二-（4-氯苯甲酰异羟肟酸）二正丁基合锡（DBDCT）的药代动力学和抗癌作用机制研究
摘要
目的合成抗癌化合物二-(4-氯苯甲酰异羟肟酸)二正丁基合锡(DBDCT),制备注射剂和脂质体两种制剂,建立其质量标准,进行体外和体内抗肿瘤活性测定,研究大鼠尾静脉注射DBDCT后的药代动力学,并通过体外肿瘤细胞培养研究其引起肿瘤细胞毒的作用机制。
方法(1)合成2种有机锡化合物,并通过红外光谱(IR)、核磁共振氢谱(~1H NMR)、碳谱(~(13)C NMR)、锡谱(~(119)Sn NMR)、质谱(MS)以及X-射线单晶衍射(X-Ray)进行结构表征。选择体外活性较强的化合物2(DBDCT)作为研究对象,制备其注射剂和脂质体两种制剂,并建立两种剂型的质量标准;(2)通过Bliss法测定小鼠静脉注射DBDCT后的半数致死量(LD_(50)),为体内抗肿瘤药效学和药代动力学研究给药剂量提供依据;(3)以肿瘤移植小鼠为模型,以顺铂(DDP)为阳性对照,研究静脉注射低、中、高三个剂量DBDCT后S_(180)、H_(22)和EAC小鼠体内的抗癌活性,同时考察实验小鼠外周血白血球数目、免疫器官胸腺和脾脏重量和指数、脏器重量和系数以及血液主要生化指标等,初步判断药物作用的毒性靶器官,采用G-CSF ELISE试验,检测DBDCT是否引起造模小鼠的炎症;(4)通过HPLC和UPLC-MS技术,研究大鼠尾静脉单剂量注射高、中、低三种剂量DBDCT注射液后原药及其主要代谢物在生物体内的吸收、分布、生物转化和排泄等过程,通过平衡透析法测定DBDCT与大鼠血浆蛋白结合率;(5)DBDCT作用后,采用MTT法测定体外培养的细胞株的增殖抑制率,并以SGC-7901为代表,采用流式细胞术(FCM)研究DBDCT对SGC-7901细胞增殖指数和细胞周期影响,通过光镜、荧光染色和电镜等方法观察细胞核形态的变化,FCM和Annexin V-FITC方法测定细胞凋亡率,DNA Lander方法检测肿瘤细胞凋亡的特异性条带,用RT-PCR法检测p53、p21、Bcl-2和Bax表达,免疫组化法检测p21和PCNA蛋白表达情况;(6)采用分光光度法测定DBDCT对SGC-7901癌细胞作用前后Caspase-3、Caspase-8和Caspase-9活性的变化;用Caspase-8抑制剂阻断DBDCT诱导SGC-7901细胞的Caspase-8途径产生的凋亡;采用新型荧光染料Flou 3-AM检测Ca~(2+)水平,以罗丹明123测定线粒体跨膜电位(△Ψm)变化,同时检测作为第二信使的活性氧(ROS)的表达,并通过Western blot分析Caspase-3、Cyt-C、Bax和Bcl-2等蛋白表达,进一步证实Caspases凋亡信号转导通路。
结果(1)得到了2种新的有机锡化合物,分别为:[(n-Bu)_2Sn(C_7H_5NO_2Cl)Cl]和[(n-Bu)_2Sn(C_7H_5NO_2Cl)_2](DBDCT)。制备了DBDCT注射剂(7.5mg/5ml)和脂质体(10mg/10ml)两种剂型,并建立了质量标准,注射剂质量标准包括性状、鉴别、检查(pH值、装量、有关物质和无菌、热源)、含量测定等;脂质体包括形态、粒径、包封率、突释效应和有机溶剂检查、含量测定等,为后续动物试验质量控制提供了保障;(2)急性毒性试验表明静脉注射DBDCT后的半数致死量LD_(50)=21.1mg/kg,LD_(50)(Feiller校正)95%的可信限为17.4～25.5mg/kg,LD_(50)的标准误为0.01;(3)体内抗肿瘤药效学实验中,DBDCT注射液组对S_(180)和H_(22)的生长显示了不同的抑制作用,中剂量和高剂量组与空白对照组相比有显著性差异(P＜0.05和P＜0.01)。各剂量组对EAC小鼠的生命延长率显示了不同的影响,三次测定低剂量组生命延长率分别为8.43%、30.97%、24.82%,中剂量组生命延长率分别为85.1%、79.2%、73.4%,高剂量组生命延长率分别为87.2%、82.6%、79.6%,中剂量组和高剂量组生命延长率远远高于阳性对照组(P＜0.001)。体内抗肿瘤活性显示出良好的剂量依赖性。采用G-CSF ELISE试验,以角叉菜胶为急性非特异性炎症模型,结果显示,实验组和阳性对照组小鼠的外周血白血球数目显著高于空白对照组,阳性对照组小鼠的G-CSF含量也显著高于空白对照组(P＜0.001),但各实验组与空白对照组相比G-CSF含量并无显著性差异(P＞0.05),提示DBDCT可能不会引起小鼠体内炎症感染、化脓菌或其毒素侵人,可能对骨髓造血系统有影响。试验中S_(180)、H_(22)和EAC小鼠的胸腺指数和脾脏指数随着DBDCT剂量的增大而降低,DBDCT可能会抑制胸腺和脾脏的发育。高剂量组DBDCT可能对肝、睾丸、胸腺、脾和卵巢有损伤;(4)大鼠尾静脉单剂量注射2、5和12mg/kg三种剂量注射液后,能迅速被分布或消除,三种剂量下分布半衰期(T_(1/2,α))很短,分别为2.3585、1.9614和2.1146min;消除半衰期(t_(1/2,β))为64.67、56.80和220.60 min。以5mg/kg的给药剂量为例对比考察了脂质体在大鼠体内的动力学参数,脂质体血药浓度可维持12h以上,药时曲线下面积AUC是相同剂量注射液的7倍以上,消除半衰期(t_(1/2β))为相同剂量注射液的12倍,清除率CL(s)比相同剂量注射液减小8倍左右,延长了DBDCT在体循环系统中的滞留时间。大鼠单次静脉注射5 mg/kg DBDCT 3min后DBDCT已迅速分布到各组织,且能够透过血脑屏障分布于脑组织,在肾上腺分布最高,其次是十二指肠,心脏、肝脏、睾丸和肾脏中均有较高的分布,在主要组织器官中无蓄积。尿、粪和胆汁中均检测不到原形药物,但尿和胆汁排泄过程中发现了多个代谢产物。DBDCT与大鼠血浆蛋白结合率高达59.6%～61.9%,表明DBDCT进入机体后大部分与血浆蛋白相结合,血中游离药物浓度较低。采用UPLC-MS对大鼠尾静脉给予5mg/kg注射液后血浆、尿液和胆汁中的出现的原药和代谢物进行了结构鉴定,初步推断了m/z575、m/z577、m/z579、m/z604、z607、m/z551、m/z663、m/z369和m/z565.7等九种化合物的结构。体内生物转化途径可能有加氢还原、乙基化和去乙基化反应、代谢产物与葡萄糖醛酸结合反应等。DBDCT在血浆、尿液和胆汁中均不稳定,逐渐脱掉配体、烷基和氯等基团而代谢转化,可能主要经肝微粒体酶脱烷基而代谢转化后大部分经肾或胆汁排出;(5)MTT细胞存活率试验显示,DBDCT能抑制白血病细胞HL60、宫颈癌细胞Hela、膀胱癌细胞T24以及胃腺癌细胞SGC-7901等细胞株的生长,作用效果具有一定的量效关系。DBDCT对SGC-7901作用不同时间(12h,24h,48,72h)后IC_(50)分别为:81.6、25.3、4.5、2.8nmol·L~(-1)。通过光镜、荧光染色和电镜观察到DBDCT处理过的SGC-7901细胞呈现凋亡细胞所特有的形态学特征,并检测到若干个凋亡小体。在琼脂糖凝胶电泳中可见凋亡特征性改变DNA梯形带。经FCM分析,从DNA组方图上可见到G_0-G_1期前亚二倍体凋亡峰。Annexin V-FITC实验显示不同浓度的DBDCT作用不同时间后SGC-7901细胞的凋亡率与对照组相比有统计学差异(P＜0.05)。FCM检测,DBDCT可能通过阻滞SGC-7901细胞G_2-M期和S期杀伤肿瘤细胞。免疫组化法检测,随着DBDCT浓度增加,p21阳性细胞数明显增加,PCNA阳性细胞数则显著减少(P＜0.01)。RT-PCR检测caspase3、p21、p53、Bcl-2和Bax基因的表达。随着DBDCT浓度和作用时间的延长,实验组p21、p53和Bax mRNA表达明显高于空白对照组(P＜0.05和P＜0.01),Bcl-2和Bcl-2/Bax的表达明显低于空白对照组(P＜0.001);(6)不同浓度DBDCT对SGC-7901作用不同时间后caspase-3、caspase-8和caspase-9酶的活性显著增加(P＜0.05和P＜0.01),证实了DBDCT诱导SGC-7901细胞凋亡与线粒体和死亡受体途径有关。采用caspase-8抑制剂不能抑制DBDCT诱导SGC-7901细胞的凋亡,确证DBDCT可通过线粒体途径诱导SGC-7901细胞凋亡。Flou 3-AM检测,细胞内Ca~(2+)浓度显著升高。罗丹明123测定,线粒体跨膜电位显著降低或消失,与DBDCT作用剂量和时间呈负相关。DBDCT迅速引起SGC-7901细胞内ROS的增加,提示DBDCT作用于细胞后可刺激肿瘤细胞ROS的产生。通过Western blot分析Caspase-3、Cyt C、Bax和Bcl-2等蛋白表达,随着DBDCT的浓度和作用时间增加,实验组Bcl-2蛋白表达下凋,而Bax、Cyt C和Caspase3等蛋白表达上调,且有明显量效和时效关系,Bel-2/Bax蛋白比值从1.30下降至0.52,与RT-PCR的结果一致,初步证实了Caspases凋亡信号转导通路。
结论(1)DBDCT注射剂和脂质体的质量标准符合要求,为后续动物试验的质量控制提供保障;(2)静脉注射DBDCT后对S_(180)、H_(22)和EAC小鼠具有较高的体内抗癌活性,主要的毒性靶器官可能是肝、睾丸、胸腺、脾和卵巢等,G-CSF ELISE试验检测DBDCT不会引起小鼠的炎症反应,可能具有提升白细胞的功能;(3)采用HPLC和UPLC-MS技术基本阐明了大鼠尾静脉注射DBDCT后在生物体内的吸收、分布、生物转化和排泄以及血浆蛋白结合率等,探讨了化合物发挥药效和毒性作用的化学物质基础;(4)确证了DBDCT能够诱导体外培养的SGC-7901肿瘤细胞发生凋亡,作用机制之一可能是通过p53信号传导通路:即p53激活Bax基因,抑制Bcl-2基因表达,通过破坏Bax与Bcl-2之间的平衡来完成p53介导的细胞凋亡信号转导机制;(5)DBDCT诱导SGC-7901细胞凋亡与线粒体途径、死亡受体途径有关。本文重点探讨了线粒体途径Caspases凋亡信号转导通路,即Caspase-8作为凋亡反应的上游调控蛋白,其活化将激活Caspase-9,Caspase-9又激活Caspase-3,细胞内游离Ca~(2+)是激活Caspase-3的先决条件之一,Ca~(2+)稳态的破坏可直接引起线粒体释放细胞色素C和一些促细胞凋亡因子。同时DBDCT诱导线粒体活性氧ROS的增加,引发Bcl-2下调和Bax上调,自由基的不断攻击引起线粒膜电位△Ψm下降,通透性增加,Cyt-C从线粒体释放后,与凋亡酶启动子结合,在脱氧三磷酸腺苷存在下激活Apaf-1。活化的Apaf-1会结合procaspase-9而启动一系列的Caspases级联反应,激活典型的线粒体凋亡通路。
Objective To synthesize antitumor diorganotin(Ⅳ) complex,di-n-butyl-(4-chlorobenzohy-droxamato) tin(Ⅳ) chloride(DBDCT),prepare its injection and liposome,establish their quality standard,study its antitumor activities in vitro and its pharmacokinetics in the plasma after single intravenous administration to rats,and investigate its antitumor cytotoxic mechanism through tumor cell culture in vitro.Methods(1)Two compounds were prepared and characterized by IR, ~1H NMR,~(13)C NMR,~(119)Sn NMR,MS and single crystal R-ray analysis.The compound 2 (DBDCT) with strong anti-tumor activity in vitro was studied.The quality standards of injection and liposome were established.(2) The median lethal dose(LD_(50)) of DBDCT injection to mice was determined by Bliss method and provided foundation for the further study of pharmacodynamics and pharmacokinetics in vivo.(3) Tumour-bear mice were used as transplantable carcinomas animal model with cis-diamminedichloroplatinum(DDP) as positive control.The weight of tumor on S_(180),H_(22) mice and the survival-extending rate of EAC mice were measured after intravenous injection at low,middle and high doses.Meanwhile,some indexes were detected such as mice peripheral blood white blood cells numbers,the immune organ weight and index number of thymus and spleen,organ weight and coefficient,blood main biochemical indicator and so on.These detections were used to preliminarily judge the active and toxic target organ of DBDCT,and G-CSF ELISE test was used to detect whether DBDCT could cause inflammation to the model mice.(4) The absorption,distribution and excretion processes of DBDCT and its main metabolites after single intravenous injection at low,middle and high doses were investigated by HPLC and UPLC-MS/MS techniques.The rat blood plasma protein binding rates of DBDCT were determined through equilibrium dialysis.(5) Growth inhibition of cell cultured in vitro were analyzed by MTT method.Flow cytometry(FCM) was used to study the effects of DBDCT on SGC-7901 cells cycle,proliferation and apoptosis. Hoechst and AO/EB staining,light and electron microscope were used to examine the nuclear changes.Apoptosis rates were determined by FCM,Annexin V-FITC methods and specific DNA ladder-shaped strap.The gene such as p53,p21,Bcl-2,Box and the proteinum(p21 and PCNA) expression changes of SGC-7901 cells treated with DBDCT were detected by RT-PCR and immunohistochemical method,respectively.(6) Using spectrophotometric method to evaluate enzymatic(caspase-3,caspase-8 and caspase-9) activities in SGC-7901 cells treated with DBDCT,simultaneously,caspase-8 inhibiter was used to block up the apoptosis of SGC-7901 cells with DBDCT treated.Apoptosis pathway would probable be illuminated.In addition,the fluorochrome 3-AM,rhodamine123 and ROS kits were applied for the studies of Ca~(2+) levels, mitochondria transmembrane potential variance(△ψm) and active oxygen ROS acted as second messenger,respectively.Meanwhile,the protein express for caspase-3,Cyt-C,Bax and Bcl-2 were analyzed by western blot method to further confirm the caspases apoptosis signal transduction pathway.Results(1) Two new diorganotin compounds were[(n-Bu)_2Sn(C_7H_(5-) NO_2Cl)Cl]and[(n-Bu)_2Sn(C_7H_5NO_2Cl)_2](DBDCT) and they were characterized by IR,~1H, ~(13)C,~(119)Sn NMR,MS spectra and single crystal X-ray analysis(DBDCT),respectively.The quality standards of DBDCT 7.5mg/5ml injection(including character,discrimination,and examination of pH,relevant materials,asepsis,heat source and assaying,etc.) and 10mg/10ml liposome(such as shape,particle diameter,envelopment ratio,unexpectedly releasing effect,organic solvent, assaying and so on) were established.It provided safeguard for the following quality control of investigations in vivo.(2) The results of acute toxicity test showed that the LD_(50) of DBDCT after intravenous in mice was 21.1mg/kg,95%confidence limit was 17.4-25.5mg/kg,and the standard error of LD_(50) was 0.01.(3) The further in vivo antitumor tests of DBDCT towards the transplantation tumor models of sarcoma carcinoma(S_(180)),hepatocellular carcinoma(H_(22)) and Ehrlich's ascites carcinoma(EAC) on mice were carried out via injection intraperitoneally with cisplatin as positive contrast drug.The results indicated that DBDCT displayed in vivo antitumor activity against the hepatocellular carcinoma H_(22) and sarcoma carcinoma S_(180) which were close to those of cisplatin,meanwhile,the survival-extending rate at middle dose and high dose on mice Ehrlich's Ascites tumor EAC was higher than that of cisplatin,and the survival-extending rates in three experiments at low-dose group were 8.4%、31.0%、24.8%(P＞0.05),at middle-dose group were 85.1%、79.2%、73.4%(P＜0.001),at high-dose group were 87.2%、82.6%、79.6%(P＜0.001),respectively.There was a good dose-effect relationship.The in vivo G-CSF ELISE tests of DBDCT towards the non-specificity inflammation models on mice were carried out with carrageenan as positive contrast drug.The results showed that the peripheral blood WBC numbers of the experimental and positive control groups were notable more than that of the blank groups,and the G-CSF contents of the positive control groups were also higher than that of the blank groups,however,the G-CSF contents of the three experimental groups were nearly the same as that of the blank groups(P＞0.05).The consequence implied that DBDCT could not cause inflammation and pyogenic bacteria to the model mice,simultaneously, and may have effect on marrow hematopoietic system.Moreover,the index numbers of thymus and spleen on S_(180),H_(22) and EAC mice decreased with DBDCT dosage increased.The results implied that DBDCT possibly restrained the development of thymus and spleen.Besides, DBDCT at high dose may damage heart,liver,testicle,ovaries,kidney and so on.(4) After single intravenous administration to rats,DBDCT could be distributed or eliminated quickly.The distribution half life T_(1/2,α) were 2.3585,1.9614,2.1146 min and the elimination half life t_(1/2β) were 64.67,56.80,220.60 min for the three dosages(2,5,12mg/kg),respectively.The pharmacokinetics parameter calculations and modeling were carried out for DBDCT lipidosome at 5mg/kg by single intravenous injection.The blood concentration of DBDCT lipidosome could maintain more than 12h,the AUC value for DBDCT lipidosome was 7 times more than that of the corresponding DDBDCT injecton at the same dosage.Meanwhile,the elimination half life(t_(1/2β)) of the lipidosome was 12 times as that of the corresponding DDBDCT injection at the same dosage,and the clearance rate CL(s) decreased approximately 8 times as that for the same dosage injection.The results elucidated that the lipidosome could lengthen resistance time of DBDCT in circulatory system.When rats were injected with 5 mg/kg dose for 3min,DBDCT could be distributed to each organ quickly and it be distributed to brain tissue lookthrough the blood brain barrier.The concentration of DBDCT in adrenal gland was the highest followed by duodenal,heart,liver,testicle and kidney,and there was no accumulation in major tissues and organs after 24h.There was also no parent drug of DBDCT in urine,manure and bile.But,in urine and bile excretion tests,multi-metabolites were detected.The rat plasma protein binding ratios with DBDCT reached up to 59.6%～61.1%,indicating that a great quantity of DBDCT was combined together plasma protein and dissociation drugs were fairly low in blood.The structures of active compound and its main metabolites in blood,urine and bile samples were characterized by UPLC-MS analysis after single intravenous administration with 5mg/kg dose to rats,and nine compounds(including m/z575,m/z577,m/z579,m/z604,z607,m/z551,m/z663, m/z369 and m/z 565.7) were identified.The biotransformation pathways in vivo perhaps comprise of hydrogen reduction,ethylization reaction,metabolic product association reaction with glucuronic acid and elimination process of ligand and ethyl.The results showed that DBDCT were instability in blood,urine and bile samples.Through hepatic microsomal enzyme, DBDCT was gradually biotransformed via stripping off ligand,alkyl and chlorine,eventually eliminated from kidney and bile.(5) The antitumor activity in vitro against tumor cell lines, human immature granulocyte leukemia(HL-60),human gastric carcinoma(SGC-7901),human Henriettacar-cinoma(Hela) and human urinary bladder(T24) were analyzed by the MTT method. DBDCT showed concentration- dependent and time-dependent antiproliferative effects.The doses causing 50%inhibition(IC_(50)) values of SGC-7901 cells treated with DBDCT for different time(12h,24h,48,72h) were 81.6,25.3,4.5,2.8nmol·L~(-1),respectively.Fluorescent staining,light and electron microscope were used to examine the nuclear morphology characteristic of SGC-7901 apoptosis cells treated with DBDCT,and several apoptotic bodies were observed.In the agarose gel electrophoresis,DNA ladder-shaped strap was also clearly observed.There were hypodiploid apoptotic peaks pre- G_0-G_1 phase of cell cycle from DNA figure by flow cytometry (FCM).Significant statistics difference(P＜0.05) was showed in apoptosis rates of SGC-7901 cells treated with DBDCT for different time with cisplatin as positive contrast drug by annexin V-FITC method.It was clearly showed that DBDCT could kill SGC-7901 tumour cells through arrestting it in the G_2/M-phase and S-phase of cell cycle.The results of immunohistochemical method showed that p21 positive cells increased obviously,however,PCNA positive cells significantly decreased with the increased concentration of DBDCT(P＜0.01).The results of RT-PCR indicated that the express of p21,p53 and bax mRNA in the experimental groups were obviously higher than these of the blank groups(P＜0.05 or P＜0.01),nevertheless,the express of Bcl-2 and Bcl-2/Bax in the experimental groups were obviously lower(P＜0.001) than these of the blank groups with the concentration and time increased.(6) The enzymatic activities of caspase-3,caspase-8 and caspase-9 of SGC-7901 cells treated with different doses for different time increased significantly(P＜0.05 or P＜0.01).Hence,this experimental results confirmed that SGC-7901 cells apoptosis induced by DBDCT was concerned with mitochondria and death receptor pathway.The mitochondria apoptosis pathway was ascertained via caspase-8 inhibiter Z-LEHD-FMK which could not inhibit the apoptosis induced by DBDCT.The concentration of Ca~(2+) in SGC-7901 cells treated with DBDCT was significantly heightened which were detected by a new fluorochrome 3-AM,the mitochondria transmembrane potential strikingly decreased or disappeared which was detected by rhodarnine123,and the increasing ROS in SGC-7901 tumor cells treated with DBDCT hinted that DBDCT could stimulate the tumor cells to produce ROS. The results of western blot analysis indicated that the expressive down-regulation of Bcl-2 protein and the expressive up-regulation of Bax,Cyt-c and Caspase-3 showing concentration-effect and time-effect relationship.The ratio of Bcl-2/Bax decrease from 1.3 to 0.52 which was consistent with the results of RT-PCR.From the above,caspase apoptosis signal transduction pathway could be tentatively confirmed.Conclusions(1) The compound 2(DBDCT) showed strong antitumor activity in vitro.The quality standards of injection and liposome of DBDCT provided ensure for the quality control for further experiments with animals.(2) DBDCT showed strong activity in vivo against S_(180),H_(22) and EAC via intravenous injection.The organs such as heart,liver,testicle,thymus,spleen,ovaries,kidney and so on perhaps were the main active and toxic target organs.G-CSF ELISE experiments indicated that DBDCT could not produce inflammatory reactions to mice and may raise the function of the white blood corpuscle.(3) The absorption,distribution,biotransformation,evacuation and protein binding rates of DBDCT and its chief metabolites were illuminated via single intravenous administration to rats by HPLC and UPLC-MS analysis,and thus,the chemical foundation of active and toxic effect was discussed.(4) One of the mechanisms of tumour cells apoptosis induced by DBDCT may be the p53 apoptosis signal conductive pathway.The balance between Bax and Bcl-2 was destroyed through activating Bax and inhibiting Bcl-2 gene mediated by p53.(5) The apoptosis of SGC-7901 cells induced by DBDCT was also related to mitochondria and death receptor pathway.In this article,mitochondria caspases signal transduction pathway was probed intensively.Caspase-8 is the upstream modulin for apoptosis and its activation could activate caspase-9 and thus lead to the activation of caspase-3,and the dissociative Ca~(2+) was one of prerequisite to activate caspase-3,and homeostatic destroying of Ca~(2+) could directly cause mitochondria to release cytochrome C and some apoptosis factor.At the same time,DBDCT induces mitochondria active oxygen ROS increasing,Bcl-2 down regulation and Bax up-regulation,and mitochondria membrane potential△ψm decreasing and permeability raising with unceasing attack of free radical.Cyt-C released from mitochondria could combine with apoptosis enzyme promoter and then activate Apaf-1 by deoxidation triphosadenine.Eventually, the activated Apaf-1 binding to procaspase-9 would start a series of caspases cascade reaction, thus activates typical mitochondria apoptosis pathway.
方法(1)合成2种有机锡化合物,并通过红外光谱(IR)、核磁共振氢谱(~1H NMR)、碳谱(~(13)C NMR)、锡谱(~(119)Sn NMR)、质谱(MS)以及X-射线单晶衍射(X-Ray)进行结构表征。选择体外活性较强的化合物2(DBDCT)作为研究对象,制备其注射剂和脂质体两种制剂,并建立两种剂型的质量标准;(2)通过Bliss法测定小鼠静脉注射DBDCT后的半数致死量(LD_(50)),为体内抗肿瘤药效学和药代动力学研究给药剂量提供依据;(3)以肿瘤移植小鼠为模型,以顺铂(DDP)为阳性对照,研究静脉注射低、中、高三个剂量DBDCT后S_(180)、H_(22)和EAC小鼠体内的抗癌活性,同时考察实验小鼠外周血白血球数目、免疫器官胸腺和脾脏重量和指数、脏器重量和系数以及血液主要生化指标等,初步判断药物作用的毒性靶器官,采用G-CSF ELISE试验,检测DBDCT是否引起造模小鼠的炎症;(4)通过HPLC和UPLC-MS技术,研究大鼠尾静脉单剂量注射高、中、低三种剂量DBDCT注射液后原药及其主要代谢物在生物体内的吸收、分布、生物转化和排泄等过程,通过平衡透析法测定DBDCT与大鼠血浆蛋白结合率;(5)DBDCT作用后,采用MTT法测定体外培养的细胞株的增殖抑制率,并以SGC-7901为代表,采用流式细胞术(FCM)研究DBDCT对SGC-7901细胞增殖指数和细胞周期影响,通过光镜、荧光染色和电镜等方法观察细胞核形态的变化,FCM和Annexin V-FITC方法测定细胞凋亡率,DNA Lander方法检测肿瘤细胞凋亡的特异性条带,用RT-PCR法检测p53、p21、Bcl-2和Bax表达,免疫组化法检测p21和PCNA蛋白表达情况;(6)采用分光光度法测定DBDCT对SGC-7901癌细胞作用前后Caspase-3、Caspase-8和Caspase-9活性的变化;用Caspase-8抑制剂阻断DBDCT诱导SGC-7901细胞的Caspase-8途径产生的凋亡;采用新型荧光染料Flou 3-AM检测Ca~(2+)水平,以罗丹明123测定线粒体跨膜电位(△Ψm)变化,同时检测作为第二信使的活性氧(ROS)的表达,并通过Western blot分析Caspase-3、Cyt-C、Bax和Bcl-2等蛋白表达,进一步证实Caspases凋亡信号转导通路。
结果(1)得到了2种新的有机锡化合物,分别为:[(n-Bu)_2Sn(C_7H_5NO_2Cl)Cl]和[(n-Bu)_2Sn(C_7H_5NO_2Cl)_2](DBDCT)。制备了DBDCT注射剂(7.5mg/5ml)和脂质体(10mg/10ml)两种剂型,并建立了质量标准,注射剂质量标准包括性状、鉴别、检查(pH值、装量、有关物质和无菌、热源)、含量测定等;脂质体包括形态、粒径、包封率、突释效应和有机溶剂检查、含量测定等,为后续动物试验质量控制提供了保障;(2)急性毒性试验表明静脉注射DBDCT后的半数致死量LD_(50)=21.1mg/kg,LD_(50)(Feiller校正)95%的可信限为17.4～25.5mg/kg,LD_(50)的标准误为0.01;(3)体内抗肿瘤药效学实验中,DBDCT注射液组对S_(180)和H_(22)的生长显示了不同的抑制作用,中剂量和高剂量组与空白对照组相比有显著性差异(P＜0.05和P＜0.01)。各剂量组对EAC小鼠的生命延长率显示了不同的影响,三次测定低剂量组生命延长率分别为8.43%、30.97%、24.82%,中剂量组生命延长率分别为85.1%、79.2%、73.4%,高剂量组生命延长率分别为87.2%、82.6%、79.6%,中剂量组和高剂量组生命延长率远远高于阳性对照组(P＜0.001)。体内抗肿瘤活性显示出良好的剂量依赖性。采用G-CSF ELISE试验,以角叉菜胶为急性非特异性炎症模型,结果显示,实验组和阳性对照组小鼠的外周血白血球数目显著高于空白对照组,阳性对照组小鼠的G-CSF含量也显著高于空白对照组(P＜0.001),但各实验组与空白对照组相比G-CSF含量并无显著性差异(P＞0.05),提示DBDCT可能不会引起小鼠体内炎症感染、化脓菌或其毒素侵人,可能对骨髓造血系统有影响。试验中S_(180)、H_(22)和EAC小鼠的胸腺指数和脾脏指数随着DBDCT剂量的增大而降低,DBDCT可能会抑制胸腺和脾脏的发育。高剂量组DBDCT可能对肝、睾丸、胸腺、脾和卵巢有损伤;(4)大鼠尾静脉单剂量注射2、5和12mg/kg三种剂量注射液后,能迅速被分布或消除,三种剂量下分布半衰期(T_(1/2,α))很短,分别为2.3585、1.9614和2.1146min;消除半衰期(t_(1/2,β))为64.67、56.80和220.60 min。以5mg/kg的给药剂量为例对比考察了脂质体在大鼠体内的动力学参数,脂质体血药浓度可维持12h以上,药时曲线下面积AUC是相同剂量注射液的7倍以上,消除半衰期(t_(1/2β))为相同剂量注射液的12倍,清除率CL(s)比相同剂量注射液减小8倍左右,延长了DBDCT在体循环系统中的滞留时间。大鼠单次静脉注射5 mg/kg DBDCT 3min后DBDCT已迅速分布到各组织,且能够透过血脑屏障分布于脑组织,在肾上腺分布最高,其次是十二指肠,心脏、肝脏、睾丸和肾脏中均有较高的分布,在主要组织器官中无蓄积。尿、粪和胆汁中均检测不到原形药物,但尿和胆汁排泄过程中发现了多个代谢产物。DBDCT与大鼠血浆蛋白结合率高达59.6%～61.9%,表明DBDCT进入机体后大部分与血浆蛋白相结合,血中游离药物浓度较低。采用UPLC-MS对大鼠尾静脉给予5mg/kg注射液后血浆、尿液和胆汁中的出现的原药和代谢物进行了结构鉴定,初步推断了m/z575、m/z577、m/z579、m/z604、z607、m/z551、m/z663、m/z369和m/z565.7等九种化合物的结构。体内生物转化途径可能有加氢还原、乙基化和去乙基化反应、代谢产物与葡萄糖醛酸结合反应等。DBDCT在血浆、尿液和胆汁中均不稳定,逐渐脱掉配体、烷基和氯等基团而代谢转化,可能主要经肝微粒体酶脱烷基而代谢转化后大部分经肾或胆汁排出;(5)MTT细胞存活率试验显示,DBDCT能抑制白血病细胞HL60、宫颈癌细胞Hela、膀胱癌细胞T24以及胃腺癌细胞SGC-7901等细胞株的生长,作用效果具有一定的量效关系。DBDCT对SGC-7901作用不同时间(12h,24h,48,72h)后IC_(50)分别为:81.6、25.3、4.5、2.8nmol·L~(-1)。通过光镜、荧光染色和电镜观察到DBDCT处理过的SGC-7901细胞呈现凋亡细胞所特有的形态学特征,并检测到若干个凋亡小体。在琼脂糖凝胶电泳中可见凋亡特征性改变DNA梯形带。经FCM分析,从DNA组方图上可见到G_0-G_1期前亚二倍体凋亡峰。Annexin V-FITC实验显示不同浓度的DBDCT作用不同时间后SGC-7901细胞的凋亡率与对照组相比有统计学差异(P＜0.05)。FCM检测,DBDCT可能通过阻滞SGC-7901细胞G_2-M期和S期杀伤肿瘤细胞。免疫组化法检测,随着DBDCT浓度增加,p21阳性细胞数明显增加,PCNA阳性细胞数则显著减少(P＜0.01)。RT-PCR检测caspase3、p21、p53、Bcl-2和Bax基因的表达。随着DBDCT浓度和作用时间的延长,实验组p21、p53和Bax mRNA表达明显高于空白对照组(P＜0.05和P＜0.01),Bcl-2和Bcl-2/Bax的表达明显低于空白对照组(P＜0.001);(6)不同浓度DBDCT对SGC-7901作用不同时间后caspase-3、caspase-8和caspase-9酶的活性显著增加(P＜0.05和P＜0.01),证实了DBDCT诱导SGC-7901细胞凋亡与线粒体和死亡受体途径有关。采用caspase-8抑制剂不能抑制DBDCT诱导SGC-7901细胞的凋亡,确证DBDCT可通过线粒体途径诱导SGC-7901细胞凋亡。Flou 3-AM检测,细胞内Ca~(2+)浓度显著升高。罗丹明123测定,线粒体跨膜电位显著降低或消失,与DBDCT作用剂量和时间呈负相关。DBDCT迅速引起SGC-7901细胞内ROS的增加,提示DBDCT作用于细胞后可刺激肿瘤细胞ROS的产生。通过Western blot分析Caspase-3、Cyt C、Bax和Bcl-2等蛋白表达,随着DBDCT的浓度和作用时间增加,实验组Bcl-2蛋白表达下凋,而Bax、Cyt C和Caspase3等蛋白表达上调,且有明显量效和时效关系,Bel-2/Bax蛋白比值从1.30下降至0.52,与RT-PCR的结果一致,初步证实了Caspases凋亡信号转导通路。
结论(1)DBDCT注射剂和脂质体的质量标准符合要求,为后续动物试验的质量控制提供保障;(2)静脉注射DBDCT后对S_(180)、H_(22)和EAC小鼠具有较高的体内抗癌活性,主要的毒性靶器官可能是肝、睾丸、胸腺、脾和卵巢等,G-CSF ELISE试验检测DBDCT不会引起小鼠的炎症反应,可能具有提升白细胞的功能;(3)采用HPLC和UPLC-MS技术基本阐明了大鼠尾静脉注射DBDCT后在生物体内的吸收、分布、生物转化和排泄以及血浆蛋白结合率等,探讨了化合物发挥药效和毒性作用的化学物质基础;(4)确证了DBDCT能够诱导体外培养的SGC-7901肿瘤细胞发生凋亡,作用机制之一可能是通过p53信号传导通路:即p53激活Bax基因,抑制Bcl-2基因表达,通过破坏Bax与Bcl-2之间的平衡来完成p53介导的细胞凋亡信号转导机制;(5)DBDCT诱导SGC-7901细胞凋亡与线粒体途径、死亡受体途径有关。本文重点探讨了线粒体途径Caspases凋亡信号转导通路,即Caspase-8作为凋亡反应的上游调控蛋白,其活化将激活Caspase-9,Caspase-9又激活Caspase-3,细胞内游离Ca~(2+)是激活Caspase-3的先决条件之一,Ca~(2+)稳态的破坏可直接引起线粒体释放细胞色素C和一些促细胞凋亡因子。同时DBDCT诱导线粒体活性氧ROS的增加,引发Bcl-2下调和Bax上调,自由基的不断攻击引起线粒膜电位△Ψm下降,通透性增加,Cyt-C从线粒体释放后,与凋亡酶启动子结合,在脱氧三磷酸腺苷存在下激活Apaf-1。活化的Apaf-1会结合procaspase-9而启动一系列的Caspases级联反应,激活典型的线粒体凋亡通路。
Objective To synthesize antitumor diorganotin(Ⅳ) complex,di-n-butyl-(4-chlorobenzohy-droxamato) tin(Ⅳ) chloride(DBDCT),prepare its injection and liposome,establish their quality standard,study its antitumor activities in vitro and its pharmacokinetics in the plasma after single intravenous administration to rats,and investigate its antitumor cytotoxic mechanism through tumor cell culture in vitro.Methods(1)Two compounds were prepared and characterized by IR, ~1H NMR,~(13)C NMR,~(119)Sn NMR,MS and single crystal R-ray analysis.The compound 2 (DBDCT) with strong anti-tumor activity in vitro was studied.The quality standards of injection and liposome were established.(2) The median lethal dose(LD_(50)) of DBDCT injection to mice was determined by Bliss method and provided foundation for the further study of pharmacodynamics and pharmacokinetics in vivo.(3) Tumour-bear mice were used as transplantable carcinomas animal model with cis-diamminedichloroplatinum(DDP) as positive control.The weight of tumor on S_(180),H_(22) mice and the survival-extending rate of EAC mice were measured after intravenous injection at low,middle and high doses.Meanwhile,some indexes were detected such as mice peripheral blood white blood cells numbers,the immune organ weight and index number of thymus and spleen,organ weight and coefficient,blood main biochemical indicator and so on.These detections were used to preliminarily judge the active and toxic target organ of DBDCT,and G-CSF ELISE test was used to detect whether DBDCT could cause inflammation to the model mice.(4) The absorption,distribution and excretion processes of DBDCT and its main metabolites after single intravenous injection at low,middle and high doses were investigated by HPLC and UPLC-MS/MS techniques.The rat blood plasma protein binding rates of DBDCT were determined through equilibrium dialysis.(5) Growth inhibition of cell cultured in vitro were analyzed by MTT method.Flow cytometry(FCM) was used to study the effects of DBDCT on SGC-7901 cells cycle,proliferation and apoptosis. Hoechst and AO/EB staining,light and electron microscope were used to examine the nuclear changes.Apoptosis rates were determined by FCM,Annexin V-FITC methods and specific DNA ladder-shaped strap.The gene such as p53,p21,Bcl-2,Box and the proteinum(p21 and PCNA) expression changes of SGC-7901 cells treated with DBDCT were detected by RT-PCR and immunohistochemical method,respectively.(6) Using spectrophotometric method to evaluate enzymatic(caspase-3,caspase-8 and caspase-9) activities in SGC-7901 cells treated with DBDCT,simultaneously,caspase-8 inhibiter was used to block up the apoptosis of SGC-7901 cells with DBDCT treated.Apoptosis pathway would probable be illuminated.In addition,the fluorochrome 3-AM,rhodamine123 and ROS kits were applied for the studies of Ca~(2+) levels, mitochondria transmembrane potential variance(△ψm) and active oxygen ROS acted as second messenger,respectively.Meanwhile,the protein express for caspase-3,Cyt-C,Bax and Bcl-2 were analyzed by western blot method to further confirm the caspases apoptosis signal transduction pathway.Results(1) Two new diorganotin compounds were[(n-Bu)_2Sn(C_7H_(5-) NO_2Cl)Cl]and[(n-Bu)_2Sn(C_7H_5NO_2Cl)_2](DBDCT) and they were characterized by IR,~1H, ~(13)C,~(119)Sn NMR,MS spectra and single crystal X-ray analysis(DBDCT),respectively.The quality standards of DBDCT 7.5mg/5ml injection(including character,discrimination,and examination of pH,relevant materials,asepsis,heat source and assaying,etc.) and 10mg/10ml liposome(such as shape,particle diameter,envelopment ratio,unexpectedly releasing effect,organic solvent, assaying and so on) were established.It provided safeguard for the following quality control of investigations in vivo.(2) The results of acute toxicity test showed that the LD_(50) of DBDCT after intravenous in mice was 21.1mg/kg,95%confidence limit was 17.4-25.5mg/kg,and the standard error of LD_(50) was 0.01.(3) The further in vivo antitumor tests of DBDCT towards the transplantation tumor models of sarcoma carcinoma(S_(180)),hepatocellular carcinoma(H_(22)) and Ehrlich's ascites carcinoma(EAC) on mice were carried out via injection intraperitoneally with cisplatin as positive contrast drug.The results indicated that DBDCT displayed in vivo antitumor activity against the hepatocellular carcinoma H_(22) and sarcoma carcinoma S_(180) which were close to those of cisplatin,meanwhile,the survival-extending rate at middle dose and high dose on mice Ehrlich's Ascites tumor EAC was higher than that of cisplatin,and the survival-extending rates in three experiments at low-dose group were 8.4%、31.0%、24.8%(P＞0.05),at middle-dose group were 85.1%、79.2%、73.4%(P＜0.001),at high-dose group were 87.2%、82.6%、79.6%(P＜0.001),respectively.There was a good dose-effect relationship.The in vivo G-CSF ELISE tests of DBDCT towards the non-specificity inflammation models on mice were carried out with carrageenan as positive contrast drug.The results showed that the peripheral blood WBC numbers of the experimental and positive control groups were notable more than that of the blank groups,and the G-CSF contents of the positive control groups were also higher than that of the blank groups,however,the G-CSF contents of the three experimental groups were nearly the same as that of the blank groups(P＞0.05).The consequence implied that DBDCT could not cause inflammation and pyogenic bacteria to the model mice,simultaneously, and may have effect on marrow hematopoietic system.Moreover,the index numbers of thymus and spleen on S_(180),H_(22) and EAC mice decreased with DBDCT dosage increased.The results implied that DBDCT possibly restrained the development of thymus and spleen.Besides, DBDCT at high dose may damage heart,liver,testicle,ovaries,kidney and so on.(4) After single intravenous administration to rats,DBDCT could be distributed or eliminated quickly.The distribution half life T_(1/2,α) were 2.3585,1.9614,2.1146 min and the elimination half life t_(1/2β) were 64.67,56.80,220.60 min for the three dosages(2,5,12mg/kg),respectively.The pharmacokinetics parameter calculations and modeling were carried out for DBDCT lipidosome at 5mg/kg by single intravenous injection.The blood concentration of DBDCT lipidosome could maintain more than 12h,the AUC value for DBDCT lipidosome was 7 times more than that of the corresponding DDBDCT injecton at the same dosage.Meanwhile,the elimination half life(t_(1/2β)) of the lipidosome was 12 times as that of the corresponding DDBDCT injection at the same dosage,and the clearance rate CL(s) decreased approximately 8 times as that for the same dosage injection.The results elucidated that the lipidosome could lengthen resistance time of DBDCT in circulatory system.When rats were injected with 5 mg/kg dose for 3min,DBDCT could be distributed to each organ quickly and it be distributed to brain tissue lookthrough the blood brain barrier.The concentration of DBDCT in adrenal gland was the highest followed by duodenal,heart,liver,testicle and kidney,and there was no accumulation in major tissues and organs after 24h.There was also no parent drug of DBDCT in urine,manure and bile.But,in urine and bile excretion tests,multi-metabolites were detected.The rat plasma protein binding ratios with DBDCT reached up to 59.6%～61.1%,indicating that a great quantity of DBDCT was combined together plasma protein and dissociation drugs were fairly low in blood.The structures of active compound and its main metabolites in blood,urine and bile samples were characterized by UPLC-MS analysis after single intravenous administration with 5mg/kg dose to rats,and nine compounds(including m/z575,m/z577,m/z579,m/z604,z607,m/z551,m/z663, m/z369 and m/z 565.7) were identified.The biotransformation pathways in vivo perhaps comprise of hydrogen reduction,ethylization reaction,metabolic product association reaction with glucuronic acid and elimination process of ligand and ethyl.The results showed that DBDCT were instability in blood,urine and bile samples.Through hepatic microsomal enzyme, DBDCT was gradually biotransformed via stripping off ligand,alkyl and chlorine,eventually eliminated from kidney and bile.(5) The antitumor activity in vitro against tumor cell lines, human immature granulocyte leukemia(HL-60),human gastric carcinoma(SGC-7901),human Henriettacar-cinoma(Hela) and human urinary bladder(T24) were analyzed by the MTT method. DBDCT showed concentration- dependent and time-dependent antiproliferative effects.The doses causing 50%inhibition(IC_(50)) values of SGC-7901 cells treated with DBDCT for different time(12h,24h,48,72h) were 81.6,25.3,4.5,2.8nmol·L~(-1),respectively.Fluorescent staining,light and electron microscope were used to examine the nuclear morphology characteristic of SGC-7901 apoptosis cells treated with DBDCT,and several apoptotic bodies were observed.In the agarose gel electrophoresis,DNA ladder-shaped strap was also clearly observed.There were hypodiploid apoptotic peaks pre- G_0-G_1 phase of cell cycle from DNA figure by flow cytometry (FCM).Significant statistics difference(P＜0.05) was showed in apoptosis rates of SGC-7901 cells treated with DBDCT for different time with cisplatin as positive contrast drug by annexin V-FITC method.It was clearly showed that DBDCT could kill SGC-7901 tumour cells through arrestting it in the G_2/M-phase and S-phase of cell cycle.The results of immunohistochemical method showed that p21 positive cells increased obviously,however,PCNA positive cells significantly decreased with the increased concentration of DBDCT(P＜0.01).The results of RT-PCR indicated that the express of p21,p53 and bax mRNA in the experimental groups were obviously higher than these of the blank groups(P＜0.05 or P＜0.01),nevertheless,the express of Bcl-2 and Bcl-2/Bax in the experimental groups were obviously lower(P＜0.001) than these of the blank groups with the concentration and time increased.(6) The enzymatic activities of caspase-3,caspase-8 and caspase-9 of SGC-7901 cells treated with different doses for different time increased significantly(P＜0.05 or P＜0.01).Hence,this experimental results confirmed that SGC-7901 cells apoptosis induced by DBDCT was concerned with mitochondria and death receptor pathway.The mitochondria apoptosis pathway was ascertained via caspase-8 inhibiter Z-LEHD-FMK which could not inhibit the apoptosis induced by DBDCT.The concentration of Ca~(2+) in SGC-7901 cells treated with DBDCT was significantly heightened which were detected by a new fluorochrome 3-AM,the mitochondria transmembrane potential strikingly decreased or disappeared which was detected by rhodarnine123,and the increasing ROS in SGC-7901 tumor cells treated with DBDCT hinted that DBDCT could stimulate the tumor cells to produce ROS. The results of western blot analysis indicated that the expressive down-regulation of Bcl-2 protein and the expressive up-regulation of Bax,Cyt-c and Caspase-3 showing concentration-effect and time-effect relationship.The ratio of Bcl-2/Bax decrease from 1.3 to 0.52 which was consistent with the results of RT-PCR.From the above,caspase apoptosis signal transduction pathway could be tentatively confirmed.Conclusions(1) The compound 2(DBDCT) showed strong antitumor activity in vitro.The quality standards of injection and liposome of DBDCT provided ensure for the quality control for further experiments with animals.(2) DBDCT showed strong activity in vivo against S_(180),H_(22) and EAC via intravenous injection.The organs such as heart,liver,testicle,thymus,spleen,ovaries,kidney and so on perhaps were the main active and toxic target organs.G-CSF ELISE experiments indicated that DBDCT could not produce inflammatory reactions to mice and may raise the function of the white blood corpuscle.(3) The absorption,distribution,biotransformation,evacuation and protein binding rates of DBDCT and its chief metabolites were illuminated via single intravenous administration to rats by HPLC and UPLC-MS analysis,and thus,the chemical foundation of active and toxic effect was discussed.(4) One of the mechanisms of tumour cells apoptosis induced by DBDCT may be the p53 apoptosis signal conductive pathway.The balance between Bax and Bcl-2 was destroyed through activating Bax and inhibiting Bcl-2 gene mediated by p53.(5) The apoptosis of SGC-7901 cells induced by DBDCT was also related to mitochondria and death receptor pathway.In this article,mitochondria caspases signal transduction pathway was probed intensively.Caspase-8 is the upstream modulin for apoptosis and its activation could activate caspase-9 and thus lead to the activation of caspase-3,and the dissociative Ca~(2+) was one of prerequisite to activate caspase-3,and homeostatic destroying of Ca~(2+) could directly cause mitochondria to release cytochrome C and some apoptosis factor.At the same time,DBDCT induces mitochondria active oxygen ROS increasing,Bcl-2 down regulation and Bax up-regulation,and mitochondria membrane potential△ψm decreasing and permeability raising with unceasing attack of free radical.Cyt-C released from mitochondria could combine with apoptosis enzyme promoter and then activate Apaf-1 by deoxidation triphosadenine.Eventually, the activated Apaf-1 binding to procaspase-9 would start a series of caspases cascade reaction, thus activates typical mitochondria apoptosis pathway.
引文
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