| Background and Objective:Vasoactive intestinal peptide receptor（VIPR）is in the G protein coupled to transmembrane receptor family,and it has been found three subtypes（VPAC1、VPAC2 and PAC1）now.Research proved that,VPAC1 is overexpressed in various cancers and metastasis,such as colon、breast、lung;However VPAC2 is expressed in benign leiomyomas only.In addition,VPAC1 has the high affinity binding sites.The Bmax is 22.557.9 times than normal.Therefore VPAC1 is an ideal target for Tumor molecular imaging and targeted therapy,which has potential application prospect.However,molecular probes for VIPR imaging are the whole VIP molecule or its analogues.And either of them can combine with three subtypes.So the shortcoming is lack of high specificity to VPAC1.In our prophase research,by phage display library screening whole-cell subtractive panning,we screened and identified a 12-mer peptide（GFRFGALHEYNS,VP2）which can high specificity target to VPAC1.AND the binding affinity to VPAC1 was significantly higher than natural ligand VIP.In our study,we get the targe peptide VP2 by Polypeptide chemical synthesis,Meanwhile amino terminal coupled bifunctional chelates（GAGG—Aba）,research the optimal conditions for 99 mTc combine G（D）AGG-Aba-VP2（TP1724）,identify its physiochemical properties,study tracer kinetics、biological distribution and dynamic imaging in normal animals,For the further research of VPAC1 target tumor imaging.Methods:1.Preparation of the 99mTc-TP1724: chemosynthesis TP1724,then combined 99 mTc by Sn Cl2 reduction method.Though paper chromatography,determination of labelling rate and get the specific activity.2.Identified biology and physiochemical properties of 99mTc-TP1724: stability testing in vitro、synthesis freeze-dried kits by one-step method、HPLC analysis、plasma protein binding testing、Cysteine displacement experiment、oil-water distribution tests.3.Tracer kinetic in vivo normal rabbits experiment: 9 Japanese rabbits were fixed on the wood lab.Each of them received 99mTc-TP1724（37 MBq,200μl）via left ear vein.Then weigh blood and measure radioactive（cpm）draw from the right ear vein at different periods of time after injected.（1.5min、5min、10min、30min、60min、120min and 240min）.After correction of reference source,Blood radioactive converse to kBq/L according to its density（1g/ml）.By the pharmacokinetics software analysis（DAS 3.1.6）,intelligent analysis results that average blood radioactive concentration–time,to choose the best compartment model、concentration-time relations and Tracer kinetic parameters.4.SPECT imaging of rabbits: Siemens Symbia T6 SPECT/CT with Low energy high resolution collimator.The parameter was set in energy peak 140 Ke V,window width 20%,matrix 128,a zoom factor of 1.0.Rabbits are fixed on Wood lab in the supine position and their abdomen in the centre of the visual field.Inject 99mTc-TP1724（74MBq）from the ear vein.Take the some images:include 1 freeze-frame/sec×60 sec,1 freeze-frame/minute×59 minute,1 flame at four different time freeze-frames（90th、120th、150th、180th and 240 th min）.By ROI,draw T-A curve of dynamic image of organic（The heart,kidneys,liver,bladder）after injection in one hour.Observe changes about the rabbit radioactive dynamic distribution during the whole of the experimenting.5.Biodistribution in Kunming mice: 35 cases of mice were allocated to 7 groups（each group 5 cases）,randomly.Inject 99mTc-TP1724（3.7 MBq,100ul）from tail vein each one.After that,cut off their heads at different time（1.5 min、5 min、10 min、30 min、60 min、120 min and 240 min after inject）,then collect the blood、heart、kidneys、liver、intestine、lung、muscle、brain and bone,immediately measure its weigh and radioactive counts（cpm）.After correction of reference source,take data turn to %ID/g（per gram tissue dose rate）.6.Rabbits inflammatory models imaging:3 cases,inject turpentine 500 ul in the interior right hind limbs,local aseptic inflammation formed after 7days.Siemens Symbia T6 SPECT/CT,matrix 256,the remaining parameters are the same as 4.a zoom factor of 1.0.Rabbits are fixed on Wood lab in the supine position and their abdomen in the centre of the visual field.Inject 99mTc-TP1724（74MBq,200ul）from ear vein,take static image 1frame at per phase（0.5h、 1h、2h、3h、4h and 5h）.use the ROI,get the radioactivity and calculate T/NT about inflammatory lesions and on the contralateral normal tissue at different time phase.Results:1.Preparation of the 99mTc-TP1724: reach 97.80% of the purity of peptide,and（96.57±0.71）% of the labeling rate,（25.52±0.29）TBq/mmol of specific activity.2.Identified the properties of biology and physiochemistry : radiochemical purity of labeled peptide is（95.74±1.53）%、（95.50±2.06）%、（93.64±2.25）%,respectively.stay in the room temperature at 1、2、4h.Freeze-dried kits have high stability（>90%）until the Twelfth week.99mTc-TP1724 incubated with blood,Sephadex G50 column chromatography show that: The major emission peak of Elution in experiment is appear the 42 nd tube,which shape basically in line with the control group.Besides we can see that 6.61% Small protein combined with emission peak emerge at the 16 th tube in the experiment group.From HPLC,the peak time between peptides and the label peptides is the same（10min）.Cysteine displacement experiment shows: 99 mTc and TP1724 chelate solid and hard to displace by Cysteine,and there are no apparent increases free 99 mTc compared to control group.Oil-water distribution coefficient of lable peptide lg P was-（1.99±0.02）.3.Tracer kinetic in vivo normal rabbits experiment: By using the specialized software,The concentration-time data of blood were t1/2α（2.64±1.32）min、t1/2β（78.36±13.38）min,CL（clearance rate）（122.15±56.33）ml/min.Labeled tracers of 99mTc-TP1724 were fitted by two compartments with weight of 1.4.SPECT imaging of rabbits:heart、liver、kidneys begin to see in first minute,then the image of Liver and kidney became weaken over time;bladder start to see five minutes and growing time.The heart and tissue can be seen until 30 min.At the same time,gallbladder appears.At the whole proceeding,stomach is always present radioactivity blank.And no unusually neck image,background image of brain.5.Kunming mice biodistribution research:after inject lable-peptides,compared to the first minute,blood and kidneys radioactivity descend 98.43% and 82.14% in 60 min,respectively;the liver radioactivity begin to reduce in 10 min;intestine begin to increase in 30 min;radioactivity of the heart、bone、lung reduce to the same with muscle in 60 min;brain always at low radioactivity.6.inflammatory models of rabbits imaging research: inflammation visualized clearly in 30 min,the T/NT about inflammation/ control side at different time（0.5h、1h、2h、3h、4h 、5 h）are 2.19±0.12、2.17±0.11、2.05±0.13、2.02±0.15、1.96±0.19、1.68±0.21,respectively.It illustrates that labeled peptide bond to inflammation is non-specific.Conclusions:1.By the indirect method,synthesis simply,high labeling rate（95%）,use directly,no need to isolate pure,prepared freeze-dried kits easily（one-step method）,save standby.2.High labeling rate and specific activity to meet their standards with receptor in vitro and in vivo research;blood clean rapidly,excretion though urinary systems mostly;high stability and well kinetic properties.lay the foundation for the further VPAC1 positive tumor imaging. |
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