Studies On Pharmacokinetics And Mechanism Of Antitumor Di-n-butyl-(4-chlorobenzohydroxamato) Tin(IV) Chloride(DBDCT)

Objective To synthesize antitumor diorganotin(â…£) complex,di-n-butyl-(4-chlorobenzohy-droxamato) tin(â…£) 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,¹³C NMR,¹¹⁹Sn 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(LD₅₀) 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 S₁₈₀,H₂₂ 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 Ca²⁺ 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(C₇H_5- NO₂Cl)Cl]and[(n-Bu)₂Sn(C₇H₅NO₂Cl)₂](DBDCT) and they were characterized by IR,¹H, ¹³C,¹¹⁹Sn 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 LD₅₀ of DBDCT after intravenous in mice was 21.1mg/kg,95%confidence limit was 17.4-25.5mg/kg,and the standard error of LD₅₀ was 0.01.(3) The further in vivo antitumor tests of DBDCT towards the transplantation tumor models of sarcoma carcinoma(S₁₈₀),hepatocellular carcinoma(H₂₂) 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 H₂₂ and sarcoma carcinoma S₁₈₀ 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 S₁₈₀,H₂₂ 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(IC₅₀) 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- G₀-G₁ 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 G₂/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 Ca²⁺ 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 S₁₈₀,H₂₂ 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 Ca²⁺ was one of prerequisite to activate caspase-3,and homeostatic destroying of Ca²⁺ 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.