Preparation And Identification Of ⁹⁹Tc^m-MAG₃-K237 And Its Experimental Studies In Anmimals

Background and objectives:Angiogenesis is closely related to the growth and metastasis of malignant tumor. Tumors with diameter more than 2 mm derive nutrition, exclude metabolic waste and facilitate metastasis through new blood vessel.The process of angiogenesis includes participation of many factors, among which vascular endothelial growth factor(VEGF)and KDR signal transduction pathway is the most important. In normal and benign tumor tissues, KDR is expressed at low level, whereas malignant tumor tissues express KDR at high level. Some immunohistochemistry studies on malignant tumors revealed that KDR was mainly expressed in the new blood vessel cell and some kind of tumor cell itself.In the tumors highly expressing some receptor, selective receptor targeting radioactive peptide has become an important kind of molecular imaging and treatment drug. The peptide labeled by nuclide emittingγ-ray is able to combine with its receptor, and then is able to noninvasively explore malignant tumor in vivo.In recent years, phage display technique and ribosome display technique have become two kinds of ways to find VEGFR specific bond. It can guarantee high chemical affinity between the peptide and its receptor. K237 was isolated from a phage-displayed peptide library, which could bind to KDR with high affinity and specificity. By interfering with VEGF-KDR interaction, the peptide K237 inhibited proliferation of cultured primary human umbilical vein endothelial cells. K237 also exerted an anti-angiogenesis activity in vivo in chick embryo chorioallantoic membrane and inhibited the growth and metastasis of breast cancer in SCID mice.This study focused on the labeling of K237 with 99Tcm and feasibility of the labeled peptide as a molecular imaging agent mediated by KDR receptor in solid malignant tumors,which would lay an experimental foundation for the nuclide-therapy study in the future.Methods:1.Preparation of ⁹⁹Tc^m-MAG₃-K237:⁹⁹Tc^m-MAG₃-K237 was prepared by way of reduction with stannous tartrate. RCP analysis of ⁹⁹Tc^m-MAG₃-K237 was demonstrated by Whatman 3MM paper chromatographic system. Orthogonal design was also applied to detect the optimized labeling condition.2.In vitro stability identification of ⁹⁹Tc^m-MAG₃-K237: Evaluate the stability of ⁹⁹Tc^m-MAG₃-K237 in vitro through a series of experiment such as stability at room temperature, displacement by Cysteine and protein precipitation by trichloroacetic acid.3.Tracer kinetics analysis in vivo of ⁹⁹Tc^m-MAG₃-K237 in healthy rabbits: Each of six rabbits was injected 37 MBq ⁹⁹Tc^m-MAG₃-K237, then draw blood from ear vein at the following time: 1.5, 3, 5, 10, 30, 60, 120, 240, 480 min. These blood sample was weighed and measured byγ-immuno-counter, and then we calculated the radioactive concentration of every sample after correction by measurement efficiency ofγ-immuno-counter. At last, we evaluated the best compartmental model and parameter by virtue of pharmacokinetics analytical software.4.Body distribution and imaging study of ⁹⁹Tc^m-MAG₃-K237 in vivo in healthy animals: 35 adult Kunming mice were involved in the study of tissue distribution, which were divided to 7 groups by random digits table. 7.4 MBq ⁹⁹Tc^m-MAG₃-K237 was administrated via caudal vein of the mice. At 1, 5, 10, 30, 60, 120, 240 min after injection, mice were sacrificed and the blood, heart, lungs, liver, kidneys, intestines, muscles, bone and brain were excised, weighed and counted for radioactivity by a gamma counter. And then we calculated the percentage of the injected radioactive dose per gram of tissue wet weigh (%ID/g) after correction by reference source. In the study of imaging in healthy animals, we injected 37 MBq ⁹⁹Tc^m-MAG₃-K237 into ear vein of rabbits. The dynamic imaging was observed by the scintigraphy with SPECT for 60 minutes and then static imaging at 90, 120, 180 and 240 min.5.Tissue distribution study of ⁹⁹Tc^m-MAG₃-K237 in vivo in tumor bearing nude mice: In the study of tissue distribution, 15 tumor bearing nude mice were divided to 5 groups. 7.4 MBq ⁹⁹Tc^m-MAG₃-K237 was administrated into the caudal vein of each mice, and then we sacrificed them and collected their blood, heart, lungs, liver, kidneys, intestines, muscles, bone, brain and tumor tissues at 10, 30, 60, 120 and 240 min. At last, we calculated the percentage of the injected radioactive dose per gram of tissue wet weigh (%ID/g) and tumor/non-tumor ratio(T/NT) at various time.6.SPECT imaging and competitive imaging by MAG₃-K237: In the study of imaging, 17.5 MBq ⁹⁹Tc^m-MAG₃-K237 was injected into the caudal vein of the mice. The dynamic distribution was observed by the scintigraphy with SPECT at 30, 60, 90, 120, 180 and 240 min. In the study of competitive imaging, mixture of 17.5 MBq ⁹⁹Tc^m-MAG₃-K237 and 0.5 mg MAG₃-K237 was administrated into the caudal vein of the mice, then competitive imaging inhibited by MAG₃-K237 was observed.Results:1.Preparation of ⁹⁹Tc^m-MAG₃-K237: ⁹⁹Tc^m-MAG₃-K237 could be successfully prepared following the steps below: added 20μg MAG₃-K237, 50μl 0.25mol/L ammonium acetate buffer solution(pH 5.2), 16μg potassium sodium tartrate, 4μg stannous tartrate, 37 MBq 99TcmO4- to the cell frozen tube, sealed the tube and boiled it under boiling water for 30 minutes. The RCP of the labeled peptide was (97.98±0.76)%, and its specific activity was (3.54±0.03)TBq/mmol.2.Identification of stability of ⁹⁹Tc^m-MAG₃-K237 in vitro: after 8 h, the RCP of ⁹⁹Tc^m-MAG₃-K237 was (96.15±0.37)%; Cysteine replacement rate was less than 1% after adding cysteine of various concentration; trichloroacetic acid precipitation test revealed the labeled peptide couldn't combine with serum protein.3.Tracer kinetics analysis in vivo of ⁹⁹Tc^m-MAG₃-K237 in healthy rabbits: tracer kinetics process in vivo of ⁹⁹Tc^m-MAG₃-K237 in healthy rabbits followed three compartment model weighed by 1/c2 and the main parameters t1/2α, , t1/2β, t1/2γand CL were 4.00±3.53 min, 24.48±9.84 min, 852.24±444.00 min and 1.83±0.41 ml/min respectively.4.Tissue distribution and imaging study of ⁹⁹Tc^m-MAG₃-K237 in vivo in healthy animals: ⁹⁹Tc^m-MAG₃-K237 was cleared quickly from the mice, it mainly distributed in the kidneys, liver and intesteins, but the distribution in liver was little after 30 min.5.Tissue distribution and imaging study of ⁹⁹Tc^m-MAG₃-K237 in vivo in tumor bearing nude mice: In the study of tissue distribution, %ID/g of tumor was highest ,(0.90±0.18) at 10 min, T/NT ratio was highest, 2.19±0.22 at 1 h, and the labeled peptide was mainly distributed in kidneys, liver and intestines. In the study of imaging, the tumor was clearest at 1 h, and the imaging was much less clear after blocked by MAG₃-K237.Conclusions:1.⁹⁹Tc^m-MAG₃-K237 was successfully prepared with high RCP(>95%) and perfect stability in vitro and in vivo.2.⁹⁹Tc^m-MAG₃-K237 had satisfactory characteristics in tracer kinetics and tissue distribution and was cleared quickly from blood and excreted quickly from kidneys and liver.3.⁹⁹Tc^m-MAG₃-K237 could concentrate in the tumor tissue with specificity in tumor bearing nude mice, which showed its bright future in the field of molecular imaging in KDR positive tumor.