抗肿瘤活性化合物呋喃二烯的骨髄抑制毒性及其初步机制研究
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摘要
目的
制备W/O/W型呋喃二烯复乳,解决呋喃二烯水溶性低、动物难以给药的问题,并评价其对S-180荷瘤小鼠的抗肿瘤活性和初步毒性作用；对呋喃二烯在小鼠体内的毒性靶器官进行确证并进行初步机制研究；研究呋喃二烯诱导SP2\0细胞凋亡的机制和信号传导通路；评价呋喃二烯复乳在大鼠体内的动力学特征、组织分布以及毒性靶器官中的蓄积；最终探讨呋喃二烯在动物体内的毒性作用、毒性靶器官、毒性靶器官中的药物蓄积以及可能的引起毒性的分子机制,为预测其他海洋来源结构类似物的毒性作用提供研究基础和理论依据。
方法
(1)采用两步乳化法制备W/O/W型呋喃二烯复乳,通过对处方组成和制备工艺进行优化,确定最优处方,通过显微镜下观察和染色两种对W/O/W型复乳进行鉴定,采用HPLC法建立呋喃二烯的体外测定方法,对复乳进行质量控制。以肿瘤移植小鼠为模型,以环磷酰胺为阳性对照药,分别研究腹腔注射和口服灌胃低、中、高三种剂量呋喃二烯复乳后对S-180荷瘤小鼠体内肿瘤生长的抑制作用,同时考察实验小鼠体重、脏器指数、外周血细胞数目、血清主要生化指标和组织病理切片的改变,评价呋喃二烯的药效和毒性作用,初步判断其毒性靶器官；
(2)以健康动物为实验对象,小鼠多剂量腹腔注射低、中、高三种剂量的呋喃二烯复乳,考察小鼠体重、脏器指数、外周血细胞数目、血清主要生化指标和组织病理切片的改变,进一步明确呋喃二烯的毒性作用和毒性靶器官。采用Western blot技术测定小鼠骨髓细胞中凋亡相关蛋白表达的变化,初步考察呋喃二烯致小鼠骨髓抑制的机制。大鼠多剂量腹腔注射低、中、高三种剂量的呋喃二烯复乳,采用超速离心法制备大鼠肝微粒体,通过对肝微粒体中蛋白含量和CYP450含量测定,初步评价呋喃二烯对大鼠肝微粒体CYP450酶的影响；采用HPLC法测定呋喃二烯在肝微粒体中的代谢率并计算酶动力学参数,通过底物探针代谢实验进一步确证其对CYP450酶系各亚型的影响作用。
(3)以大鼠为实验对象,采用HPLC-APCI-MS/MS技术,测定大鼠分别尾静脉注射、腹腔注射以及口服灌胃低、中、高三种不同剂量呋喃二烯复乳后的血药浓度,计算药物动力学参数；采用外翻肠囊法分别测定呋喃二烯原药和复乳的累积透过量及肠段残留量；测定大鼠单剂量尾静脉注射、腹腔注射以及口服灌胃呋喃二烯复乳后的组织分布,并对大鼠多剂量腹腔注射和口服灌胃后药物在毒性靶器官中的蓄积进行了监测。
(4)以SP2/0为实验细胞株,采用MTT法测定呋喃二烯对体外培养细SP2/0细胞的增殖抑制率,并计算IC50；通过倒置显微镜观察细胞形态的变化；采用流式细胞技术测定呋喃二烯诱导SP2/0细胞的凋亡率、DNA含量以及对细胞周期的影响。采用Western blot技术检测凋亡相关蛋白Bax、Bcl-2、Caspase-9、 Cyt-c、Fas、Fas-L、Caspase-8以及Caspase-3等表达的变化。采用RT-PCR技术检测凋亡相关基因Bax、Bcl-2、Caspase-3、8、9mRNA的变化,通过时效和量效关系说明呋喃二烯诱导SP2/0细胞凋亡的信号转导通路。
结果
(1)两步乳化法制得的W/O/W复乳粒径大小均匀,分散性好,质量稳定,标示含量为99%±2.1%,符合制剂质量要求,可用于动物给药的制剂。腹腔注射呋喃二烯低、中、高三种不同剂量的复乳后,S-180荷瘤小鼠的肉瘤生长均得到一定程度的抑制,抑瘤率分别为18.55%±3.5%、19.82%±2.7%和29.82%±3.4%,与空白复乳组比较具有显著性差异(p<0.05)。高剂量组的抑瘤率与阳性对照组环磷酰胺(33.53%±3.5%)比较无显著性差异(p>0.05)。口服灌胃三种不同剂量的复乳后,与空白复乳组比较,只有高剂量组(20.76%±4.5%)具有显著性差异(p<0.05)。说明腹腔注射组具有一定的抑瘤率,而口服灌胃组的作用较弱。腹腔注射呋喃二烯可显著抑制S-180腹水瘤小鼠腹围的增长(p<0.01),但不能显著延长小鼠的生存时间,预测可能是其毒性引起的小鼠死亡。脏器指数与空白对照组比较,脾脏指数明显升高(p<0.01),其次为肝脏指数(p<0.05)。外周血细胞计数结果显示呋喃二烯可引起小鼠全血血细胞的变化,其中白细胞的降低最为明显(p<0.01),高剂量组还可同时引起红细胞(p<0.05)和血小板的减少(p<0.01)。血清生化指标测定结果显示呋喃二烯可引起血清中ALT、AST、BUN和SCR水平的变化,其中以AST水平的升高最为显著(p<0.01)。组织切片结果显示呋喃二烯给药组小鼠脾脏组织结构被破坏,红髓内出现幼稚粒系细胞髓外造血,提示骨髓造血系统可能是其毒性靶器官。
(2)多剂量腹腔注射呋喃二烯低、中、高三种不同剂量的复乳后可引起健康小鼠全血血细胞的变化,其中以白细胞的降低最为明显(p<0.01),高剂量组还可同时引起血小板减低(p<0.05)。同时可引起骨髓造血系统损伤相关血清生化指标EPO、TPO及GM-CSF水平的变化,其中以GM-CSF水平的降低最为明显(p<0.01),高剂量组还可同时引起TPO水平的降低(p<0.05)。肝损伤相关血清生化指标ALT、AST、ALP、MDA及SOD的变化不大,只有高剂量组可引起ALT、AST、ALP和MDA的升高(p<0.05)。脏器指数以脾脏指数的升高最为明显(p<0.01),其次为肝脏指数(p<0.05)。组织切片显示呋喃二烯给药组小鼠的骨髓髓腔内细胞数目减少,且脾脏出现明显的粒系髓外造血,肝脏也出现轻微的粒系髓外造血,提示存在明显的骨髓抑制作用,其他脏器的组织学变化不大。小鼠骨髓细胞Western blot检测结果显示,呋喃二烯给药组可上调Bax的表达、下调Bcl-2的表达,腹腔注射呋喃二烯低、中、高三种不同剂量的复乳后Bax/Bcl-2的比值依次为：2.22±0.65、5.32±1.56、6.03±2.43,与空白对照组比较p<0.01,存在一定的剂量相关性,且出现明显的Cleaved capase-3条带,说明呋喃二烯通过诱导骨髓细胞的凋亡导致骨髓抑制。
多剂量腹腔注射呋喃二烯低、中、高三种不同剂量的复乳后,超速离心法制备肝微粒体,与空白对照组比较,呋喃二烯中、高剂量组可引起肝微粒体中蛋白含量和CPY450含量的减少(p<0.05)。并且呋喃二烯在经中、高剂量处理过的肝微粒中具有明显的代谢抑制现象,Km和Vmax的值均有一定程度的降低(p<0.05),提示呋喃二烯对CYP450可能存在一定的抑制作用。底物探针实验结果显示：S-美芬妥英和苯妥英在经高剂量呋喃二烯处理过的肝微粒体中的代谢速度与空白对照组比较具有显著性差异(p<0.01和p<0.05),其他底物的代谢速度与空白对照组比较无统计学意义,提示呋喃二烯可能对CYP2B6和2C9存在一定的抑制作用。
(3)大鼠尾静脉单剂量注射低、中、高三种不同剂量的呋喃二烯复乳后,其药动学特征符合二室开放模型,t1/2。分别为3.9±2.3、4.3±1.2和4.8±2.1min, t1/2β分别为73.2±7.9、64.2±14.7、69.3±12.1min,表明药物在大鼠体内的分布和消除都较快。曲线下面积(AUC)分别为83231.34±9784.3、151481.88±14573.1和834672.65±67481.43min·ng·mL-1,采用最小二乘法对AUC和剂量进行相关性考察,直线方程为y=16845x-8510.6(r=0.987),表明呋喃二烯在大鼠体内呈现线性药动学特征。单剂量腹腔注射或者口服灌胃给予大鼠低、中、高三种不同剂量呋喃二烯复乳后,低、中剂量基本测不到血药浓度,高剂量组所测得的血药浓度由于数值很小,无法进行药动学参数的求算。外翻肠囊法评价药物吸收的实验结果显示,呋喃二烯原药组在小肠各段60min的累积透过量分别为956±324、657±126、542±148ng,其在小肠各段的残留分别为8654±1654、7684±957、7342±1256ng；呋喃二烯复乳组在小肠各段60min的累积透过量分别为3456±534、3024±368、2687±286ng,其在小肠各段的残留分别为2459±625、2861±589、2103±574ng,累积透过量按十二指肠、空肠、回肠依次减少。组织分布结果显示,大鼠尾静脉单剂量注射呋喃二烯复乳后迅速在体内广泛分布,在脾脏中的浓度最高,其次按肝脏、心脏、肺脏、肾脏、小肠和脑依次减少。单剂量腹腔注射或口服给药后,在小肠中的浓度最高,其次按为脾脏、心脏、肝脏、肺脏、肾脏和脑依次减少。药物蓄积结果显示：大鼠多剂量腹腔注射呋喃二烯复乳后,毒性靶器官中的药物含量在逐渐升高,脾脏中的含药量从第1天的25.3±3.3rng·g-1升高到第7天的43.1±4.1ng·g-1,骨髓中的含药量从第1天的1.6±0.4ng·g-1升高到第7天的5.1±0.5ng·g-1,胸腺中的含药量从第1天的0.9±0.2ng·g-1升高到第7天的2.5±0.2ng·g-1。
(4)MTT法检测细胞存活率试验结果显示,呋喃二烯能抑制SP2/0细胞的生长,具有一定的量效关系,经计算IC50为5.2μg·mL-1。通过倒置显微镜观察到呋喃二烯处理过的SP2/0细胞呈现凋亡的形态学特征。经FCM分析,Annexin V-FITC法实验显示不同浓度的呋喃二烯作用不同时间后SP2/0细胞的凋亡率与对照组相比有统计学差异(p<0.05),并且从DNA组方图上可见到Go-G1期前亚二倍体凋亡峰。Western blot检测结果显示,随着呋喃二烯作用浓度的增加,细胞中Bax/Bcl-2的比值由1.83+0.6、8.35±4.3升至14.6±5.6,线粒体内的Cty-c由0.7±0.2、0.440.3降至0.5±0.2,胞浆内的Cty-c由2.6±0.7、5.9±1.3升至8.3±2.5,细胞中Fas由1.14±0.42、1.57±0.63升至1.73±0.48,Fas-L由0.39±0.09、0.58+0.26升至0.68+0.32,具有明显的浓度依赖性,并出现了清晰的Cleaved caspase-9、8和3条带。随着呋喃二烯作用时间的延长,细胞中Bax/Bcl-2的比值由4.26±1.4、10.99±3.5升至14.6±5.6,线粒体内的Cty-c由0.5±0.2、0.3+0.1降至0.2±0.1,胞浆内的Cty-c由5.3±0.8、6.3±1.7升至8.3±2.5,细胞中Fas由0.69±0.21、0.88±0.24升至1.73±0.48,Fas-L由0.38+0.14、0.43±0.21升至0.68±0.32,具有明显的时间依赖性,也出现清晰的Cleaved caspase-9、8和3条带；Bax, bcl-2, caspase-8, caspase-9,caspase-3的mRNA表达水平随呋喃二烯的浓度和作用时间的不同而变化,具有一定的时效与量效关系。说明呋喃二烯可通过线粒体途径和死亡受体诱导SP2/0细胞凋亡。
结论
(1)W/O/W型呋喃二烯复乳制备工艺简单,质量稳定,为动物体内的药效学、药动学及毒理学研究提供保障；腹腔注射呋喃二烯复乳对S-180荷瘤小鼠具有一定的抗肿瘤活性,同时主要毒性靶器官可能是骨髓造血系统和肝脏；
(2)确证了呋喃二烯在健康小鼠体内的毒性靶器官主要为骨髓造血系统,可通过上调Bax、下调Bcl-2、破坏Bax和Bcl-2之间的平衡诱导骨髓细胞凋亡。呋喃二烯对肝脏的毒性主要表现在对CYP2B6和2C9有一定的抑制作用。
(3)大鼠尾静脉单剂量注射呋喃二烯复乳后,其药动学符合二室模型,消除半衰期较短,呈现线性动力学特征。经血管外途径给药后血药浓度低的原因可能是乳滴经毛细淋巴管途径进入大循环后迅速转运至组织,因此不影响呋喃二烯发挥药效以及产生毒性。呋喃二烯在毒性靶器官中的蓄积也是其毒性发生、发展的原因之一。
(4)呋喃二烯不引起小鼠骨髓瘤(SP2/0)细胞周期的变化,其诱导SP2/0细胞凋亡是线粒体途径和死亡受体途径共同作用的结果。
Objective
To prepare water-in-oil-in-water multiple emulsions for furanodiene in order to solve difficulties of poor soluble and animal administration, investigate the antitumor activity and preliminary toxicity to S180carcinoma in vivo, and confirm the toxic target organs in mice and the preliminary toxic mechanism. To study the mechanism on furanodiene induced SP2\0apoptosis and its signal transduction pathway, evaluate the pharmacokinetics, tissue distribution of furanodiene multiple emulsions in rats and accumulate in toxic target organs.
Methods
(1) A Two-step emulsification method was used to prepare the W/O/W multiple emulsions. The prescription and preparation of emulsions were optimized. Both the primary and multiple emulsions were identified through microscope-observation and eosin staining method. The HPLC method was established and used to the quality control of furanodiene multiple emulsions in vitro. S-180sarcoma transplanted mice as animal model and cyclophosphamide as positive control, the tumor growth inhibition rates were measured after intraperitoneal injection and oral at low, middle and high doses. Meanwhile, the weight change, viscera index, blood cell count, serum biochemical indexes and tissue pathological slices were used to evaluate the toxicity.
(2) The healthy mice were used as experiment objects with the changes of their weight, viscera index, blood cell count, serum biochemistry and tissue pathological slices as evaluation indexes, the toxic effects and target organs were confirmed after intraperitoneal injection at low, middle and high multiple doses. In order to investigate the preliminary mechanism of furanodiene induced myelosuppression, the changes of apoptosis related protein expression were measured by Western blot technology. Liver microsomes of rats which treated with furanodiene multiple emulsions at low, middle and high multiple doses by intraperitoneal injection were prepared by using ultracentrifuge method. The concentration of protein in liver microsome determined by the BCA method and the content of cytochrome P450detected by the Omura and Sato method were used to evaluate the effect of furanodiene on CYP450preliminarily. In order to calculate the enzyme kinetics parameters, a HPLC method for determination of furanodiene in rat liver microsomes was established and the in vitro metabolic rates were studied by incubation with rat liver microsomes. The effect of furanodiene on CYP450subtypes was confirmed by the specific substrate experiments.
(3) The plasma concentrations of furanodiene were determined by HPLC-APCI-MS/MS method after intravenous, intraperitoneal injection and oral furanodiene multiple emulsions at low, middle and high single dose in rats, respectively. Pharmacokinetics parameters were calculated by Winnolin software. The cumulative penetration and intestinal residue of furanodiene and its multiple emulsions were determined by the everted gut sacs method. Tissue distribution of furanodiene were studied in rats after oral, intraperitoneal and intravenous injection furanodiene multiple emulsions with single dose. Meanwhile, drug accumulation were monitored in the toxic target organs after oral and intraperitoneal injection furanodiene multiple emulsions with mutable doses, respectively.
(4) The growth inhibitions of SP2/0cell cultured in vitro were analyzed by MTT method, than IC50was calculated. Cell morphology changes were observed by using light microscope. Flow cytometry (FCM) was used to study the apoptosis rates, content of DNA and effects of furanodiene on SP2/0cells cycle. The changes of apoptosis related protein expression such as Bax, Bcl-2, Caspase-9, Cyt-c, Fas, Fas-L, Caspase-8and Caspase-3, were measured by Western blot technology. Apoptosis signal transduction pathway was elucidated by the time/dose-effect relationship of protein expression.
Results
(1) Furanodiene W/O/W multiple emulsions prepared by two-step emulsification method had even particle size, good dispersion and stable quality. The labeled content was99%±2.1%, which fitted to the requirement of formulation quality. Anti-tumor activity results showed that furanodiene could inhibit the growth of S-180sarcoma after intraperitoneal injection at low, middle and high doses and the tumor growth inhibition rates were18.55%±3.5%,19.82%±2.7%and29.82%±3.4%, which showed significant difference compared with multiple emulsions control group (p<0.05). The tumor growth inhibition rate of high dose group was similar to the positive control drug (cyclophosphamide,33.53%±3.5%, p>0.05). There was significant difference only between high dose group (20.76%±4.5%) and multiple emulsions control group after oral at low, middle and high doses (p<0.05), which indicated the poor activity of oral administration group. The survival-extending rate results showed that furanodiene could significantly inhibit the abdominal circumference growth of S-180ascetics'tumor beating mice, but could not prolong the life of mice, which suggested the toxicity lead to death. Spleen index compared with blank control group, was significantly increased (p<0.01), meanwhile, liver index was increased too (p<0.05). Blood cell count results showed that furanodiene could cause leukopenia (p<0.01). Erythropenia (p<0.05) and thrombocytopenia (p<0.01) occurred in the high dose group. Serum biochemical index results showed that AST level was significantly increased (p<0.01). Tissue pathological slices results suggested that spleen tissue structure was damaged and founded extramedullary hematopoiesis. It indicated that the toxic target organ might be bone marrow hematopoietic system.
(2) Blood cell count results showed that furanodiene could cause leukopenia (p<0.01) in healthy mice after intraperitoneal injection at low, middle and high multiple doses. Thrombocytopenia (p<0.05) occurred in the high dose group. Serum biochemical index results showed that GM-CSF level was significantly decreased (p<0.01). TPO level was decreased in the high dose group (p<0.05). ALT, AST, ALP and MDA level compared with blank control group were increased in the high dose group (p<0.05). Spleen index compared with blank control group, was significantly increased (p<0.01), meanwhile, liver index was increased too (p<0.05). Tissue pathological slices results suggested that cell numbers were decreased in the marrow cavity, spleen was founded extramedullary hematopoiesis and liver also was founded slight extramedullary hematopoiesis. Western blot results showed that furanodiene could increase the ratio of Bax/Bcl-2via up-regulation the protein expression of Bax, down-regulation the protein expression of Bcl-2. The ratio of Bax/Bcl-2was2.22±0.65,5.32±1.56and6.03±2.43, respectively. Compared with blank control group, there was significant difference in low, middle and high dose group (p<0.01) and the dose-effect relationship was good. The clear bands of cleaved caspase-3suggested that furanodiene induced bone marrow apoptosis lead to myelosuppression.
Furanodiene could decrease the content of protein and CYP450in rat liver microsome treated with furanodiene multiple emulsions at low, middle and high multiple doses by intraperitoneal injection. Compared with blank control group, there was significant difference in the middle and high dose group (p<0.05). Meanwhile, metabolic inhibition of furanodiene was founded in the middle and high dose group. Compared with blank control group, the enzyme kinetics parameters (Km and Vmax) decreased significantly (p<0.05), which showed furanodiene could probably inhibit CYP450. The specific substrate experiments results showed that the metabolic rates, compared with blank control group, S-mephenytoin and phenytoin in rat liver microsomes treated with high multiple doses had significant difference(p<0.01,p<0.05), which indicated the inhibitory effect of furanodiene on CYP2B6and2C9.
(3) The concentration-time curve of furanodiene in rats after intravenous injection furanodiene multiple emulsions at a low, middle and high single dose is fitted to a two-compartment model. T1/2α was3.9±2.3,4.3±1.2and4.8±2.1min and T12β was73.2±7.9,64.2±14.7and69.3±12.1min, which showed the rapid distribution and elimination in rats. AUC was83231.34±9784.3,151481.88±14573.1and834672.65±67481.43min-ng-mL"1, respectively. A dose proportionality study indicated that there was good correlation between AUC and dose. The linear equation was y=16845x-8510.6(r=0.987) which showed that the pharmacokinetics of furanodiene in rats was of the linear pharmacokinetic characteristic. The plasma concentration of furanodiene in rats after intraperitoneal injection and oral furanodiene multiple emulsions at a low, middle and high single dose, was too low to calculate the pharmacokinetic parameters. Everted gut sacs experiment results showed that the cumulative penetration of furanodiene group was3456±534,3024±368and2687±286ng and the intestinal residue was8654±1654,7684±957and7342±1256ng in60min at the order of duodenum, jejunum and ileum. The cumulative penetration of furanodiene multiple emulsions group was3456±534,3024±368and2687±286ng and the intestinal residue was2459±625,2861±589and2103±574ng in60min at the order of duodenum, jejunum and ileum. Tissue distribution results showed that furanodiene underwent a rapid and wide distribution in tissues within the time course examined. The highest tissue concentrations after intravenous injection were found in the spleen, followed by liver, heart, lung, kidney small intestine and brain. The highest tissue concentrations after oral or intraperitoneal injection were found in the small intestine, followed by spleen, heart, liver, lung, kidney and brain. Drug accumulation results showed that the drug content in the toxic target organs increased with the extension of administration days. The drug content in spleen was25.3±3.3ng·g-1on the first day and43.1±4.1ng·g-1on the seventh day. The drug content in the bone marrow was1.6±0.4ng·g-1on the first day and5.1‘0.5ng·g-1on the seventh day. The drug content in the thymus was0.9±0.2ng·g-1on the first day and2.5±0.2ng·g-1on the seventh day.
(4) MTT results showed that furanodiene could inhibit the growth of SP2/0cell cultured in vitro with a good dose-dependent relationship. The calculated IC50was5.2μng-mL"1. Nuclear morphology characteristic of SP2/0apoptosis cells treated with furanodiene was observed by using light microscope. There were hypodiploid apoptotic peaks pre-G0-G1phase of cell cycle from DNA figure by flow cytometry (FCM). Significant statistics difference (p<0.05) was showed in apoptosis rates of SP2/0cells treated with furanodiene for different time and different concentration by annexin V-FITC method. Western blot results showed that the ratio of Bax/Bcl-2increased form1.83±0.6,8.35±4.3to14.6±5.6with the increase of furanodiene concentration, Cyt-C in the mitochondria decreased form0.7±0.2,0.4±0.3to0.5±0.2, Cyt-C in the cytoplasm increased form2.6±0.7,5.9±1.3to8.3±2.5, Fas increased form1.14±0.42,1.57±0.63to1.73±0.48and Fas-L increased form0.39±0.09,0.58±0.26to0.68±0.32. The ratio of Bax/Bcl-2increased form4.26±1.4,10.99±3.5to14.6±5.6with the extension of furanodiene treated time,Cyt-c in the mitochondria decreased form0.5±0.2,0.3±0.1to0.2±0.1, Cyt-c in the cytoplasm increased form5.3±0.8,6.3±1.7to8.3±2.5, Fas increased form0.69±0.21,0.88±0.24to1.73±0.48and Fas-L increased form0.38±0.14,0.43±0.21to0.68±0.32. All of the results showed that the apoptosis related protein expression had good time-/concentration-dependent relationship. The clear bands of cleaved caspase-9, cleaved caspase-8and cleaved caspase-3suggested that furanodiene induced SP2/0cell apoptosis via the combined actions of mitochondrial pathway and death receptor pathway.
Conclusions
(1) The preparation technology of furanodiene W/O/W multiple emulsions are simple, practical and easily controlled, which can provide guarantee for the study on pharmacodynamics, pharmacokinetics and toxicology. Furanodiene shows strong activity in vivo against S180mice-via intraperitoneal injection, and its main toxic target organs may be bone marrow hematopoietic system and liver.
(2) The toxic target organ confirmed in healthy mice is bone marrow hematopoietic system. Toxic mechanism may be that furanodiene can induce apotosis of bone marrow cells via up-regulation the protein expression of Bax, down-regulation the protein expression of Bcl-2and break balance between Bax and Bcl-2. Moreover, furanodiene shows significant thepatotoxicity via inhibitory effect on CYP2B6and2C9.
(3) The half-life of furanodiene is short and elimination is rapid. The concentration-time curve in rats after intravenous injection furanodiene multiple emulsions with single dose is fitted to a two-compartment model and the linear pharmacokinetic characteristic. The reason of low concentration after extravascular administration is probably that the transport of emulsion drops may via lymph circulation instead of blood circulation, which can not affect the efficacy and toxicity. Drug accumulation in toxic target organs may be a key reason which plays an important role in the toxic occurrence and development.
(4) Furanodiene does not exhibit effect on SP2/0cell cycle; however, it could induce SP2/0cell apoptosis via the combined actions of mitochondrial pathway and death receptor pathway.
制备W/O/W型呋喃二烯复乳,解决呋喃二烯水溶性低、动物难以给药的问题,并评价其对S-180荷瘤小鼠的抗肿瘤活性和初步毒性作用；对呋喃二烯在小鼠体内的毒性靶器官进行确证并进行初步机制研究；研究呋喃二烯诱导SP2\0细胞凋亡的机制和信号传导通路；评价呋喃二烯复乳在大鼠体内的动力学特征、组织分布以及毒性靶器官中的蓄积；最终探讨呋喃二烯在动物体内的毒性作用、毒性靶器官、毒性靶器官中的药物蓄积以及可能的引起毒性的分子机制,为预测其他海洋来源结构类似物的毒性作用提供研究基础和理论依据。
方法
(1)采用两步乳化法制备W/O/W型呋喃二烯复乳,通过对处方组成和制备工艺进行优化,确定最优处方,通过显微镜下观察和染色两种对W/O/W型复乳进行鉴定,采用HPLC法建立呋喃二烯的体外测定方法,对复乳进行质量控制。以肿瘤移植小鼠为模型,以环磷酰胺为阳性对照药,分别研究腹腔注射和口服灌胃低、中、高三种剂量呋喃二烯复乳后对S-180荷瘤小鼠体内肿瘤生长的抑制作用,同时考察实验小鼠体重、脏器指数、外周血细胞数目、血清主要生化指标和组织病理切片的改变,评价呋喃二烯的药效和毒性作用,初步判断其毒性靶器官；
(2)以健康动物为实验对象,小鼠多剂量腹腔注射低、中、高三种剂量的呋喃二烯复乳,考察小鼠体重、脏器指数、外周血细胞数目、血清主要生化指标和组织病理切片的改变,进一步明确呋喃二烯的毒性作用和毒性靶器官。采用Western blot技术测定小鼠骨髓细胞中凋亡相关蛋白表达的变化,初步考察呋喃二烯致小鼠骨髓抑制的机制。大鼠多剂量腹腔注射低、中、高三种剂量的呋喃二烯复乳,采用超速离心法制备大鼠肝微粒体,通过对肝微粒体中蛋白含量和CYP450含量测定,初步评价呋喃二烯对大鼠肝微粒体CYP450酶的影响；采用HPLC法测定呋喃二烯在肝微粒体中的代谢率并计算酶动力学参数,通过底物探针代谢实验进一步确证其对CYP450酶系各亚型的影响作用。
(3)以大鼠为实验对象,采用HPLC-APCI-MS/MS技术,测定大鼠分别尾静脉注射、腹腔注射以及口服灌胃低、中、高三种不同剂量呋喃二烯复乳后的血药浓度,计算药物动力学参数；采用外翻肠囊法分别测定呋喃二烯原药和复乳的累积透过量及肠段残留量；测定大鼠单剂量尾静脉注射、腹腔注射以及口服灌胃呋喃二烯复乳后的组织分布,并对大鼠多剂量腹腔注射和口服灌胃后药物在毒性靶器官中的蓄积进行了监测。
(4)以SP2/0为实验细胞株,采用MTT法测定呋喃二烯对体外培养细SP2/0细胞的增殖抑制率,并计算IC50；通过倒置显微镜观察细胞形态的变化；采用流式细胞技术测定呋喃二烯诱导SP2/0细胞的凋亡率、DNA含量以及对细胞周期的影响。采用Western blot技术检测凋亡相关蛋白Bax、Bcl-2、Caspase-9、 Cyt-c、Fas、Fas-L、Caspase-8以及Caspase-3等表达的变化。采用RT-PCR技术检测凋亡相关基因Bax、Bcl-2、Caspase-3、8、9mRNA的变化,通过时效和量效关系说明呋喃二烯诱导SP2/0细胞凋亡的信号转导通路。
结果
(1)两步乳化法制得的W/O/W复乳粒径大小均匀,分散性好,质量稳定,标示含量为99%±2.1%,符合制剂质量要求,可用于动物给药的制剂。腹腔注射呋喃二烯低、中、高三种不同剂量的复乳后,S-180荷瘤小鼠的肉瘤生长均得到一定程度的抑制,抑瘤率分别为18.55%±3.5%、19.82%±2.7%和29.82%±3.4%,与空白复乳组比较具有显著性差异(p<0.05)。高剂量组的抑瘤率与阳性对照组环磷酰胺(33.53%±3.5%)比较无显著性差异(p>0.05)。口服灌胃三种不同剂量的复乳后,与空白复乳组比较,只有高剂量组(20.76%±4.5%)具有显著性差异(p<0.05)。说明腹腔注射组具有一定的抑瘤率,而口服灌胃组的作用较弱。腹腔注射呋喃二烯可显著抑制S-180腹水瘤小鼠腹围的增长(p<0.01),但不能显著延长小鼠的生存时间,预测可能是其毒性引起的小鼠死亡。脏器指数与空白对照组比较,脾脏指数明显升高(p<0.01),其次为肝脏指数(p<0.05)。外周血细胞计数结果显示呋喃二烯可引起小鼠全血血细胞的变化,其中白细胞的降低最为明显(p<0.01),高剂量组还可同时引起红细胞(p<0.05)和血小板的减少(p<0.01)。血清生化指标测定结果显示呋喃二烯可引起血清中ALT、AST、BUN和SCR水平的变化,其中以AST水平的升高最为显著(p<0.01)。组织切片结果显示呋喃二烯给药组小鼠脾脏组织结构被破坏,红髓内出现幼稚粒系细胞髓外造血,提示骨髓造血系统可能是其毒性靶器官。
(2)多剂量腹腔注射呋喃二烯低、中、高三种不同剂量的复乳后可引起健康小鼠全血血细胞的变化,其中以白细胞的降低最为明显(p<0.01),高剂量组还可同时引起血小板减低(p<0.05)。同时可引起骨髓造血系统损伤相关血清生化指标EPO、TPO及GM-CSF水平的变化,其中以GM-CSF水平的降低最为明显(p<0.01),高剂量组还可同时引起TPO水平的降低(p<0.05)。肝损伤相关血清生化指标ALT、AST、ALP、MDA及SOD的变化不大,只有高剂量组可引起ALT、AST、ALP和MDA的升高(p<0.05)。脏器指数以脾脏指数的升高最为明显(p<0.01),其次为肝脏指数(p<0.05)。组织切片显示呋喃二烯给药组小鼠的骨髓髓腔内细胞数目减少,且脾脏出现明显的粒系髓外造血,肝脏也出现轻微的粒系髓外造血,提示存在明显的骨髓抑制作用,其他脏器的组织学变化不大。小鼠骨髓细胞Western blot检测结果显示,呋喃二烯给药组可上调Bax的表达、下调Bcl-2的表达,腹腔注射呋喃二烯低、中、高三种不同剂量的复乳后Bax/Bcl-2的比值依次为：2.22±0.65、5.32±1.56、6.03±2.43,与空白对照组比较p<0.01,存在一定的剂量相关性,且出现明显的Cleaved capase-3条带,说明呋喃二烯通过诱导骨髓细胞的凋亡导致骨髓抑制。
多剂量腹腔注射呋喃二烯低、中、高三种不同剂量的复乳后,超速离心法制备肝微粒体,与空白对照组比较,呋喃二烯中、高剂量组可引起肝微粒体中蛋白含量和CPY450含量的减少(p<0.05)。并且呋喃二烯在经中、高剂量处理过的肝微粒中具有明显的代谢抑制现象,Km和Vmax的值均有一定程度的降低(p<0.05),提示呋喃二烯对CYP450可能存在一定的抑制作用。底物探针实验结果显示：S-美芬妥英和苯妥英在经高剂量呋喃二烯处理过的肝微粒体中的代谢速度与空白对照组比较具有显著性差异(p<0.01和p<0.05),其他底物的代谢速度与空白对照组比较无统计学意义,提示呋喃二烯可能对CYP2B6和2C9存在一定的抑制作用。
(3)大鼠尾静脉单剂量注射低、中、高三种不同剂量的呋喃二烯复乳后,其药动学特征符合二室开放模型,t1/2。分别为3.9±2.3、4.3±1.2和4.8±2.1min, t1/2β分别为73.2±7.9、64.2±14.7、69.3±12.1min,表明药物在大鼠体内的分布和消除都较快。曲线下面积(AUC)分别为83231.34±9784.3、151481.88±14573.1和834672.65±67481.43min·ng·mL-1,采用最小二乘法对AUC和剂量进行相关性考察,直线方程为y=16845x-8510.6(r=0.987),表明呋喃二烯在大鼠体内呈现线性药动学特征。单剂量腹腔注射或者口服灌胃给予大鼠低、中、高三种不同剂量呋喃二烯复乳后,低、中剂量基本测不到血药浓度,高剂量组所测得的血药浓度由于数值很小,无法进行药动学参数的求算。外翻肠囊法评价药物吸收的实验结果显示,呋喃二烯原药组在小肠各段60min的累积透过量分别为956±324、657±126、542±148ng,其在小肠各段的残留分别为8654±1654、7684±957、7342±1256ng；呋喃二烯复乳组在小肠各段60min的累积透过量分别为3456±534、3024±368、2687±286ng,其在小肠各段的残留分别为2459±625、2861±589、2103±574ng,累积透过量按十二指肠、空肠、回肠依次减少。组织分布结果显示,大鼠尾静脉单剂量注射呋喃二烯复乳后迅速在体内广泛分布,在脾脏中的浓度最高,其次按肝脏、心脏、肺脏、肾脏、小肠和脑依次减少。单剂量腹腔注射或口服给药后,在小肠中的浓度最高,其次按为脾脏、心脏、肝脏、肺脏、肾脏和脑依次减少。药物蓄积结果显示：大鼠多剂量腹腔注射呋喃二烯复乳后,毒性靶器官中的药物含量在逐渐升高,脾脏中的含药量从第1天的25.3±3.3rng·g-1升高到第7天的43.1±4.1ng·g-1,骨髓中的含药量从第1天的1.6±0.4ng·g-1升高到第7天的5.1±0.5ng·g-1,胸腺中的含药量从第1天的0.9±0.2ng·g-1升高到第7天的2.5±0.2ng·g-1。
(4)MTT法检测细胞存活率试验结果显示,呋喃二烯能抑制SP2/0细胞的生长,具有一定的量效关系,经计算IC50为5.2μg·mL-1。通过倒置显微镜观察到呋喃二烯处理过的SP2/0细胞呈现凋亡的形态学特征。经FCM分析,Annexin V-FITC法实验显示不同浓度的呋喃二烯作用不同时间后SP2/0细胞的凋亡率与对照组相比有统计学差异(p<0.05),并且从DNA组方图上可见到Go-G1期前亚二倍体凋亡峰。Western blot检测结果显示,随着呋喃二烯作用浓度的增加,细胞中Bax/Bcl-2的比值由1.83+0.6、8.35±4.3升至14.6±5.6,线粒体内的Cty-c由0.7±0.2、0.440.3降至0.5±0.2,胞浆内的Cty-c由2.6±0.7、5.9±1.3升至8.3±2.5,细胞中Fas由1.14±0.42、1.57±0.63升至1.73±0.48,Fas-L由0.39±0.09、0.58+0.26升至0.68+0.32,具有明显的浓度依赖性,并出现了清晰的Cleaved caspase-9、8和3条带。随着呋喃二烯作用时间的延长,细胞中Bax/Bcl-2的比值由4.26±1.4、10.99±3.5升至14.6±5.6,线粒体内的Cty-c由0.5±0.2、0.3+0.1降至0.2±0.1,胞浆内的Cty-c由5.3±0.8、6.3±1.7升至8.3±2.5,细胞中Fas由0.69±0.21、0.88±0.24升至1.73±0.48,Fas-L由0.38+0.14、0.43±0.21升至0.68±0.32,具有明显的时间依赖性,也出现清晰的Cleaved caspase-9、8和3条带；Bax, bcl-2, caspase-8, caspase-9,caspase-3的mRNA表达水平随呋喃二烯的浓度和作用时间的不同而变化,具有一定的时效与量效关系。说明呋喃二烯可通过线粒体途径和死亡受体诱导SP2/0细胞凋亡。
结论
(1)W/O/W型呋喃二烯复乳制备工艺简单,质量稳定,为动物体内的药效学、药动学及毒理学研究提供保障；腹腔注射呋喃二烯复乳对S-180荷瘤小鼠具有一定的抗肿瘤活性,同时主要毒性靶器官可能是骨髓造血系统和肝脏；
(2)确证了呋喃二烯在健康小鼠体内的毒性靶器官主要为骨髓造血系统,可通过上调Bax、下调Bcl-2、破坏Bax和Bcl-2之间的平衡诱导骨髓细胞凋亡。呋喃二烯对肝脏的毒性主要表现在对CYP2B6和2C9有一定的抑制作用。
(3)大鼠尾静脉单剂量注射呋喃二烯复乳后,其药动学符合二室模型,消除半衰期较短,呈现线性动力学特征。经血管外途径给药后血药浓度低的原因可能是乳滴经毛细淋巴管途径进入大循环后迅速转运至组织,因此不影响呋喃二烯发挥药效以及产生毒性。呋喃二烯在毒性靶器官中的蓄积也是其毒性发生、发展的原因之一。
(4)呋喃二烯不引起小鼠骨髓瘤(SP2/0)细胞周期的变化,其诱导SP2/0细胞凋亡是线粒体途径和死亡受体途径共同作用的结果。
Objective
To prepare water-in-oil-in-water multiple emulsions for furanodiene in order to solve difficulties of poor soluble and animal administration, investigate the antitumor activity and preliminary toxicity to S180carcinoma in vivo, and confirm the toxic target organs in mice and the preliminary toxic mechanism. To study the mechanism on furanodiene induced SP2\0apoptosis and its signal transduction pathway, evaluate the pharmacokinetics, tissue distribution of furanodiene multiple emulsions in rats and accumulate in toxic target organs.
Methods
(1) A Two-step emulsification method was used to prepare the W/O/W multiple emulsions. The prescription and preparation of emulsions were optimized. Both the primary and multiple emulsions were identified through microscope-observation and eosin staining method. The HPLC method was established and used to the quality control of furanodiene multiple emulsions in vitro. S-180sarcoma transplanted mice as animal model and cyclophosphamide as positive control, the tumor growth inhibition rates were measured after intraperitoneal injection and oral at low, middle and high doses. Meanwhile, the weight change, viscera index, blood cell count, serum biochemical indexes and tissue pathological slices were used to evaluate the toxicity.
(2) The healthy mice were used as experiment objects with the changes of their weight, viscera index, blood cell count, serum biochemistry and tissue pathological slices as evaluation indexes, the toxic effects and target organs were confirmed after intraperitoneal injection at low, middle and high multiple doses. In order to investigate the preliminary mechanism of furanodiene induced myelosuppression, the changes of apoptosis related protein expression were measured by Western blot technology. Liver microsomes of rats which treated with furanodiene multiple emulsions at low, middle and high multiple doses by intraperitoneal injection were prepared by using ultracentrifuge method. The concentration of protein in liver microsome determined by the BCA method and the content of cytochrome P450detected by the Omura and Sato method were used to evaluate the effect of furanodiene on CYP450preliminarily. In order to calculate the enzyme kinetics parameters, a HPLC method for determination of furanodiene in rat liver microsomes was established and the in vitro metabolic rates were studied by incubation with rat liver microsomes. The effect of furanodiene on CYP450subtypes was confirmed by the specific substrate experiments.
(3) The plasma concentrations of furanodiene were determined by HPLC-APCI-MS/MS method after intravenous, intraperitoneal injection and oral furanodiene multiple emulsions at low, middle and high single dose in rats, respectively. Pharmacokinetics parameters were calculated by Winnolin software. The cumulative penetration and intestinal residue of furanodiene and its multiple emulsions were determined by the everted gut sacs method. Tissue distribution of furanodiene were studied in rats after oral, intraperitoneal and intravenous injection furanodiene multiple emulsions with single dose. Meanwhile, drug accumulation were monitored in the toxic target organs after oral and intraperitoneal injection furanodiene multiple emulsions with mutable doses, respectively.
(4) The growth inhibitions of SP2/0cell cultured in vitro were analyzed by MTT method, than IC50was calculated. Cell morphology changes were observed by using light microscope. Flow cytometry (FCM) was used to study the apoptosis rates, content of DNA and effects of furanodiene on SP2/0cells cycle. The changes of apoptosis related protein expression such as Bax, Bcl-2, Caspase-9, Cyt-c, Fas, Fas-L, Caspase-8and Caspase-3, were measured by Western blot technology. Apoptosis signal transduction pathway was elucidated by the time/dose-effect relationship of protein expression.
Results
(1) Furanodiene W/O/W multiple emulsions prepared by two-step emulsification method had even particle size, good dispersion and stable quality. The labeled content was99%±2.1%, which fitted to the requirement of formulation quality. Anti-tumor activity results showed that furanodiene could inhibit the growth of S-180sarcoma after intraperitoneal injection at low, middle and high doses and the tumor growth inhibition rates were18.55%±3.5%,19.82%±2.7%and29.82%±3.4%, which showed significant difference compared with multiple emulsions control group (p<0.05). The tumor growth inhibition rate of high dose group was similar to the positive control drug (cyclophosphamide,33.53%±3.5%, p>0.05). There was significant difference only between high dose group (20.76%±4.5%) and multiple emulsions control group after oral at low, middle and high doses (p<0.05), which indicated the poor activity of oral administration group. The survival-extending rate results showed that furanodiene could significantly inhibit the abdominal circumference growth of S-180ascetics'tumor beating mice, but could not prolong the life of mice, which suggested the toxicity lead to death. Spleen index compared with blank control group, was significantly increased (p<0.01), meanwhile, liver index was increased too (p<0.05). Blood cell count results showed that furanodiene could cause leukopenia (p<0.01). Erythropenia (p<0.05) and thrombocytopenia (p<0.01) occurred in the high dose group. Serum biochemical index results showed that AST level was significantly increased (p<0.01). Tissue pathological slices results suggested that spleen tissue structure was damaged and founded extramedullary hematopoiesis. It indicated that the toxic target organ might be bone marrow hematopoietic system.
(2) Blood cell count results showed that furanodiene could cause leukopenia (p<0.01) in healthy mice after intraperitoneal injection at low, middle and high multiple doses. Thrombocytopenia (p<0.05) occurred in the high dose group. Serum biochemical index results showed that GM-CSF level was significantly decreased (p<0.01). TPO level was decreased in the high dose group (p<0.05). ALT, AST, ALP and MDA level compared with blank control group were increased in the high dose group (p<0.05). Spleen index compared with blank control group, was significantly increased (p<0.01), meanwhile, liver index was increased too (p<0.05). Tissue pathological slices results suggested that cell numbers were decreased in the marrow cavity, spleen was founded extramedullary hematopoiesis and liver also was founded slight extramedullary hematopoiesis. Western blot results showed that furanodiene could increase the ratio of Bax/Bcl-2via up-regulation the protein expression of Bax, down-regulation the protein expression of Bcl-2. The ratio of Bax/Bcl-2was2.22±0.65,5.32±1.56and6.03±2.43, respectively. Compared with blank control group, there was significant difference in low, middle and high dose group (p<0.01) and the dose-effect relationship was good. The clear bands of cleaved caspase-3suggested that furanodiene induced bone marrow apoptosis lead to myelosuppression.
Furanodiene could decrease the content of protein and CYP450in rat liver microsome treated with furanodiene multiple emulsions at low, middle and high multiple doses by intraperitoneal injection. Compared with blank control group, there was significant difference in the middle and high dose group (p<0.05). Meanwhile, metabolic inhibition of furanodiene was founded in the middle and high dose group. Compared with blank control group, the enzyme kinetics parameters (Km and Vmax) decreased significantly (p<0.05), which showed furanodiene could probably inhibit CYP450. The specific substrate experiments results showed that the metabolic rates, compared with blank control group, S-mephenytoin and phenytoin in rat liver microsomes treated with high multiple doses had significant difference(p<0.01,p<0.05), which indicated the inhibitory effect of furanodiene on CYP2B6and2C9.
(3) The concentration-time curve of furanodiene in rats after intravenous injection furanodiene multiple emulsions at a low, middle and high single dose is fitted to a two-compartment model. T1/2α was3.9±2.3,4.3±1.2and4.8±2.1min and T12β was73.2±7.9,64.2±14.7and69.3±12.1min, which showed the rapid distribution and elimination in rats. AUC was83231.34±9784.3,151481.88±14573.1and834672.65±67481.43min-ng-mL"1, respectively. A dose proportionality study indicated that there was good correlation between AUC and dose. The linear equation was y=16845x-8510.6(r=0.987) which showed that the pharmacokinetics of furanodiene in rats was of the linear pharmacokinetic characteristic. The plasma concentration of furanodiene in rats after intraperitoneal injection and oral furanodiene multiple emulsions at a low, middle and high single dose, was too low to calculate the pharmacokinetic parameters. Everted gut sacs experiment results showed that the cumulative penetration of furanodiene group was3456±534,3024±368and2687±286ng and the intestinal residue was8654±1654,7684±957and7342±1256ng in60min at the order of duodenum, jejunum and ileum. The cumulative penetration of furanodiene multiple emulsions group was3456±534,3024±368and2687±286ng and the intestinal residue was2459±625,2861±589and2103±574ng in60min at the order of duodenum, jejunum and ileum. Tissue distribution results showed that furanodiene underwent a rapid and wide distribution in tissues within the time course examined. The highest tissue concentrations after intravenous injection were found in the spleen, followed by liver, heart, lung, kidney small intestine and brain. The highest tissue concentrations after oral or intraperitoneal injection were found in the small intestine, followed by spleen, heart, liver, lung, kidney and brain. Drug accumulation results showed that the drug content in the toxic target organs increased with the extension of administration days. The drug content in spleen was25.3±3.3ng·g-1on the first day and43.1±4.1ng·g-1on the seventh day. The drug content in the bone marrow was1.6±0.4ng·g-1on the first day and5.1‘0.5ng·g-1on the seventh day. The drug content in the thymus was0.9±0.2ng·g-1on the first day and2.5±0.2ng·g-1on the seventh day.
(4) MTT results showed that furanodiene could inhibit the growth of SP2/0cell cultured in vitro with a good dose-dependent relationship. The calculated IC50was5.2μng-mL"1. Nuclear morphology characteristic of SP2/0apoptosis cells treated with furanodiene was observed by using light microscope. There were hypodiploid apoptotic peaks pre-G0-G1phase of cell cycle from DNA figure by flow cytometry (FCM). Significant statistics difference (p<0.05) was showed in apoptosis rates of SP2/0cells treated with furanodiene for different time and different concentration by annexin V-FITC method. Western blot results showed that the ratio of Bax/Bcl-2increased form1.83±0.6,8.35±4.3to14.6±5.6with the increase of furanodiene concentration, Cyt-C in the mitochondria decreased form0.7±0.2,0.4±0.3to0.5±0.2, Cyt-C in the cytoplasm increased form2.6±0.7,5.9±1.3to8.3±2.5, Fas increased form1.14±0.42,1.57±0.63to1.73±0.48and Fas-L increased form0.39±0.09,0.58±0.26to0.68±0.32. The ratio of Bax/Bcl-2increased form4.26±1.4,10.99±3.5to14.6±5.6with the extension of furanodiene treated time,Cyt-c in the mitochondria decreased form0.5±0.2,0.3±0.1to0.2±0.1, Cyt-c in the cytoplasm increased form5.3±0.8,6.3±1.7to8.3±2.5, Fas increased form0.69±0.21,0.88±0.24to1.73±0.48and Fas-L increased form0.38±0.14,0.43±0.21to0.68±0.32. All of the results showed that the apoptosis related protein expression had good time-/concentration-dependent relationship. The clear bands of cleaved caspase-9, cleaved caspase-8and cleaved caspase-3suggested that furanodiene induced SP2/0cell apoptosis via the combined actions of mitochondrial pathway and death receptor pathway.
Conclusions
(1) The preparation technology of furanodiene W/O/W multiple emulsions are simple, practical and easily controlled, which can provide guarantee for the study on pharmacodynamics, pharmacokinetics and toxicology. Furanodiene shows strong activity in vivo against S180mice-via intraperitoneal injection, and its main toxic target organs may be bone marrow hematopoietic system and liver.
(2) The toxic target organ confirmed in healthy mice is bone marrow hematopoietic system. Toxic mechanism may be that furanodiene can induce apotosis of bone marrow cells via up-regulation the protein expression of Bax, down-regulation the protein expression of Bcl-2and break balance between Bax and Bcl-2. Moreover, furanodiene shows significant thepatotoxicity via inhibitory effect on CYP2B6and2C9.
(3) The half-life of furanodiene is short and elimination is rapid. The concentration-time curve in rats after intravenous injection furanodiene multiple emulsions with single dose is fitted to a two-compartment model and the linear pharmacokinetic characteristic. The reason of low concentration after extravascular administration is probably that the transport of emulsion drops may via lymph circulation instead of blood circulation, which can not affect the efficacy and toxicity. Drug accumulation in toxic target organs may be a key reason which plays an important role in the toxic occurrence and development.
(4) Furanodiene does not exhibit effect on SP2/0cell cycle; however, it could induce SP2/0cell apoptosis via the combined actions of mitochondrial pathway and death receptor pathway.
引文
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