【作者】 常艳；

【导师】 朱宝泉； 钱蓓丽；

【作者基本信息】 上海医药工业研究院 ， 药理学， 2005， 博士

【摘要】 现代人类生存的环境是一个化学物质纷繁的大千世界。在一定条件下,许多外源化学物质都能通过呼吸道、胃肠道或皮肤进入体内并损害机体,成为毒物。这其中又有数以千计的化学物质可以通过母体胎盘屏障,导致胚胎毒性和致畸性。二十世纪六十年代发生的“反应停事件”使人们更加深刻地认识到发育毒性评价的重要性,此后,美国食品药品监督管理局率先于1966年提出了人体用药物安全性评价的生殖发育毒性试验指南,随后世界各国均以此指南为基础制定了有关农药、化学物质、药物和食品等的生殖发育毒性试验指南。由于各国的试验指南各不相同,限制了药品在各国间的开发利用,为此欧洲、美国和日本三方共同发起人用药品注册技术要求国际协调会议(ICH)。到目前为止,已召开过六届大会,达成多项共识。 胚胎发育毒性和致畸性评价主要是在怀孕动物器官发生期内给予受试物,评价受试物对母体、胚胎-胎仔的毒性和致畸性。该试验周期长,费用高,由于剖检取出的胎仔均计入试验动物总数中,因此与一般毒性试验相比,试验使用的动物数量非常大。随着官方和民众对于减少试验动物使用的呼声日益高涨,进行胚胎发育毒性体外替代试验势在必行。 本研究参照ICH的技术要求对四种候选新药进行了规范化的生殖和发育毒性评价,建立了体外胚胎毒性筛检方法,并对七种已知胚胎毒性物质和二种候选新药的体外胚胎毒性进行了评价。第一部分新药生殖发育毒性评价 为适应新形势的要求,并早日实现与国际接轨,我们参照ICH的技术要求,进行了候选新药佛波双酯注射液、槐果碱注射液、三七素和河鲀Ⅰ型胶原蛋白提取物的生殖和发育毒性研究,并按照GLP要求,对各项检测指标制定了标准化和规范化的操作规更多还原

【Abstract】 Today we live in a world surrounded by numerous kinds of chemicals. Many xenobiotics are able to enter the human body through the respiratory tract, the gastrointestinal tract or the skin, and to cause toxic effects. Chemicals which can pass the placenta barrier will cause embryotoxicity and teratogenicity. The thalidomide disaster, occurring in the 1960’s, emphasized the importance and necessity of reproductive and developmental toxicity assessment. In 1966, FDA established the guidelines of reproductive and developmental studies of drugs for human use. Subsequently, many countries have instituted reproductive and developmental toxicity guidelines for pesticides, chemicals, drugs and food additives. The registration of new drugs in various countries has been limited by different countries having different guidelines. Thus, Europe, Japan and the US have sponsored the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) to facilitate drug testing. During six ICH meetings much harmonization between the guidelines of those countries has been achieved.The developmental toxicity testing according to current international guidelines involves exposure of pregnant animals to drugs during organogenesis and the subsequent assessment of toxic effects in dams and their fetuses. These tests are very time consuming, laborious, expensive, and require relatively large numbers of animals, which becomes even more pronounced as the offspring in the late fetal stages is also counted as experimental animals. Furthermore, there is an increasing political and public demand for a reduction in the use of laboratory animals. This situation has urged research into alternative methods for developmental toxicity testing.The present investigations include in vivo reproductive and developmental toxicity evaluation of four drug candidates and establishment and application of an in vitro embryo toxicity screening test. Seven model chemicals and two drug candidates were tested using in vitro embryotoxicity screening test.Part 1: In vivo reproductive and developmental toxicity evaluation of drug candidatesFour drug candidates, Phobol diester, Sophocarpine, SH-1 and AGUAP were tested in the in vivo reproductive and developmental toxicity study in rats according to ICH guidelines and in compliance to Good Laboratory Practice, GLP. Standard Operation Procedures, SOPs, have been worked out for each method.1. MethodsIn the fertility and early embryo developmental toxicity study male rats were treated for 28 days before cohabitation, during 14 days cohabitation and until termination. Female rats were treated for 14 days before cohabitation, during 14 days cohabitation and from days 0 to 7 post coitum. Male rats were sacrificed after completion of the cohabitation period. Sperm analysis or histopathology evaluation of testis and epididymis was performed for each male rat. Female rats were Caesarean-sectioned on gestation day 15. The uterus of each animal was examined for the numbers of live and dead fetuses and resorptions, and the ovaries were examined for the numbers of corpora lutea.In the embryo-fetal developmental toxicity and teratogenicity study chemicals were administered to pregnant rats during the period of organogenesis (gestation days 6-15). Surviving rats were Caesarean-sectioned on gestation day 20. The uterus of each animal was examined for the numbers of live and dead fetuses and resorptions, and the ovaries were examined for the numbers of corpora lutea. Live fetuses were weighed and evaluated for external, visceral and skeletal abnormalities. 2. Results 2.1 The fertility and early embryo developmental toxicity studySophocarpine, an extract from Radix Sophorae Flavescentis, induced no adverse effects on male fertility, but caused an increase of resorptions and postimplantation losses, thus showing adverse effects on early embryo development. AGUAP, a soluble protein extracted from fish bone, showed no adverse effects on male fertility and postimplantation embryo development.However, AGUAP caused a decrease in the number of corpora lutea and an increase of preimplantation losses, thus showing adverse effects on ovulation and implantation. Further research needs to be done to elucidate the mechanisms.2.2 The embryo-fetal developmental toxicity and teratogenicity studyPhorbol diester, extracted from croton oil, showed no adverse effects on rat embryo-fetal development or teratogenicity. Sophocarpine, an extract from Radix Sophorae Flavescentis, showed no adverse effects on embryo-fetal development and no teratogenicity. SH-1, which is the active component of Radix Notoginseng to treat hemostasis caused an increase of late resorptions and fetal deaths using dosages with or without accompanying parent toxicity. AGUAP, a soluble protein extracted from fish bone, showed no adverse effects on rat embryo-fetal development and no teratogenicity.Part 2: Establishment and application of an in vitro embryo toxicity screening testAlternative methods for developmental toxicology testing can be roughly subdivided into three types of systems with increasing complexity: cell cultures, organ cultures, and embryo cultures. Cell cultures have the advantage of ease to perform and minimal or no animal use, while on the other hand they have the disadvantage of being too simplistic. Whole embryo culture systems have the advantage of representing embryogenesis in full complexity from cellular proliferation and differentiation to pattern formation. Based on the characteristic of embryotoxic chemical, the predict accuracy will be increased significantly when investigating the toxic effect on cell differentiation in whole embryo culture and the toxic effect on 3T3 cell proliferation simultaneously.1. MethodsThe rat postimplantation whole embryo culture was used. Embryos, 9.5d or 10.5d, were isolated from pregnant rats. The embryos were cultured in immediately centrifuged rat serum with different concentrations of chemical for 48 hours at 37°C under continuous rotation. The embryos were gassed four times with increasing oxygen concentrations. After 48 hours in culture the embryos were evaluated according to Brown’s method. ICnoec and ICmai were deduced from dose-response curves. IC503T3 was deduced from 3T3 cell toxicity study. The prediction model suggested by ECVAM was used.The effect of different animal sera and culture mediums and DMSO concentrations onembryonic development was evaluated and comparisons of in vivo and in vitro embryo development were performed in the post-implantation whole embryo culture in rats. Screening for potential embryo toxicity of seven model chemicals was performed using whole embryo cultures. Two new drug candidates, SH-1 and voriconazole, were also evaluated in this in vitro screening model.2. Results2.1 Comparison of different experimental conditions2.1.1 Comparison of different animal sera and culture mediumsEmbryos did not develop in fetal bovine serum, horse serum or goat serum. Embryos did develop in RPMI 1640 or DMEM with 50% immediately centrifuged rat serum, but the growth parameters were significantly lower than in the 100% rat serum group.2.1.2 Toxic effects of the solvent, DMSODMSO, a widely used organic vehicle in in vitro tests, has previously been demonstrated to be toxic to various types of cells. DMSO at concentrations of 0.5% and above was shown to produce significant embryo toxicity and malformations. As an appropriate final concentration 0.125% DMSO was used.2.1.3 Comparison of in vitro and in vivo embryonic developmentGrowth parameters such as yolk sac diameter, crown-rump length, head length and somite number of in vitro 11.5d embryos showed no significant differences to in vivo embryos. The morphology score of organ anlage showed no significant difference to in vivo data. Growth parameters and morphology score of in vitro 12.5d embryos were significantly reduced compared with in vivo data. However, in both 11.5d and 12.5d embryos different kinds of malformations could be detected after treatment with 5-fluorouracil and retinoic acid. Both are appropriate in vitro alternative tests for embryotoxicity.2.2 Results of seven model chemicalsThe test chemicals, which have been used in validation studies of developmental toxicity tests and with existing in vivo developmental toxicity data or human data, were penicillin G, isoniazid, busulphan, 5-fluorouracil, methotrexate, hydroxyurea and retinoic acid.ICnoec and ICmai were deduced from dose-response curves of the seven model chemicals. Using the prediction model suggested by ECVAM, only isoniazid was misclassified and notconsistent with the in vitro data drawn from micromass and embryo stem cell tests. The prediction accuracy was in total 85.7%, and for the five strong embryotoxic chemicals 100%. 2.3 Comparison of in vivo and in vitro results of drug candidates, SH-1 and voriconazoleThe embryotoxic effects of SH-1 and voriconazole were evaluated in Sprague-Dawley rats in vivo and in whole embryo cultures. In in vivo tests of SH-1, an increase in late resorptions and fetal deaths was observed at each dose, and a decrease of live fetuses was observed in the high and middle dose groups. No teratogenicity was noted. In vitro, SH-1 caused significant decreases in growth parameters and morphology score. Flexion defects and development retardation of the brain were observed at 250 mg/mL. Five out of nine embryos died at the highest concentration, 1000 mg/mL. The main embryotoxic effects included development retardation of the brain and neural tube, lack of allantois fusion and flexion defects. Thus, both the in vivo and in vitro data showed adverse effects induced by SH-1 on embryo development.In in vivo tests of voriconazole some external, visceral, and skeletal malformations and an increase in resorptions and fetal deaths were observed. The main external malformation type was cleft palate. In vitro, voriconazole mainly caused malformations in branchial bars. The branchial bars will develop into maxillary and mandibular processes. Thus, the malformation of branchial bars indicated that voriconazole has adverse effects on occlusal surface development. Good consistency was shown between in vivo and in vitro data.Conclusions1) Reproductive and developmental toxicity evaluation methods according to ICH guidelines and GLP were established.2) Reproductive and developmental toxicity evaluation of plant and biotechnology-derived products must be strengthened.3) The rat whole embryo culture method was shown to be a valuable in vitro alternative screening method with 100% prediction accuracy for five tested strong embryo toxicants.更多还原