Study On The Preparation Of⁹⁹Tc^m-TP1326Targeting At α_vβ₃Receptor And Testing In Animals

Background and objectives:Angiogenesis is the physiological process involving the growth of new blood vesselsfrom pre-existing vessels, which is important to the tumor growth, invasion and metastasis.While integrin is closely related to the process of angiogenesis in tumor growth.Integrin is a kind of cell surface receptor that mediates the attachment between a celland the tissues that surround it, such as other cells or the extracellular matrix （ECM）. It is atransmembrane glycoprotein including two different subunits, one α and one β chain. Also,it is one of the most important adhesion molecule attached to the cell surfaces. There are atleast twenty different integrins consisting of eighteen α and eight β subunits. One of them,integrin α_vβ₃, plays an important role during the process of tumor growth, migration andangiogenesis. Statistic shows that it has a high level expression on the surface of varietiesof tumor cells and newborn vascular endothelial cells but not on mature cells and mostnormal tissues and organs. All of the items mentioned above make the integrin α_vβ₃be anew target of inhibition research against tumor angiogenesis.Small molecular circular peptide containing RGD protein sequence is an efficientinhibiter against α_vβ₃receptor. Furthermore, the inhibition efficiency of the circular RGDpeptide with two disulfide bonds is higher than other kinds of RGD peptide. And parts ofthose small molecular peptide used as a inhibiter against integrin have entered phase IIItrial to treat malignant tumor. It has been a hot area of research that small molecularcircular peptide containing RGD protein sequence labeled with radionuclide is valuable inmolecular imaging and targeted therapy.This research is designed to get the target peptide—TP1326, after modifying acircular nine peptide named RGD-4CK having two disulfide bonds with GAGG-Aba, andto explore the best labeling condition, physicochemical properties, tracer kinetics anddistribution in animals, imaging results in normal and tumor-burdened animals in order topave the way for targeted receptor imaging research. Methods:1. Synthesize TP1326: Using stannous chloride as the reductant, TP1326synthesizedby chemosynthesis is labeled with⁹⁹Tc^m. The labeling rate and specific activity of thelabeled mixture are measured by chromatography.2. Identify the physicochemical properties of⁹⁹Tc^m-TP1326: The stability test in vitro,HPLC analysis, seralbumin binding test and oil-water distribution test are used.3. Tracer kinetics test: The best compartment model, matched curves andpharmacokinetic parameters are obtained with the help of pharmacokinetic analysissoftware （DAS3.1.0） through determining nine rabbits’ blood radioactive concentration atdifferent time phases （1.5,3,5,10,30,60,120,240,480min） after intravenous injection of⁹⁹Tc^m-TP1326（200μl,37MBq）.4. Rabbits imaging research: Fix nine rabbits at supine position respectively. Afterintravenous injection of labeled peptide （200μl,74MBq）, SPECT imaging is applied toobserve the dynamic distribution in main organs of the rabbits. Obtain following images:1frame/s×60s,1frame/min×59min,1frame at different time phases （1.5th,2.0nd,2.5th,3.0rd,4.0thh）.5. Biodistribution experiment: Divide35healthy mice randomly into7groups equally.Then inject labeled solution （200μl,7.4MBq） from the tail vein. Finally, collect samples（blood, heart, lung, liver, kidney, intestine, muscle, bone and brain） to calculate thePercentage Injected Dose per gram （%ID/g）.6. The imaging research of tumor-burdened nude mice: After intravenous injection oflabeled peptide （100μl,7.4MBq） from the tail vein of the nude mice bearing humanmelanoma B16, SPECT imaging is applied to observe the dynamic distribution in tumorand other organs. And T/NT ratio is measured through ROI technique. Obtain one frame atdifferent time phases （0.5th,1.0st,1.5th,2.0nd,3.0rd,4.0thh）.7. The competitive inhibition imaging research: In this research, non-labeled peptidewith a concentration300times higher （100μg） is injected one hour in advance in order toevaluate the specificity of this peptide.Results:1. Synthesize TP1326: The purity of TP1326is97.31%. The labeling rate is （95.86±0.83）%. The specific activity is （38.62±0.32） TBq/mmol. 2. Identify the physicochemical properties of⁹⁹Tc^m-TP1326: After being conserved atroom temperature for four hours, the radiochemical purity is （91.76±2.75）%. According tothe HPLC analysis, the retention time of the labeled peptide is the same as the peak time ofthe eluent—around the14thminute. In the seralbumin binding test, the shapes of the curves（labeled peptide and mixture） are alike. The oil-water distribution coefficient is－（1.76±0.06）.3. Tracer kinetics test: The dynamics of the labeled peptide in rabbits fits for thetwo-compartment model, and the main parameters, t1/2αand t1/2β, are （3.115±0.771） min and（76.978±22.460） min respectively.4. Rabbits imaging research: The SPECT images of rabbits show that the bloodradioactivity disappears fast, and most radioactivities are eliminated through kidneys; thereis no obvious nuclide accumulation in bowels, stomaches, thyroids or brains.5. Biodistribution experiment: The biodistribution in normal mice shows that theradioactivity in hearts, livers and blood disappears fast over time. Meanwhile, both themuscle and brain present low radioactive background.6. The imaging research of tumor-burdened nude mice: The tumor image is clear at the30thminute after injection. And its highest T/NT ratio is5.26at the first hour.7. The competitive inhibition imaging research: After injection of non-labeled peptidein advance, the T/NT ratio decreases to2.83at the first hour. And the inhibition ratio at thefirst hour is46.2%.Conclusions:1. TP1326can be labeled easily with a high labeling rate and stability and there is noneed for purification.2. The blood elimination through renal system reaches a fast speed.3. The labeled peptide can concentrate on tumor tissues specifically with highresolution images and high T/NT ratios.