Studies On The Chemical Modifications Of Endostatin And The Bioactivities And Pharmacokinetics Of The Modified Derivatives

Endostatin(ES),an endogenous angiogenesis inhibitor,could cut off the nutrition supply and metabolic pathway of tumor to develop an inhibitory effect on tumor growth and metabasis by inhibitting angiogenesis.Compared with chemotherapy drugs,ES has many advantages such as its low toxic and side effects, broad-spectrum action,low drug resistance in repeated therapy,etc.ES(Endostar) has been approved by SFDA for the treatment of non-small-cell lung cancer in China. However,the clinical application of endostatin was hindered by high-dose requirements(300mg/m²Â·d),manufacturing constraints,relative instability and short half-life of the recombinant ES protein.If the stability could be improved,or the half-life could be prolonged,it will be useful to improve the curing effects of ES and enlarge its clinical use.In this topic,three different modifiers,polyethylene glycol (PEG),low molecular weight heparin(LMWH) and polysulfated heparin(PSH) were used to modify ES,and the secondary structure,stability,bioactivity, pharmacokinetics and tissue distribution of ES and its modified products were studied. All the results were summarized as follows.1 Study on the purification and chemical modification of ES with three modifiers and heat stability of the modified ES derivativesSupematant of Pichia yeast strain containing human ES gene was submitted to CMâ…¡ion exchange chromatography and Superdex 75 gel filtration for purification. The sample showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE).Its inhibitory activity on human umbilical vein endothelial cell(HUVEC) proliferation was also studied by MTT method. PEG was activated by cyanuric chloride and the modification was carried out in pH 9.0,10mmol/L sodium tetraborate buffer with activated PEG and ES in.LMWH was activated by sodium metaperiodate,and the modification was carried out in pH 9.5,0.3mol/L Na₂CO₃-NaHCO₃ buffer with activated LMWH and ES in.PSH was also activated by sodium metaperiodate,and the modification was carried out in pH 9.5,0.3mol/L Na₂CO₃-NaHCO₃ buffer with activated PSH and ES in.SDS-PAGE, free amino group determination and retained activity determination were used to monitoring the reaction.The results showed that,one ES molecule could bind three molecules of modifiers,and the most satisfied reaction time of PEG modification, LMWH modification and PSH modification was 24 h,48 h and 48 h,respectively.ES modified products,PEG-ES,LMWH-ES and PSH-ES,were purified by Superdex 75 gel filtration and the heat stability were studied under 25℃and 37℃.The results showed that modified products had higher heat stability compared with native ES,and the stabilities were in the order of:at 25℃,PEG-ES,PSH-ES>LMWH-ES>ES;at 37℃,PEG-ES>PSH-ES,LMWH-ES>ES.2 Secondary structure analysis of ES and its modified productsCircular dichroism spectra(CD) method was employed to study the secondary structure of ES and its modified products.The results showed that,β-turn of PEG-ES showed a obvious change from 16.20%to 23.21%compared with ES;Secondary structure of LWMH-ES has little change compared with ES;β-antiparallel of PSH-ES showed a change from 1.17%to 3.52%compared with ES,the other structure components had no obvious change.In all,secondary structure of ES's modified products had little change compared with native ES.3 Targeting of ES derivatives to HUVEC3.1 Flow cytometry analysis of the binding ability of ES conjugates to HUVECThe binding ability of ES conjugates to cells was determined by flow cytometry.The results indicated that fluorescence intensity increased after incubation with FITC-labeled ES derivatives,and that of PSH-ES-FITC was the highest.The fluorescence intensity of HUVEC incubated with FITC-ES for 1 h was only 18.47%;while incubated with FITC-PEG-ES, FITC-LMWH-ES and FITC-PSH-ES for 1 h,the fluorescence intensities were 48.01%,54.08%%and 57.15%respectively.3.2 Study on intracellular distribution of FITC-labeled ES and ES derivatives in HUVEC by confocal laser scanning microscopyHUVEC were incubated with 200μg/mL FITC-ES and ES derivatives at 37℃for 1 h or 2 h.The images were obtained by confocal laser scanning microscopy.One hour after incubation,the fluorescent signals of FITC-labeled native ES were weak, and that of FITC-labeled PEG-ES were slightly stronger than that of native ES.In contrast,FITC labeled LMWH-ES and PSH-ES were observed to have much stronger fluorescent signals than native ES.Two hours after incubation,all the fluorescent signals were enhanced compared with that of 1 h incubation,and FITC labeled PEG-ES,LMWH-ES and PSH-ES showed higher fluorescent signals than ES.4 Antiangiogenesis and antitumor activity of ES and its derivativesPurified ES and its derivatives were used in chorioallantoic membrane(CAM) model to study their antiangiogenesis activity.Distilled water was chosen as a blank control,and basic fibroblast growth factor(bFGF) was chosen as a negative control. The results showed that,purified ES could significantly inhibit bFGF induced CAM angiogenesis,the blood vessel number of ES(0.5μg/embryo) was(11.3±1.24), compared with(25.2±3.55) of bFGF(P <0.05).The inhibitory effects showed a concentration dependent manner.ES derivatives,PEG-ES,LMWH-ES and PSH-ES, could also significantly inhibit bFGF induced CAM angiogenesis with vessel number of(11.8±1.38),(10.8±2.14) and(8.6±0.53),respectively.LMWH-ES and PSH-ES showed higher activity than ES.Choroidal neovascularization(CNV) model was prepared on C57BL/6 mice by laser photocoagulation to evaluate the effects of endostatin and its derivatives on choroidal neovascularization in vivo.Western bloting was employed to study the effects of ES and its derivatives on the expression of vascular endothelial growth factor(VEGF) and pigment epithelium derived factor(PEDF) in chorioid tissues. Three days after treatment,the CNV area of PBS,ES,PEG-ES,LWMH-ES and PSH-ES were 18.14×10³Î¼m²,10.65×10³Î¼m²,8.92×10³Î¼m²,9.02×10³Î¼m² and 8.97×10³Î¼m²,respectively;Seven days after treatment,the CNV area of PBS,ES, PEG-ES,LWMH-ES and PSH-ES were 23.28×10³Î¼m²,14.20×10³Î¼m²,11.93×10³Î¼m²,12.74×10³Î¼m² and 12.15×10³Î¼m²,respectively.The results showed that endostatin and its derivatives could significantly inhibit endothelial cell proliferation in vitro(P<0.05),suppress choroidal neovascularization by down regulating the expression of VEGF and up regulating the expression of PEDF in chorioid tissues.ES and its derivatives could significantly inhibit zebra fish angiogenesis.At concentration of 5μg/ml,10μg/ml and50μg/ml,the inhibitory ratio were 13.21%, 22.22%and 41.74%for ES,18.62%,34.23%and 49.25%for PEG-ES,18.92%, 31.53%and 46.85%for LMWH-ES,19.22%,39.64%and 55.25%for PSH-ES, respectively.Compared with ES,its derivatives showed high activity.The results of antitumor activity study of ES and its derivatives in S₁₈₀ mice model showed that the tumor inhibitory ratios of ES,PEG-ES,LMWH-ES and PSH-ES were 40.50%,51.24%,61.98%and 65.29%,respectively.ES derivatives showed higher antitumor activity in S₁₈₀ model.5 Pharmacokinetics and tissue distribution of ES and its derivativesES and its derivatives were labeled by ¹²⁵I and their pharmacokinetics and tissue distribution were studied.Elimination halflife of PEG-ES,LMWH-ES and PSH-ES were 43.88 h,39.54 h and 35.58 h respectively,and they were all longer than that of ES(8.81 h).Their distribution halflife were also longer than that of ES.The AUCs and AUMCs of ES derivatives were higher than that of ES,and MRTs of ES derivatives were longer than that of ES.A higher distribution in liver was obtained after administration of PSH-ES compared with that of ES.Tissue distribution of PEG-ES and LMWH-ES was almost the same as that of ES.The main results and conclusions were as follows:(1) ES was successfully modified by PEG,LWMH and PSH,and pure PEG-ES, LMWH-ES and PSH-ES were obtained.The modified products of ES had higher heat stability than ES.(2) Secondary structure analysis by CD showed that,after chemical modification, the secondary structure of ES had little change,except theβ-turn increased in PEG-ES.(3) Chemical modification with PEG,LMWH or PSH,ES could increase HUVEC targeting properties of ES.(4) ES and its derivatives could significantly inhibit HUVEC proliferation, angiogenesis in CAM,CNV and zebra fish models and tumor growth in S₁₈₀bearing mice,and the derivatives had better effects than ES.(5) Compared with ES,halflifes of the three ES derivatives were all longer and the tissue distribution showed little change.