended dose and the 5 * recommended dose group was 3 D and 5 d, the number of somatic cells in the milk was reduced to about 200 thousand /mL after the administration of 10 d, indicating that the drug could cause the increase of the number of somatic cells in the milk of dairy cows and the end of the drug effect in a short time. After that, the number of somatic cells of cows will drop to the level before administration, and the stimulation intensity can be taken by cows. The drug is safe in clinical use.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.7

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