本文關(guān)鍵詞： 核苷類抗乙肝病毒藥物 ZBH201001 臨床前安全性評價 Beagle犬 單次給藥毒性研究 重復(fù)給藥毒性研究　出處：《廣西醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：ZBH201001是軍事醫(yī)學(xué)科學(xué)院毒物藥物研究所研究人員在阿米福韋化學(xué)結(jié)構(gòu)的基礎(chǔ)上設(shè)計合成的一種核苷類抗乙肝病毒藥物。本研究依據(jù)藥物臨床前安全性評價相關(guān)指導(dǎo)原則,以Beagle犬為研究對象,分別進(jìn)行單次給藥毒性研究和重復(fù)給藥毒性研究,觀察動物出現(xiàn)的毒性反應(yīng),了解ZBH201001的急性毒性作用,以及重復(fù)給藥毒性作用的靶器官及其損害的可逆性,確定未觀察到損害作用的劑量(NOAEL),為其臨床試驗的用藥劑量和用藥安全劑量范圍提供參考,同時為該新藥臨床申請和新藥注冊申請?zhí)峁⿺?shù)據(jù)支持。方法：(1)Beagle犬經(jīng)口急性毒性試驗：采用近似致死劑量法,單次經(jīng)口灌胃給藥,給藥劑量分別為118、264、594、890、1334、2000 mg/kg體重。毒性檢測指標(biāo)包括一般臨床觀察、體重、攝食量、體溫、心電圖、血液學(xué)、血液生化、凝血指標(biāo)以及尿液化學(xué),給藥觀察3周后,對所有動物進(jìn)行組織病理學(xué)檢查。(2) Beagle犬經(jīng)口重復(fù)給藥1個月毒性試驗：實驗設(shè)6.0、12.0、24.0mg/kg體重組及玉米油溶劑對照組,每天經(jīng)口給藥1次,給藥期30天,給藥結(jié)束后恢復(fù)4周。連續(xù)給藥9天后,因各劑量組動物均出現(xiàn)了明顯的毒性反應(yīng),故將劑量調(diào)低為3.0、6.0與9.0 mg/kg體重,繼續(xù)給藥至實驗結(jié)束。實驗期間每日進(jìn)行一般臨床觀察與體重、攝食量測定,給藥中期、給藥結(jié)束與恢復(fù)期分別進(jìn)行體溫、心電圖、血液學(xué)、血液生化、凝血指標(biāo)與尿液化學(xué)檢測,并于給藥結(jié)束與恢復(fù)期結(jié)束進(jìn)行組織病理學(xué)檢查。結(jié)果：(1)Beagle犬經(jīng)口急性毒性試驗：Beagle犬單次經(jīng)口灌胃給藥,594 mg/kg體重及以上劑量組動物給藥后籠旁觀察顯示,給藥后和/或藥后次日可見動物嘔吐,偶見稀便。隨后觀察到動物攝食狀況普遍較差、體重持續(xù)降低；約自第3周起,動物攝食狀況逐漸好轉(zhuǎn),體重逐漸增加。890 mg/kg體重劑量組1只雌性動物于給藥后第12天意外死亡,重復(fù)給予該劑量雄性動物存活。118 mg/kg及264 mg/kg組給藥后觀察到動物攝食減少,體重輕度降低。較高劑量組動物給藥后個別時間點個別血液學(xué)指標(biāo)、尿液化學(xué)指標(biāo)出現(xiàn)異常變化,體溫、心電圖等參數(shù)未見有毒理學(xué)意義的改變。組織病理學(xué)檢查：890 mg/kg組意外死亡動物尸檢除體型消瘦外未見明顯異常,鏡下檢查見肺嚴(yán)重水腫、出血、肺泡上皮脫落,胃腸系統(tǒng)出現(xiàn)病變；其余6只存活動物剖殺,體表和內(nèi)臟檢查均未見異常。1334 mg/kg體重組動物肝臟灶性肝細(xì)胞空泡變性、散在壞死,結(jié)腸出血、上皮細(xì)胞壞死、炎性細(xì)胞浸潤；2000mg/kg體重組動物胃黏膜壞死、水腫；其他動物各臟器未見明顯異常。(2) Beagle犬經(jīng)口重復(fù)給藥1個月毒性試驗：結(jié)果顯示,以24.0、12.0、6.0 mg/kg體重劑量給藥9天后,雌、雄動物均表現(xiàn)出胃腸毒性反應(yīng),包括稀便、動物攝食狀況差、體重降低等體征,并呈現(xiàn)較明顯的量效與時效關(guān)系。因此將劑量將低為9.0、6.0與3.0 mg/kg體重繼續(xù)給藥,我們觀察到高劑量9.0 mg/kg體重組動物攝食量、體重持續(xù)降低,并陸續(xù)有稀便和/或血便等現(xiàn)象,并呈現(xiàn)消瘦、活動減少等臨床體征；3.0、6.0mg/kg組動物體重雖仍低于同期對照組,但基本保持在穩(wěn)定的水平,未進(jìn)一步降低。無論劑量調(diào)整前還是調(diào)整后,高、中、低劑量組動物的攝食量變化表現(xiàn)出與體重變化基本相一致的變化趨勢。各期高、中、低劑量組心電圖、體溫及眼科檢查均未見有毒理學(xué)意義的異常變化。給藥中期與給藥結(jié)束后血液學(xué)檢查提示高劑量組動物APTT明顯降低,并且這種變化在恢復(fù)期恢復(fù)正常,提示受試藥物可能會影響動物的內(nèi)源性凝血系統(tǒng)。各期尿液分析高、中、低劑量組未見異常改變。組織病理學(xué)檢查,中劑量和/或高劑量組對消化管與胃腸系統(tǒng)具有明顯的毒性作用,中劑量和/或高劑量組動物胸腺、睪丸、前列腺、胰腺萎縮,骨髓紅系造血抑制、脂肪化,肝細(xì)胞腫脹、變性。上述各組織/臟器病變在恢復(fù)期均減輕和/或恢復(fù),說明其毒性具有一定的可恢復(fù)性。給藥結(jié)束高、中、低劑量組均有部分動物出現(xiàn)肺水腫、肺泡上皮脫落、炎性細(xì)胞浸潤等病變,恢復(fù)期結(jié)束各組動物未見上述病變；考慮到口服給予供試品混懸液時動物偶有嗆咳反應(yīng),并且肺部病變的嚴(yán)重程度及發(fā)生率無量效關(guān)系,因此認(rèn)為所觀察到的肺部病變與給藥操作有關(guān)。結(jié)論：在本試驗條件下,(1)Beagle犬經(jīng)口急性毒性試驗MTD2000mg/kg體重,最小毒性反應(yīng)劑量為118 mg/kg體重,胃腸道可能是ZBH201001對Beagle犬急性毒性作用的靶器官/靶組織。(2)Beagle犬經(jīng)口重復(fù)給藥1個月毒性試驗,中劑量和/或高劑量可引起食管、肝臟、胸腺、睪丸、前列腺、胰腺、骨髓、腎臟、胃腸系統(tǒng)一定的毒性反應(yīng),并可能影響內(nèi)源性凝血系統(tǒng),并且這些毒性反應(yīng)停藥后可恢復(fù)；因此,ZBH201001對Beagle犬重復(fù)給藥的NOAEL為3.0 mg/kg體重(人臨床擬用劑量以10 mg/日計,約相當(dāng)于臨床擬用劑量的18倍)。
[Abstract]:Objective: ZBH201001 is the Military Medical Science Academy of the PLA Drug Research Institute researchers design a synthesis of nucleoside anti HBV drugs based on the chemical structure of Amie. On the basis of tenofovir drug preclinical safety evaluation guidelines in Beagle dogs as the research object, respectively for single dose toxicity study and repeated toxicity of drugs the toxic reaction of the animal, the acute toxicity of ZBH201001, reversibility and repeated drug toxicity and target organ damage, determine the observed damage dose (NOAEL), to provide reference for the clinical trials of the dosage and safety dose range, and provide data support for the clinical application and new drug application. Methods: (1) Beagle canine acute toxicity test: the approximate lethal dose, single oral gavage Drug dosage was 11826459489013342000 mg/kg weight. Toxicity indexes included clinical observation, average weight, food intake, body temperature, electrocardiogram, hematology, blood biochemistry, blood coagulation index and urine chemistry, 3 weeks after the administration of observation, pathological examination of all animal. (2) Beagle dogs by mouth repeated dose toxicity test: 1 months experiment set 6.0,12.0,24.0mg/kg and body weight of corn oil solvent control group, daily oral administration of 1 times, delivery period of 30 days after the end of dosing recovery for 4 weeks. After 9 days of medication, for each dose group animal showed obvious toxicity, it will be the dose was 3.0,6.0 and 9 mg/kg by weight, continued administration until the end of the experiment. During the experiment, daily general clinical observation and determination of body weight, food intake, drug administration and the end of the mid recovery period were temperature, ECG, hematology, blood biochemistry, coagulation Blood and urine chemical detection, and recovery period to the end of dosing and end for histopathological examination. Results: (1) Beagle canine acute toxicity test: Beagle canine single oral gavage, and more than 594 mg/kg body weight dose group after administration of animal cage observation showed that the drug after the drug and / or the day after the visible animal vomiting, occasionally stools. Then observed animal feeding conditions are generally poor, continue to reduce weight; since about third weeks, animal feeding conditions gradually improved, the body weight increased.890 mg/kg body weight dose group of 1 female animal in twelfth days after administration of accidental death, repeated administration the dose of the male animal survival.118 mg/kg and 264 mg/kg were observed after the administration of animal food intake, body weight decreased slightly. The higher dose group after administration of animal individual time points of individual blood index, urine chemical indicators of abnormal ECG, body temperature. There is no toxicological significance map and other parameters change. Histopathological examination: 890 mg/kg group in addition to the accidental death of animal body weight and autopsy showed no abnormalities, endoscopic examination showed severe pulmonary edema, hemorrhage, alveolar epithelial shedding, gastrointestinal system disease; the remaining 6 were killed only activities, superficial and visceral examination showed no abnormal.1334 mg/kg recombinant animal liver focal liver cell degeneration, scattered necrosis, bleeding in the colon, epithelial cell necrosis, inflammatory cell infiltration; 2000mg/kg recombinant animal gastric mucosa necrosis, edema; other animal organs had no obvious abnormalities. (2) Beagle dogs by mouth repeated administration of 1 month toxicity test: the results showed that after 9 days of treatment, the 24.0,12.0,6.0 dose of mg/kg body weight of female and male animal showed gastrointestinal toxicity, including stools, animal feeding condition, weight loss and other symptoms, and showed obvious dose effect With the aging relationship. So the dose will continue to give medicine for low 9.0,6.0 and 3 mg/kg weight, we observed high dose 9 mg/kg recombinant animal food intake, body weight decreased, and there were rare and / or bloody stool phenomenon, and showed clinical signs such as weight loss, reduced activity; 3.0,6.0mg/kg group was lower than the weight of an animal compared with the control group, but remained at a stable level, not further reduced. No dose adjustment before or after adjustment, high, low dose group showed a trend of animal food intake and body weight change change accord. The high, low dose group, ECG, abnormal changes in toxicological significance body temperature and ophthalmic examination showed no medication. The middle and afteradministration hematology examination showed high dose group animal APTT decreased significantly, and the change in the recovery period returned to normal, suggesting that subjects may affect The endogenous coagulation system. Analysis of the animal urine during the high and low dose group showed no abnormal changes. Histopathological examination, middle dose and / or high dose group has obvious toxic effect on the digestive tract and gastrointestinal system, middle dose and / or high dose group of animal thymus, testis, prostate, pancreatic atrophy. Bone marrow hematopoietic suppression, fat, liver cell swelling, degeneration. The tissue / organ diseases during the recovery period were reduced and / or recovery, the toxicity can be recovered. The end of administration, and pulmonary edema occurred in low dose group were part of an animal, alveolar epithelial shedding, inflammatory cell infiltration lesions were not found in the end of the recovery period of animal disease; considering the oral administration of the sample suspension when the animal occasionally cough, and the severity of lung disease and there was no dose-response relationship, so that the observed lung Department of administration and operation of the lesions. Conclusion: under the experimental conditions, (1) Beagle dogs were acute toxicity test of MTD2000mg/kg weight, minimum dose toxicity was 118 mg/kg body weight, the gastrointestinal tract may be the target organ / ZBH201001 on acute toxicity of Beagle dog target tissue. (2) Beagle canine oral repeat for 1 months drug toxicity test, dose and / or high doses can cause the esophagus, liver, thymus, testis, prostate, pancreas, bone marrow, kidney toxicity, gastrointestinal system, and may affect the endogenous coagulation system, and the toxic reaction after drug withdrawal can be recovered; therefore, ZBH201001 repeat in Beagle dogs administration of NOAEL for 3 mg/kg weight (clinical dose of 10 mg/ to date, equivalent to about clinical dose of 18 times).

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R965

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