艾地普林临床前毒理学研究

发布时间：2018-04-20 11:03

  本文选题：艾地普林 + 急性毒性　； 参考：《华中农业大学》2014年硕士论文

【摘要】：二氨基嘧啶类药物是一类本身有抗菌活性且具有抗菌增效作用的药物,常见的有甲氧苄啶、二甲氧苄啶、巴喹普林和奥美普林等。艾地普林(Aditoprim, ADP)是本类的新药,在国内由本实验室率先开展系统研究。本课题立足实验室前期研究成果,通过系统的毒理学试验研究,对艾地普林的安全性进行评价,为后期的临床研究提供参考资料。1急性毒性试验选用SPF级Wistar大鼠和昆明小鼠雌雄若干,采用上下法(UDP)进行试验。经口灌喂艾地普林混悬液染毒,短期观察决定上下剂量顺序,存活动物观察14天,记录中毒反应及死亡情况,最终利用AOT 425软件计算结果。试验得出Wistar大鼠口服艾地普林的半数致死量(LD5o)为1400 mg/kg b.w.,95%可信限为954.9到1750mg/kg b.w.;在昆明小鼠的LD50为1130 mg/kg b.w.,95%可信限为859.2到1900 mg/kgb.w.。艾地普林急性毒性分级为低毒。2遗传毒性试验细菌回复突变试验试验选用鼠伤寒沙门氏菌株分别为TA97a, TA98,TA100,TA102和TA1535,艾地普林剂量分别为1.0、0.5、0.25、0.125和0.0625μg/皿,同时设阳性对照组和阴性对照组。在加和不加S9代谢活化系统条件下检测受试物的致突变性。试验采用平皿掺入法,计数各皿回变菌落数,计算各剂量组与阴性对照组回变菌落数比值。结果显示,不论加与不加S9代谢活化系统,艾地普林在0.0625-1.0μg/皿的剂量范围内,各菌株回变菌落数与阴性对照组的比值均小于2,结果为阴性。说明艾地普林在本试验条件下不具有致突变性。小鼠骨髓细胞微核试验选用7-8周龄SPF级昆明小鼠随机分组,每组雌雄各5只,艾地普林剂量分别为141.5、282.5和565 mg/kg b.w.,同时设置阴性对照组和阳性对照组。经口染毒2次(阳性对照组一次腹腔注射),间隔24 h,第二次给药6h后,小鼠脱颈椎处死,采股骨骨髓样制片。每只动物计嗜多染红细胞(PCE)1000个计算含微核的嗜多染红细胞(MNPCE)数。在计数PCE时,计数见到的正染红细胞(NCE),求出PCE/NCE的比值。结果显示,与阴性对照组相比,艾地普林各剂量组微核发生率没有显著上升(p0.05)。说明艾地普林在体内微核试验结果中为阴性,不具体内致突变能力。体外哺乳动物细胞染色体畸变试验 以中国仓鼠肺成纤维细胞(V79)为研究对象,艾地普林剂量分别为300、150、75和37.5μg/mL,并设阴性对照组和阳性对照组。试验分别在加和不加S9代谢活化系统条件下进行,不加S9时,受试物分别作用6h和18 h；加S9时,受试物作用6 h,培养至24 h制片。每一剂量组计数200个中期分裂相细胞,计算细胞染色体畸变率。试验结果显示,艾地普林各剂量组在加与不加S9时,细胞畸变率与阴性对照组没有显著差异(p0.05),且均小于5%,结果判定为阴性。表明艾地普林在本试验条件下不会引起V79细胞的染色体畸变。体外哺乳动物细胞基因突变试验以中国仓鼠卵巢细胞(CHO-K1)为研究对象,在加与不加S9代谢活化系统条件下,观察艾地普林对CHO-K1细胞的致突变作用。艾地普林的浓度分别为300、150、75和37.5 μg/mL,同时设阴性对照组和阳性对照组。经处理6h后,采用克隆形成法观察艾地普林对CHO-K1细胞的毒性作用,并在此基础上观察其对CHO-K1细胞hgprt基因位点突变频率的影响。结果显示,不论加与不加S9代谢活化系统,艾地普林在37.5-300 μg/mL的剂量范围内,对CHO-K1细胞hgprt基因的突变率与阴性对照组相比差异不显著(p0.05)。说明,在本试验条件下,艾地普林对CHO-K1细胞无诱变作用。小鼠睾丸染色体畸变试验选用体重30 g左右SPF级昆明小鼠随机分组,艾地普林剂量分别为141.5、282.5和565 mg/kg b.w.,同时设置阴性对照组和阳性对照组,保证采样时每组至少5只存活。经口染毒(阳性对照组腹腔注射),每天一次,连续5天,第12天处死小鼠。处死前6h注射秋水仙素,采睾丸制备染色体片,每只动物分析100个中期分裂相细胞,计算细胞染色体畸变率。试验结果显示,与阴性对照组相比,艾地普林各剂量组细胞染色体畸变率没有显著差异(p0.05),说明艾地普林不具有体内的致生殖细胞染色体畸变效应。390天喂养试验选用5-6周龄SPF级Wistar大鼠作为试验动物,通过混饲方式染毒13周,最高剂量组有2周恢复期,剂量设置分别为艾地普林(ADP)0、20、100和1000 mg/kg饲料。空白对照组和艾地普林1000 mg/kg饲料组每组雌雄各20只；艾地普林20和100 mg/kg饲料组每组雌雄各15只,饲养条件符合国际动物福利规定。每日观察并在每周固定时间称量大鼠体重及饲料剩余,计算摄食量及饲料利用率。分别在试验45天和90天宰杀各组雌雄大鼠各5只和10只,取血样进行血液学和血清生化指标检验,尸体作病理剖检,主要脏器称重后作组织病理学检查。剩余大鼠饲喂空白饲料,两周恢复期后作上述同样处理。试验结果显示,在艾地普林最高剂量1000 mg/kg饲料组,大鼠体重显著低于空白组p0.05),艾地普林1000 mg/kg饲料组雌性大鼠肝脏、肾脏脏体比显著大于空白对照组(p0.05)；雄性大鼠睾丸(带附睾)脏体比显著大于空白对照组p0.05)。血常规和血清生化检验结果显示,与空白组相比,艾地普林100和1000 mg/kg饲料组总蛋白(TP)、白蛋白(ALB)、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)出现显著升高(p0.05),且超出正常生理范围。组织病理学检查发现,高剂量组大鼠肝脏门管区出现大量炎性细胞浸润,周围有肝细胞变性坏死,病变典型且发生率显著高于空白组。艾地普林20 mg/kg饲料组在以上观察指标中未出现异常,每天实际摄入药物量为1.44 mg/kg b.w.。综合各项指标结果表明,艾地普林对Wistar大鼠亚慢性毒性作用的靶器官为肝脏,最大未观察到有害作用剂量(NOAEL)为1.44 mg/kg b.w.4二代繁殖试验试验动物选用SPF级Wistar大鼠,设空白对照组和艾地普林试验组,剂量为艾地普林20、100和1000 mg/kg饲料。第一代(Fo代)每组雌鼠25只(以保证每组有20只受孕雌鼠),雄鼠18只；第二代(F1代)每组雌鼠25只,雄鼠20只。连续染毒13周后,各组按雌雄2：1或1：1进行交配。整个试验期间对F0代和F1代大鼠一般毒性的观察同90天喂养试验,另外统计繁殖性能相关的各项指标。试验结果显示,艾地普林1000 mg/kg饲料组F0代和F1代大鼠体重显著性低于空白对照组；F1代窝平均活仔数和第21天平均仔重显著低于空白对照组p0.05),F2a代窝平均活仔数和第4和21天平均仔重显著低于空白对照组(p0.05)。各组雌雄大鼠主要脏器病理组织学检查结果同90天喂养试验一致,生殖器官病理组织学检查均正常。以上结果表明艾地普林在高剂量下(1000 mg/kg饲料)有轻微的母体毒性,并对后代有轻微的发育毒性。大鼠生殖发育毒性的NOAEL为7.89 mg/kg b.w..5喂养致畸试验喂养致畸试验联合二代繁殖试验Fl代进行,在F2a代断奶后,Fl代雌鼠与雄鼠再次交配,在妊娠第20天时进行剖腹检查畸胎。试验结果显示艾地普林1000 mg/kg饲料组平均着床数、出生活胎数、胎仔体重以及体长和尾长均显著低于空白对照(p0.05)。各组均未出现明显的与药物相关的胎儿内脏畸形或骨骼畸形。结果表明艾地普林在1000 mg/kg饲料剂量时有轻微的母体毒性以及胚胎毒性,但未表现出致畸效应。所以艾地普林致畸试验NOAEL为8.75 mg/kg b.w.。6计算每日容许摄入量(ADI)及安全浓度(SC)综合各项试验结果,得到ADP的NOAEL为1.44 mg/kg b.w.,按照FDA给出的计算公式,得出ADP的ADI为0.00144 mg/kg b.w.,在动物可食性组织(肌肉、肝脏、肾脏和脂肪)中的SC分别为0.29,0.86,1.73和1.73 mg/kg。本课题完成了艾地普林的急性毒性、遗传毒性、亚慢性毒性以及繁殖和致畸毒性试验,得到了艾地普林一般毒性、繁殖毒性和致畸毒性的NOAEL,确定了艾地普林毒性作用的靶器官,证实了艾地普林不具有致突变性。所有试验均参考国内外相关的权威指南,并结合药物的自身特点合理选择试验组合,本研究成果可为艾地普林后期的临床研究提供相关的科学资料。
[Abstract]:Two amino pyrimidine is a class of drugs that have antibacterial activity and have an antibacterial synergistic effect. It is common to have trimethoprim, two trimethoprim, basquine and olmopin. Aditoprim (ADP) is a new drug in this class. Fruit, the safety of alprinoline was evaluated through systematic toxicological test, and a reference material for the later clinical study was provided for.1 acute toxicity test of SPF grade Wistar rats and Kunming mice and male and female mice. The experiment was carried out by using the upper and lower methods (UDP). In order, the survival animals were observed for 14 days, recording the toxic reaction and death, and finally using AOT 425 software. The results showed that half of the lethal dose (LD5o) of oral alprinoline in Wistar rats was 1400 mg/kg b.w., 95% confidence limit was 954.9 to 1750mg/kg b.w., and the LD50 in Kunming mice was 1130 mg/kg b.w., and 95% confidence limit was 859.2 to 1900 mg/k. Gb.w.. Alprin's acute toxicity grade was classified as low toxic.2 genotoxicity test for bacterial response mutation test. The strains of Salmonella typhimeni were selected as TA97a, TA98, TA100, TA102 and TA1535 respectively. The dosage of alprin was 1.0,0.5,0.25,0.125 and 0.0625 UA respectively, and the positive control group and negative control group were set up at the same time. Test the mutagenicity of the subject matter under the condition of the chemical system. The test used the method of Petri dish incorporation to count the number of the colonies in each dish, and calculated the ratio of the number of the back colonies in the negative control group. The results showed that the number of the strains and the negative colonies of all the strains were in the dose range of the 0.0625-1.0 g/ dish, regardless of the addition or or without the metabolic activation system. The ratio of the control group was less than 2, and the results were negative. It showed that alprinoline had no mutagenicity under the test conditions. The micronucleus test of bone marrow cells of mice was randomly divided into 7-8 weeks old SPF Kunming mice, 5 rats in each group, and 141.5282.5 and 565 mg/ kg b.w. respectively, and the negative control group and the positive pair were set up at the same time. After 2 times (one abdominal injection of positive control group), 24 h interval and second doses of 6h, the mice were removed from the cervical vertebra and took the femur myeloid production. Each animal calculated the number of polychromatic erythrocytes (MNPCE) containing micronucleus (PCE). The number of positive red cells (NCE) was counted and PCE/NCE was counted at the count of PCE. The ratio. Results showed that the incidence of micronucleus in every dose group of alprunin did not rise significantly (P0.05), indicating that alprunp was negative in the results of micronucleus test in vivo and had no specific mutagenicity. In vitro mammalian cell chromosome aberration test was studied by Chinese hamster lung fibroblast cells (V79). The dosage of ground Prine was 300150,75 and 37.5 g/mL respectively, and the negative control group and positive control group were set up. The experiment was carried out under the condition of adding or without S9 metabolism activation system, and the subjects acted as 6h and 18 h without S9. When adding S9, the subjects acted 6 h and cultured to 24 h. Each dose group counted 200 metaphase cells, calculated The cell aberration rate. The results showed that the cell aberration rate was not significantly different from that of the negative control group (P0.05) when the dose group was added or not with S9 (P0.05), and the results were less than 5%, and the results were negative. It showed that alprinoline did not cause chromosome aberration of V79 cells in this test. The mutation test was conducted in Chinese hamster ovary cells (CHO-K1). The mutagenic effect of alprin on CHO-K1 cells was observed under the condition of adding or without S9 metabolism activation. The concentration of alprin was 300150,75 and 37.5 mu g/mL respectively, and the negative control group and positive control group were set up at the same time. After the treatment of 6h, the clone formation method was used to observe the results. The effect of alprin on CHO-K1 cells was observed and the effect on the mutation frequency of HGPRT gene loci in CHO-K1 cells was observed on this basis. The results showed that the mutation rate of the HGPRT based factor of CHO-K1 cells was not significantly different from that of the negative control group, regardless of the addition or non S9 metabolic activation system, in the dose range of 37.5-300 micron g/mL. (P0.05). In this experiment, it was indicated that alprin had no mutagenesis on CHO-K1 cells. The test of testicular chromosome aberration in mice was randomly divided into 30 g SPF Kunming mice, and the dose of alprin was 141.5282.5 and 565 mg/kg b.w. respectively. At the same time, the negative control group and the positive control group were set up to ensure that each group was at least 5 in each group. Only surviving. The mice were sacrificed once a day for 5 days and twelfth days after 5 days. The chromosomes were prepared by injection of colchicine and testis before death. 100 metaphase mitotic cells were analyzed in each animal and the chromosome aberration rate was calculated. The results showed that the dose of alprinoline was compared with the negative control group. There was no significant difference in chromosome aberration rate in the group (P0.05), indicating that alprin did not have the effect of chromosomal aberration in the germ cells in the body. The.390 day feeding test of 5-6 weeks old SPF Wistar rats was selected as experimental animal, and it was poisoned by mixed feeding. The maximum dose group had 2 weeks of recovery period, and the dosage was set to alprinoline (ADP) 0,2, respectively. 0100 and 1000 mg/kg feed. 20 in each group of the blank control group and alprin 1000 mg/kg feed group; 15 in each group of alprin 20 and 100 mg/kg feed group. The feeding conditions were in accordance with the international animal welfare regulations. The weight and feed surplus of rats were weighed daily and at the fixed time of the week, and the intake and feed utilization rate were calculated. After 45 days and 90 days of trial, 5 and 10 rats were slaughtered in each group. Blood samples were taken for hematology and serum biochemical indexes. The corpses were examined for pathological examination. After the main organs were weighed, the histopathological examination was performed. The remaining rats were fed blank feed and the same treatment was made after two weeks of recovery. The results showed that in the alprin, the test results showed that in the best alprin The weight of the rats in the high dose 1000 mg/kg diet group was significantly lower than that of the blank group P0.05). The liver of the female rats in the alprin 1000 mg/kg diet group was significantly greater than that in the blank control group (P0.05), and the ratio of the testis (epididymis) in the male rats was significantly greater than that of the blank control group P0.05). The results of blood routine and serum biochemical test showed that the group was with the blank group. In comparison, the total protein (TP), albumin (ALB), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the 100 and 1000 mg/kg groups of alprinoline increased significantly (P0.05) and exceeded the normal physiological range. Histopathological examination found that a large number of inflammatory cells were infiltrated in the liver portal area of the high dose group and there were liver around them. The cell degeneration and necrosis were significantly higher than that in the blank group. There were no abnormalities in the 20 mg/kg feed group of alprinoline. The actual intake of drugs was 1.44 mg/kg b.w.. Every day. The target organ of alprin on the subchronic toxicity of Wistar rats was the liver, and the maximum was not observed. The harmful effect dose (NOAEL) was 1.44 mg/kg b.w.4 two generation test animals selected SPF Wistar rats, blank control group and alprin test group, the dose of alprinol 20100 and 1000 mg/kg feed. The first generation (Fo generation) female rats in each group of 25 (to ensure that each group of 20 pregnant females), male rats 18; second generation (F1 generation) each group of females. 25 rats and 20 male rats. After 13 weeks of continuous exposure, each group was copulated according to male and female 2:1 or 1:1. The general toxicity of F0 and F1 rats was observed during the whole trial and 90 days of feeding test, and the indexes related to reproductive performance were also measured. The results showed that the weight of F0 and F1 generation rats of alprin 1000 mg/ kg feed group was significantly lower In the blank control group, the average number of live litter in the F1 generation and the average weight of the twenty-first days was significantly lower than that of the blank control group (P0.05). The average number of live litter and the average weight of the fourth and twenty-first days in the F2a generation were significantly lower than that in the blank control group (P0.05). The results of the main organ histopathology examination of the female and male rats were the same as that of the 90 day feeding test, and the reproductive organs were histopathologically histopathologically studied. The results showed that the above results showed that alprinol had slight maternal toxicity at high dose (1000 mg/kg feed) and slightly developmental toxicity to offspring. The NOAEL of reproductive development toxicity of rats was 7.89 mg/kg b.w..5 feeding teratogenicity test combined with two generation reproduction test Fl generation, and after F2a generation weaned, Fl generation female mice and The male rats were again mating and undergoing a caesarean section at twentieth days of pregnancy. The results showed that the average implantation number, the number of living fetuses, the fetal weight, the body length and the tail length of the alprinole 1000 mg/kg diet group were significantly lower than that of the blank control (P0.05). The results of all groups did not appear to be evidently related to the visceral deformities or skeletal deformities associated with the drug. It showed that alprin had slight maternal toxicity and embryo toxicity at 1000 mg/kg feed dose, but did not show teratogenic effect. So the alpriner teratogenicity test NOAEL was 8.75 mg/kg b.w..6 for daily allowable intake (ADI) and safety concentration (SC) comprehensive test fruit, and NOAEL of ADP was 1.44 mg/kg b.w., according to FDA. The calculation formula showed that the ADI of ADP was 0.00144 mg/kg b.w., and the SC in animal edible tissues (muscle, liver, kidney and fat) was 0.29,0.86,1.73 and 1.73 mg/kg., respectively. The acute toxicity, hereditary toxicity, subchronic toxicity and reproductive and teratogenic toxicity test of alpralin were completed, and the general toxicity and propagation of alprin was obtained. The NOAEL of the toxic and teratogenic toxicity identified the target organs of the toxic effect of alprinoline, and confirmed that alprinoline did not have mutagenicity. All the tests refer to the relevant authoritative guidelines at home and abroad, and choose the test combination in combination with the characteristics of the drug. The results of this study can provide relevant clinical research for the later stage of alprinolin. Scientific data.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96
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本文编号：1777487