【作者】 王志军；

【导师】 王茂强；

【作者基本信息】 中国人民解放军军医进修学院 ， 影像医学与核医学， 2011， 博士

【摘要】 目的：(1)评估经导管动脉内给予索拉非尼的安全性、可行性和效果。(2)评估携带索拉非尼的新型载药微球(DC-Bead)对正常肝脏组织功能影响及缓释性能的研究。材料与方法：第一部分：(1)体外研究：分别将碘油-索拉非尼与碘油-顺铂混匀,并在此基础上分别加盐水制成乳剂,静置20分钟后观察乳剂的物理变化,比较索拉非尼与常规化疗药顺铂在碘油载体中的稳定性。(2)体内研究：选用新西兰大白兔24只,随机的分为Ⅰ、Ⅱ、IⅢI组,每组8只,Ⅰ组实验组(碘油-索拉非尼组),Ⅱ组(单纯碘油组)和Ⅲ组(单纯索拉非尼组)为对照组,分别经导管肝动脉注入碘油-索拉非尼乳剂、单纯碘化油注射液及单纯索拉非尼盐水溶液。采用高效液相色谱法测定Ⅰ、Ⅲ两组给药前、给药后10min、20min、1h、2h、4h、8h、16h、32h、48h外周血索拉非尼血药浓度；观察实验动物的生理(呼吸频率、心率、肛温、体重)指标、测血常规、肝、肾、心脏血液指标；术后3天、1周、3周、6周分别处死每组动物2只,对肝脏、心脏、肾脏、肺、脑、胆囊、肠管进行病理检查。第二部分：(1)体外研究：选取不同大小(300-500μm和500-700μm)的DC-Bead颗粒各5瓶,分别置于不同浓度的索拉非尼-75%乙醇溶液中-a瓶：50mg/20ml、b瓶:100mg/20ml、c瓶:100mg/40ml、d瓶：200mg/40ml、e瓶250mg/50ml。静置24h后测定不同颗粒大小DC-Bead的最大载药量,为体内研究提供实验参数。(2)体内研究：选择16只杂种犬,按随机数字法随机分为四组,每组4只,分别为A组：DC-Bead(500-700μm)-索拉非尼组、B组DC-Bead(300-500μm)-索拉非尼组、C组单纯DC-Bead(300-500μm)组、D组碘油-索拉非尼组。分别经肝动脉右支进行栓塞处理。采用高效液相色谱法测其给药前、给药后10min、30min、1h、4h、16h、1天、2天、4天、7天、10天、14天不同时期的外周血索拉非尼浓度。术后3天、1周、2周、3周观察栓塞前、后肝脏功能变化。分别于术后3天、1周、2周、3周处死四组动物,处理后肝组织局部索拉非尼浓度测定,并进行病理检查。结果：第一部分：(1)体外研究：索拉非尼为不溶于水和碘油的物质；与常规可溶于水的化疗药顺铂相比,碘油-索拉非尼乳剂在水溶液中更稳定；(2)体内研究：①外周血索拉非尼浓度：动脉栓塞术后Ⅰ组2h后外周血药浓度达高峰。Ⅲ组介入术后20min达高峰。Ⅰ组CmaX(最大药物浓度)与AUC(曲线下面积)分别为2.46±0.101μg/ml和945.72±52.3μg/mL.min,III组的Cmax与AUC分别为3.78±0.180 ug/ml,AUC546.98±21.1μg/mL.min,二者均具有显著统计学差异(p值均<0.05)。②Ⅰ、Ⅱ组动物栓塞术后3天呼吸频率、心率、AST/ALT、白细胞和中性百分比、肛温、体重均较术前有差异性(P值均<0.05)。Ⅲ组栓塞术前、术后呼吸频率、心率、肛温较术前无明显变化(P>0.05)。Ⅲ组术后3天体重较前下降,术后1周、3周、6周体重增加；③Ⅰ、Ⅱ、Ⅲ组栓塞术后CK、CK-MB、DB、ALB、Cr、BUN、RBC、PLT均较前无明显变化；④病理学：Ⅰ组、Ⅱ组栓塞术后3天、1周以局灶性出血性、凝固性坏死为主要表现,栓塞后3周可见局部肉芽组织形成,可见增生胆管、血管,未见胆管坏死等表现。Ⅲ组栓塞术后肝脏组织无明显变化。三组动物心、肺、肾、脑、肠管道、胆囊等组织病理学检查均未见异常病理学改变。第二部分：(1)体外研究：索拉非尼可溶解于75%乙醇中,其最佳载药浓度为100mg/20ml。相同时间和最大浓度情况下500-700μm的DC-Bead载药量为63.9±21.7mg/ml,300-500μm的DC-Bead载药量为57.6±14.8mg/ml,二者有统计学差异(P<0.05)。(2)体内研究：①A组Cmax.AUC分别为0.3±0.06μg/mL和664.82±36.7μg/mL min。B组Cmax.AUC分别为0.38±0.04μg/mL和1264.82±56.6μg/mL min,D组Cmax.AUC分别为1.24±0.109μg/mL和1728.97±67.90μg/mL min.A和D比较,二者具有统计学差异(P<0.05),A和B比较,二者有统计学差异(P<0.05),即与颗粒小的DC-Bead相比,颗粒大的DC-Bead携带的索拉非尼Cmax更小；②组织中药物浓度：介入术后3天,D组组织中已测不出药物浓度,而A、B组组织中药物浓度明显升高,介入术后1周组织内仍可测到索拉非尼药物浓度；③肝功能变化：四组介入术后3天转氨酶均升高,以B、C组最明显(P=0.02<0.05)。介入后1周D组基本恢复正常,而B、C组转氨酶仍然较高具有统计学意义(p<0.05)。四组介入术后DB、ALB较前无明显变化。④肝脏病理学变化：四组介入术后1周肝脏均存在不同程度的损伤,坏死程度及范围从高到低顺序依次为B、C、A、D。DC-Bead中颗粒越小,坏死程度越重。而碘油栓塞后坏死为局灶性的。介入术后3周,坏死部位开始出现局部肉芽组织增生、可见巨噬细胞。合并局部胆管、门静脉、微小动脉增生。DC-Bead携带索拉非尼栓塞后4周出现血管内膜增厚、周围可见血管出血及巨噬细胞。B组有一实验动物介入术后局部可见胆管损伤,镜下见巨噬细胞吞噬大量胆色素。初步结论：(1)以碘油为载体,经导管肝动脉内导入索拉非尼粉剂技术可行、安全,具有一定的缓释效果；与常规乳化化疗药物相比,碘油-索拉非尼乳剂具有较好的稳定性。(2)DC-Bead可携载索拉非尼,但需要一定条件,即需要能有效溶解的溶剂；(3)DC-Bead携载索拉非尼缓释效应确切,且优于碘油的携载作用；(4)DC-Bead携载索拉非尼做肝动脉栓塞安全的,但存在一些问题,如对损伤肝功能恢复有一定影响。(5)小颗粒DC-Bead携载DC-Bead性能较好,小颗粒DC-Bead可导致胆管损伤。更多还原

【Abstract】 OBJECTIVE:(1)To assess the safety, feasibility and efficacy of combination transcatheter arterial embolization wih lipiodol and sorafenib.(2) To determine the slow-release effect of DC-Bead loaded-sorafenib and its impact on the normal liver of dogs. MATERIALS AND METHODSPARTⅠ.(1)In vitro study:The emulsions of sorafenid-lipiodol and cisplatin-lipiodol were prepared by mixing the drug (4mg),lipiodol (1 ml) and normal sodium. After placement for 20 minutes, the stability of sorafenid and cisplatin in lipiodol were observed..(2)In vivo study:24 New Zealand rabbits were randomly divided into three groups:groupⅠ(Lipiodol-sorafenid), groupⅡ(Lipiodol) and group III (Sorafenib). groupⅠandⅡwere treated by transcatheter selective hepatic arterial embolization with emulsions of lipiodol and sorafenib or with only lipiodol,while groupⅢwas given hepatic arterial infusion with sorafenib. Sorafenib concentration in plasma was determined by HPLC (high performance liquid chromatography) in 0 min,20min,1h,2h,4h,8h,16h,32h and 48h respectively.The breathing rate,heart rate,rectal temperature and body weight were measured,as well the.blood routine test and the function of liver, kidney,and heart.Two animals of each group were respectively killed in the 3rd day,1st,3rd and 6th week after treatment.Histopathologic study was done to liver,heart,kinney,lung,brain,gallbladder and intestine.PARTⅡ.(1) To obtain the maximal drug-loading of DC-Bead, different sizes of DC-Bead (300-500μm and 500-700μm) were tried. five bottles of different sizes of DC-Bead were added into 75% salution of sorafenid-alcohol with different concentrations:Bottle a,50mg/20ml; Bottle b, 100mg/20ml; Bottle c,100mg/40ml; Bottle d,200mg/40m; Bottle e,250mg/50ml.(2)In vivo study:16 dogs were randomly divided into four groups [group A, Sorafenib-DC-Bead(500-700nm); group B, Sorafenib-DC-Bead (300-500μm); group C, DC-Bead(300-500μm); group D, Lipiodol-sorafenib] and four dogs in each group.Each group was treated with TAE with emulsion mentioned above. Sorafenib plasma concentration was determined with HPLC in 0 min,30min,lh,4h,16h,ld,2d,4d,7d and 10 day respectively.The function of liver and kidney were observed.Experimental animals were killed and were subjected to a comprehensive necropsy that involved detailed examination of the external surface of the body and all orifices as well as the cranial,thoracic, and abdominal cavities and contents. Particular attention was paid to the liver and associated tissue and vasculature, lungs, gallbladder and bile ducts, and heart.Tissues were embedded,sectioned, and stained with hematoxylin and eosin for histopathologic assessment..RESULT:PART I:(1)In vitro study:Sorafenib was not dissolved into water or lipiodol.Compared with DDP, the emulsion of sorafenib and lipiodol was more stable in water.(2) In vivo study:①The peak sorafenib concentration (Cmax)and AUC(Area under curve) in plasma in group I was 2.46±0.101μg/ml and 945.72±52.3μg/mL.min respectively,while in group III which was 3.78±0.180 ug/ml and 546.98±21.1μg/mL.min. Compared with group III,the Cmax and AUC of group I had a significant statistics difference(p<0.05).②The breathing rate,heart rate,rectal temperature and AST/ALT,WBC,NEU% of groupⅠand groupⅢhas a significant statistics difference(p<0.05) in the 3rd day.③CK,CK-MB,DB,Cr, BUN,RBC,PLT in plasma did not change in all group.④Local necrosis was seen in group I and groupⅡin the 3rd day and 1st week,but they did not seem to be different.Group III showed no necrosis.Granulation tissue with bile dut,portal vein and micriovessels hyperplasia were seen in local necrosis area in the 3rd week.No pathological changes were found in brain,heart,kiney, intestine and gallbladder.PRATⅡ:(1) In vitro stduy:Sorafenib can be dissovled into 75% alchohol and the best concentraion for drug-loading was 100mg/20ml. (2)In vivo study:①Compared with group D,the Cmax and AUC in plasma in group A and B has a significant statistics difference(p<0.05).②Sorafenib concentarion in liver tissue could be determined in group A and B in the 3rd day and even afer one week while it could not be determined in group D.③ALT/AST in all groups increased significantly in the 3rd day after treatment.But in group B and C it changed more(P<0.05).AST/ALT returned to normal in the 1st week in group D,while in group B and C they were in a relatively high level(P<0.05).DB/ALB in all groups did not change.④The liver was injuried differently in all groups in the 3rd day and 1st week.To the tetail,group B>goup C>group A>group D.Smaller size of DC-Bead would cause more severe injury.⑤Granulation tissue with bile dut,portal vein and micriovessels hyperplasia were seen in local necrosis area.⑥One dog in group B suffered from bile duct injury.Conclusion:(1)TAE with emulsions of lipiodol and sorafenib is feasible,safe and has some slow-release effect.It seems to be more stable in water than DDP when mixed with lipiodol.(2) Sorafenib can be loaded in DC-Bead in a certain condition, such as 75% alcohol.Compared with emulsion with sorafenib and lipiodol,DC-Bead has a definite sow-release function and it is superior to lipiodol.(3)TAE with DC-Bead loaded with sorafenib is safe and feasible,but it may have an effect on the recover of injured liver.(4)Smaller particle size of DC-Bead with sorafenib may cause bile duct injury.更多还原