|
Background and Purpose angiogenesis and tumor growth and metastasis is closely related to tumor diameter greater than 2 mm, through neovascularization conveying enough nutrients and remove metabolic waste, and for facilitating the transfer. Angiogenesis need the participation of a variety of factors, wherein the vascular endothelial growth factor (vascular endothelial growth factor, VEGF) signaling pathway is the major rate-limiting aspects, and VEGFR-2 (KDR), play a biological role of angiogenesis main receptor types or low expression in normal tissue and benign tumors do not express in malignant tissues showed high expression. Immunohistochemical study for a variety of malignant tumors show, KDR expression in neovascular endothelial cells, some tumor cells themselves high expression of the receptor. Highly expressed in tumor cell membrane receptors, the selective receptor-targeted radioactive peptides has become an important class of molecular imaging and therapeutic radiopharmaceuticals. This radionuclide labeled polypeptide of the emitted gamma rays with the receptor binding, thus achieving a non-invasive detection of tumor expression of the receptor object. In recent years, phage display technology and the emergence of the ribosome display technology conjugates provide a new way to find VEGFR specificity. The technology known receptors in the high-throughput phage peptide libraries or ribosomal peptide library screening to ensure the screened peptide receptor with high specificity and high affinity binding. K237 is the application of the technology to filter out dodecapeptide study confirmed that the K237 can be blocked by a combination of VEGF and KDR inhibition of VEGF-mediated human umbilical vein endothelial cells (HUVEC) proliferation and angiogenesis chorioallantoic membrane and can inhibit the growth and metastasis of breast cancer cells in SCID mice BICR-H1 Department. This study by 99Tcm labeling of K237, exploring the feasibility of molecular imaging agent of malignant solid tumors as KDR receptor-oriented, and to provide an experimental basis for the future to carry out the internal exposure to radionuclides treatment research. Method 1. 99 Tc m -MAG 3 the-K237 Preparation: the tartaric acid stannous reduction method 99 Tc m -MAG 3 -K237, 3MM chromatography paper chromatography determination of radiochemical purity of orthogonal test design method of screening the best labeling conditions. 2. 99 Tc m -MAG 3 -K23 in vitro stability of identification: room temperature stability, cysteine ??replacement and trichloroacetic acetic acid protein precipitation in vitro stability experiments, evaluation 99 of Tc m -MAG 3 -K237 of the physical and chemical properties. 3. 99 Tc m -MAG 3 -K237 in healthy rabbits tracer kinetics analysis: Take six Japanese male with big ears rabbit, by ear vein injection of 37 MBq 99 Tc m -MAG 3 -K237 solution, and then were 1.5,3,5,10 , 30,60,120,240,480 min from the ear vein blood, weighing and measuring the radioactivity count, the results of measuring efficiency correction, calculating the radioactive concentration (KBq / L). Pharmacokinetic analysis software to to the DAS 2.1.1 version to determine optimal atrioventricular model and derived pharmacokinetic parameters. 4. 99 Tc m -MAG 3 -K237 health Biodistribution and imaging studies: the 7.4 MBq 99 the Tc m -MAG 3 -K237 solution was diluted to 500 ml were taken 1 ml added to the 5 amp exemption tube, the measured radioactivity counts (cpm) are multiplied by the number 500, i.e. the radioactive count of the reference source. Take 35 adult Kunming mice, according to the random number table is divided into seven groups of five. By intravenous injection of 7.4 MBq 99 Tc m -MAG 3 -K237 solution, respectively 1,5,10,30,60,120 240 min eyeballs France blood, and then the mice were sacrificed by cervical dislocation, the collection of the heart, lung, liver, kidney, intestine, muscle, bone, brain, were weighed and measured the radioactivity count, calculated by the reference source after correction per gram of tissue is the percent injected dose rate (% ID / g). The rabbit fixed on wooden board the injection 99 3 -K237 solution of 37 MBq Tc m -MAG 1 / min acquisition immediately preset in 90,120,180,240 min, 60 min, and timed 2 min acquisition image of a dynamic changes observed distribution of radioactivity of each organ. The Tc m of of 5. 99 -MAG 3 -K237 charge HepG2 in nude mice in vivo distribution studies: 7.4 MBq 99 Tc m -MAG 3 -K237 solution was diluted to 500 ml, respectively, added to 1 ml of the the 5 amp exemption tube, the measured radioactivity counts (cpm) The average number is multiplied by 500, i.e. the radioactive count of the reference source. Take 15 Dutch HepG2 nude mice were divided into five groups, according to the random number table. By intravenous injection of 7.4 MBq 99 Tc m -MAG 3 -K237 solution, respectively, by 10,30,60,120,240 min enucleated blood was collected, and then the mice were sacrificed by cervical dislocation, the collection of the heart, lung, liver, kidney, intestine, muscle, bone, brain, tumors, respectively, weighing and measuring the radioactive counts (cpm) after correction by the reference source Calculate the percentage of the injected dose (% ID / g), and calculate each phase of the T / NT ratio per gram of tissue. 6. 99 the Tc m -MAG 3 -K237-bearing nude mice imaging and competition combined with imaging studies: Take charge HepG2 nude mice fixed wooden board, tail vein injection 99 the Tc m to 3 -K237 solution after 17.5 MBq, respectively, at 30, 60, 90, 120,180,240 min each a preset timing 2 min capture images, to observed tumor tissue distribution of radioactivity dynamic change. Other single tumor-bearing nude mice, intravenous injection of mixed with the 0.5 mg MAG 3 K237's in 99 Tc m -MAG to 3 -K237 solution 17.5 MBq observed the MAG 3 -K237 on the inhibition of tumor imaging. Results of 1. 99 the Tc m -MAG 3 -K237 Preparation: 99Tcm labeled MAG 3 -K237 (20μg ) the best reaction conditions: 0.25 mol / L ammonium acetate buffer (pH 5.2), 16μg of potassium sodium tartrate, 4μg tartaric acid stannous of 37 MBq 99TcmO4-a total reaction volume of 182μl, nitrogen, sealed under the conditions in the boiling water bath was heated for 30 min. Whatmann 3MM paper chromatography, γ counter measuring mark mixture each radioactive components than in the saline, aqueous ammonia and acetone, and three kinds of mobile phase-shift value (Rf value). Prepared 99 Tc m -MAG 3 -K237's mark was (97.98 ± 0.76)%, the direct method to calculate the specific activity The degree of (3.54 ± 0.03) TBq / mmol. 99 the Tc m -MAG 3 -K23 in vitro stability: markers room temperature for 8 h, the mark is still more than 95 %, (96.15 ± 0.37)%; different concentrations of cysteine ??substitution experiments 99 Tc m -MAG 3 -K237 is not 99Tcm changes were less than 1%; trichloroacetic acid precipitation experiments showed 99 the Tc m -MAG 3 -K237 serum protein had no significant combination. 3. 99 Tc m -MAG 3 -K237 in healthy rabbits tracer kinetics analysis: the labeled peptides rabbits power learning process in line with the weight of the three-compartment model for 1/c2, t1/2α 4.00 ± 3.53 min to 24.48 ± 9.84 min t1/2β t1/2γ to 852.24 ± 444.00 min clearance (CL) was 1.83 ± 0.41 ml / min. 4. 99 Tc m -MAG 3 -K237 in healthy animal biodistribution and imaging: markers in healthy mice in vivo clearance rapidly, kidney, liver and intestinal distribution more, but the distribution in the liver after 30 min rarely. 5. 99 Tc m -MAG 3 -K237-bearing nude mice in vivo distribution and imaging: Dutch HepG2 nude mice distribution display, 10 min phase tumor tissue uptake of radioactive highest for tumor and contralateral muscle radioactivity ratio (T / NT) 1 h (0.90 ± 0.18) (% ID / g); reached the highest value, 2.19 ± 0.22; kidney, liver and intestinal Road radioactive distribution more. ABN of HepG2 nude SPECT imaging, tumor 1 h developing the most clear the the tumor image significantly Dodge -the MAG 3 K237 blocking. The conclusions of the study prepared 99 Tc m -MAG 3 -K237 markup rate gt; 95% Needless separation and purification direct application having good in vivo stability. 2. 99 Tc m -MAG 3 -K237 vivo distribution and tracer dynamics in healthy animals, fast blood clearance excreted through the kidneys and liver double. 99 Tc m -MAG 3 -K237-specific uptake in nude mice bearing human tumors, tumor imaging clearly, and T / NT ratio, has a potential value of tumor the KDR molecular imaging agent.
|