本文選題：艾地普林 + 急性毒性��； 參考：《華中農(nóng)業(yè)大學》2014年碩士論文

【摘要】：二氨基嘧啶類藥物是一類本身有抗菌活性且具有抗菌增效作用的藥物,常見的有甲氧芐啶、二甲氧芐啶、巴喹普林和奧美普林等。艾地普林(Aditoprim, ADP)是本類的新藥,在國內(nèi)由本實驗室率先開展系統(tǒng)研究。本課題立足實驗室前期研究成果,通過系統(tǒng)的毒理學試驗研究,對艾地普林的安全性進行評價,為后期的臨床研究提供參考資料。1急性毒性試驗選用SPF級Wistar大鼠和昆明小鼠雌雄若干,采用上下法(UDP)進行試驗。經(jīng)口灌喂艾地普林混懸液染毒,短期觀察決定上下劑量順序,存活動物觀察14天,記錄中毒反應及死亡情況,最終利用AOT 425軟件計算結(jié)果。試驗得出Wistar大鼠口服艾地普林的半數(shù)致死量(LD5o)為1400 mg/kg b.w.,95%可信限為954.9到1750mg/kg b.w.;在昆明小鼠的LD50為1130 mg/kg b.w.,95%可信限為859.2到1900 mg/kgb.w.。艾地普林急性毒性分級為低毒。2遺傳毒性試驗細菌回復突變試驗試驗選用鼠傷寒沙門氏菌株分別為TA97a, TA98,TA100,TA102和TA1535,艾地普林劑量分別為1.0、0.5、0.25、0.125和0.0625μg/皿,同時設陽性對照組和陰性對照組。在加和不加S9代謝活化系統(tǒng)條件下檢測受試物的致突變性。試驗采用平皿摻入法,計數(shù)各皿回變菌落數(shù),計算各劑量組與陰性對照組回變菌落數(shù)比值。結(jié)果顯示,不論加與不加S9代謝活化系統(tǒng),艾地普林在0.0625-1.0μg/皿的劑量范圍內(nèi),各菌株回變菌落數(shù)與陰性對照組的比值均小于2,結(jié)果為陰性。說明艾地普林在本試驗條件下不具有致突變性。小鼠骨髓細胞微核試驗選用7-8周齡SPF級昆明小鼠隨機分組,每組雌雄各5只,艾地普林劑量分別為141.5、282.5和565 mg/kg b.w.,同時設置陰性對照組和陽性對照組。經(jīng)口染毒2次(陽性對照組一次腹腔注射),間隔24 h,第二次給藥6h后,小鼠脫頸椎處死,采股骨骨髓樣制片。每只動物計嗜多染紅細胞(PCE)1000個計算含微核的嗜多染紅細胞(MNPCE)數(shù)。在計數(shù)PCE時,計數(shù)見到的正染紅細胞(NCE),求出PCE/NCE的比值。結(jié)果顯示,與陰性對照組相比,艾地普林各劑量組微核發(fā)生率沒有顯著上升(p0.05)。說明艾地普林在體內(nèi)微核試驗結(jié)果中為陰性,不具體內(nèi)致突變能力。體外哺乳動物細胞染色體畸變試驗 以中國倉鼠肺成纖維細胞(V79)為研究對象,艾地普林劑量分別為300、150、75和37.5μg/mL,并設陰性對照組和陽性對照組。試驗分別在加和不加S9代謝活化系統(tǒng)條件下進行,不加S9時,受試物分別作用6h和18 h；加S9時,受試物作用6 h,培養(yǎng)至24 h制片。每一劑量組計數(shù)200個中期分裂相細胞,計算細胞染色體畸變率。試驗結(jié)果顯示,艾地普林各劑量組在加與不加S9時,細胞畸變率與陰性對照組沒有顯著差異(p0.05),且均小于5%,結(jié)果判定為陰性。表明艾地普林在本試驗條件下不會引起V79細胞的染色體畸變。體外哺乳動物細胞基因突變試驗以中國倉鼠卵巢細胞(CHO-K1)為研究對象,在加與不加S9代謝活化系統(tǒng)條件下,觀察艾地普林對CHO-K1細胞的致突變作用。艾地普林的濃度分別為300、150、75和37.5 μg/mL,同時設陰性對照組和陽性對照組。經(jīng)處理6h后,采用克隆形成法觀察艾地普林對CHO-K1細胞的毒性作用,并在此基礎上觀察其對CHO-K1細胞hgprt基因位點突變頻率的影響。結(jié)果顯示,不論加與不加S9代謝活化系統(tǒng),艾地普林在37.5-300 μg/mL的劑量范圍內(nèi),對CHO-K1細胞hgprt基因的突變率與陰性對照組相比差異不顯著(p0.05)。說明,在本試驗條件下,艾地普林對CHO-K1細胞無誘變作用。小鼠睪丸染色體畸變試驗選用體重30 g左右SPF級昆明小鼠隨機分組,艾地普林劑量分別為141.5、282.5和565 mg/kg b.w.,同時設置陰性對照組和陽性對照組,保證采樣時每組至少5只存活。經(jīng)口染毒(陽性對照組腹腔注射),每天一次,連續(xù)5天,第12天處死小鼠。處死前6h注射秋水仙素,采睪丸制備染色體片,每只動物分析100個中期分裂相細胞,計算細胞染色體畸變率。試驗結(jié)果顯示,與陰性對照組相比,艾地普林各劑量組細胞染色體畸變率沒有顯著差異(p0.05),說明艾地普林不具有體內(nèi)的致生殖細胞染色體畸變效應。390天喂養(yǎng)試驗選用5-6周齡SPF級Wistar大鼠作為試驗動物,通過混飼方式染毒13周,最高劑量組有2周恢復期,劑量設置分別為艾地普林(ADP)0、20、100和1000 mg/kg飼料�？瞻讓φ战M和艾地普林1000 mg/kg飼料組每組雌雄各20只；艾地普林20和100 mg/kg飼料組每組雌雄各15只,飼養(yǎng)條件符合國際動物福利規(guī)定。每日觀察并在每周固定時間稱量大鼠體重及飼料剩余,計算攝食量及飼料利用率。分別在試驗45天和90天宰殺各組雌雄大鼠各5只和10只,取血樣進行血液學和血清生化指標檢驗,尸體作病理剖檢,主要臟器稱重后作組織病理學檢查。剩余大鼠飼喂空白飼料,兩周恢復期后作上述同樣處理。試驗結(jié)果顯示,在艾地普林最高劑量1000 mg/kg飼料組,大鼠體重顯著低于空白組p0.05),艾地普林1000 mg/kg飼料組雌性大鼠肝臟、腎臟臟體比顯著大于空白對照組(p0.05)；雄性大鼠睪丸(帶附睪)臟體比顯著大于空白對照組p0.05)。血常規(guī)和血清生化檢驗結(jié)果顯示,與空白組相比,艾地普林100和1000 mg/kg飼料組總蛋白(TP)、白蛋白(ALB)、丙氨酸氨基轉(zhuǎn)移酶(ALT)和天門冬氨酸氨基轉(zhuǎn)移酶(AST)出現(xiàn)顯著升高(p0.05),且超出正常生理范圍。組織病理學檢查發(fā)現(xiàn),高劑量組大鼠肝臟門管區(qū)出現(xiàn)大量炎性細胞浸潤,周圍有肝細胞變性壞死,病變典型且發(fā)生率顯著高于空白組。艾地普林20 mg/kg飼料組在以上觀察指標中未出現(xiàn)異常,每天實際攝入藥物量為1.44 mg/kg b.w.。綜合各項指標結(jié)果表明,艾地普林對Wistar大鼠亞慢性毒性作用的靶器官為肝臟,最大未觀察到有害作用劑量(NOAEL)為1.44 mg/kg b.w.4二代繁殖試驗試驗動物選用SPF級Wistar大鼠,設空白對照組和艾地普林試驗組,劑量為艾地普林20、100和1000 mg/kg飼料。第一代(Fo代)每組雌鼠25只(以保證每組有20只受孕雌鼠),雄鼠18只；第二代(F1代)每組雌鼠25只,雄鼠20只。連續(xù)染毒13周后,各組按雌雄2：1或1：1進行交配。整個試驗期間對F0代和F1代大鼠一般毒性的觀察同90天喂養(yǎng)試驗,另外統(tǒng)計繁殖性能相關的各項指標。試驗結(jié)果顯示,艾地普林1000 mg/kg飼料組F0代和F1代大鼠體重顯著性低于空白對照組；F1代窩平均活仔數(shù)和第21天平均仔重顯著低于空白對照組p0.05),F2a代窩平均活仔數(shù)和第4和21天平均仔重顯著低于空白對照組(p0.05)。各組雌雄大鼠主要臟器病理組織學檢查結(jié)果同90天喂養(yǎng)試驗一致,生殖器官病理組織學檢查均正常。以上結(jié)果表明艾地普林在高劑量下(1000 mg/kg飼料)有輕微的母體毒性,并對后代有輕微的發(fā)育毒性。大鼠生殖發(fā)育毒性的NOAEL為7.89 mg/kg b.w..5喂養(yǎng)致畸試驗喂養(yǎng)致畸試驗聯(lián)合二代繁殖試驗Fl代進行,在F2a代斷奶后,Fl代雌鼠與雄鼠再次交配,在妊娠第20天時進行剖腹檢查畸胎。試驗結(jié)果顯示艾地普林1000 mg/kg飼料組平均著床數(shù)、出生活胎數(shù)、胎仔體重以及體長和尾長均顯著低于空白對照(p0.05)。各組均未出現(xiàn)明顯的與藥物相關的胎兒內(nèi)臟畸形或骨骼畸形。結(jié)果表明艾地普林在1000 mg/kg飼料劑量時有輕微的母體毒性以及胚胎毒性,但未表現(xiàn)出致畸效應。所以艾地普林致畸試驗NOAEL為8.75 mg/kg b.w.。6計算每日容許攝入量(ADI)及安全濃度(SC)綜合各項試驗結(jié)果,得到ADP的NOAEL為1.44 mg/kg b.w.,按照FDA給出的計算公式,得出ADP的ADI為0.00144 mg/kg b.w.,在動物可食性組織(肌肉、肝臟、腎臟和脂肪)中的SC分別為0.29,0.86,1.73和1.73 mg/kg。本課題完成了艾地普林的急性毒性、遺傳毒性、亞慢性毒性以及繁殖和致畸毒性試驗,得到了艾地普林一般毒性、繁殖毒性和致畸毒性的NOAEL,確定了艾地普林毒性作用的靶器官,證實了艾地普林不具有致突變性。所有試驗均參考國內(nèi)外相關的權威指南,并結(jié)合藥物的自身特點合理選擇試驗組合,本研究成果可為艾地普林后期的臨床研究提供相關的科學資料。
[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.

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R96
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本文編號：1777487