Rhenium-188 Radiolabeled RGD-4CK Using Prethinning Method: Preparation, Characterization And Studies In Mice And Rabbits

Background and objectives:Angiogenesis, the process that results in the formation of new vessels by sprouting of pre-existing vessels, is involved in tumor growth, infiltration and metastasis. Tumor angiogenesis is a complex, multi-step process that requires activation of a number of key molecules. There is growing evidence that the diversity of expression of integrins in tumor neovascular endothelial cells is critical for tumor angiogenesis. The integrins, the same as vascular endothelial cell growth factor receptor (VEGFR), are considered as a hallmark for tumor angiogenesis and are valuable in tumor therapy studies.The integrins are a family of transmembrane glycoproteins consisting of two subunits,αandβ, when combined together, form about 20 heterodimers in vertebrates. Integrinαvβ3 is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartate (RGD) tripeptide sequence. These include fibronectin(FN), vitronectin(VN), thrombospondin-1(TSP-1), osteoponin(OPN), von Willibrand's factor(vWF), etc. Integrinαvβ3 plays a significant role in tumor-induced angiogenesis and metastasis. It is expressed at low levels on mature endothelial cells, but it is overexpressed on the activated endothelial cells of tumor neovasculature and some tumor cells. Being characterized by genetic stability, tumor newly-formed endothelial cells are prone to access pharmaceuticals on account of its direct attachment to blood circulation, which constitutes an advantage for drug targeting because it is unlikely that such cells will acquire mutations and become resistant. Based on the structure-activity relationship studies of a series of integrinαvβ3 antagonists, the cyclic RGD-containing peptide with two disulfide bonds accounts for most of theα_v integrin inhibiting activity, whereas the cyclic peptide with one disulfide bond is about 20-fold less potent and the linear peptide is about 200-fold less potent. And besides, the cyclic RGD-containing antagonist reveals a significant high binding specificity than that of the linear peptide. Recently, the RGD-containing peptides applied to anti-angiogenesis cancer therapy are being studied in phase II clinical trial. And radiolabeled integrinα_vβ₃ antagonists, which allow to target the expression of integrinα_vβ₃, becomes more promising in molecular nuclear medicine on oncology.Our previous preparation of ⁹⁹Tc^m-RGD-4CK using direct labeling method had demonstrated that ⁹⁹Tc^m-RGD-4CK is a promising tumor receptor-targeting imaging agent for reason of its rapid and convenient labeling, high radiochemical purity(RCP), favorable stability in vitro, appropriate tracer kinetic characteristics in vivo.Based on the analysis, the present study was designed to prepare ¹⁸⁸Re-radiolabeling RGD-4CK using direct labeling method and integrallty investigated the most appropriate labeling method, physical and chemical characteristics, tracer kinetic features and distribution in vivo.Methods:1.RGD-4CK was directly radiolabeled using prethinning method. RCP analysis of ¹⁸⁸Re-RGD-4CK had been demonstrated by Whatman 3MM paper chromatographic system. A group of orthogonal test was arranged and carried out to study the effect of radiolabeling conditions, and the range analysis and ANOVA(analysis of variance) was employed to distinguish the influential degree of factors included in this test.2.Stability analysis in vitro, e.g., serum protein binding assay and cysteine challenge testing (CCT), together with high performance liquid chromatography (HPLC) were carried out to evaluate physical and chemical characteristics of 188RGD-RGD-4CK.3.9 male Japanese flap-eared rabbits were intravenously administered via left ear vein with 37 MBq ¹⁸⁸Re-RGD-4CK.Blood samples were withdrawn via right ear vein at 1.5, 3.0, 5.0, 10, 30, 60, 90, 120, 180, 360, 600, 1020 min after administration. The samples were wet weighted, the radioactivity of which was measured using aγ-scintillation counter. The results were presented as megabecquerels per litre (MBq/l) after the corrected counts were expressed in terms of a reference source. The obtained data were processed at the aid of program DAS version 2.0 and the compartment model was judged by combining the actual expression ofα_vβ₃ receptor in vivo.4.25 C57BL/6 mice bearing subcutaneous B16/F10(α_vβ₃+) malignant melanoma xenografts were randomly divided into 5 groups, each with 5 mice. Animals were sacrificed and dissected at 0.5, 1, 3, 8, 16 h after injection of 0.74 MBq of ¹⁸⁸Re-RGD-4CK into the tail vein. Blood, tumor, major organs, and tissues were collected and wet weighted. The radioactivity in the tissues was measured using aγ-scintillation counter. The results were presented as the percentage injected dose per gram (%ID/g). For quantitation purposes the corrected counts were expressed in terms of a reference source. T/NT values were calculated and expressed as mean±SD.5.The scintigraphy of ¹⁸⁸Re-RGD-4CK was performed in 2 male Japanese flap-eared rabbits respectively. 74 MBq of ¹⁸⁸Re-RGD-4CK was intravenously injected to each rabbit. The dynamic distribution was observed by the scintigraphy with SPECT combining time-activity curve(T-A curve) of the region of interest(ROI).Results:1.Comprehensive range analysis on all the used factors and their levels showed that the best condition for radiolabeling RGD-4CK with ¹⁸⁸Re was specified as potassium sodium tartrate 2.83μmol, stannous chloride 4.43μmol, ascorbic acid 500μg, prethinning temperature 60℃, prethinning time 4 h, prethinning pH 2.5, labeling temperature 95℃, labeling time 40 min, respectively. Similar RCP results (96.49%±0.68%) were obtained in eight independent experiments on this optimum condition.2.The retention time was essentially identical as the radioactive peak of eluent from an analytical HPLC. The RCP was still over 90% after keeping 16 hours at room temperature. The amount of free ¹⁸⁸ReO4- merely increased 1.56% after incubation with 400 mmol/L cysteine for l h at 37℃. There was no obvious combination between ¹⁸⁸Re-RGD-4CK and serum protein.3 . Tracer kinetic features in normal rabbits was fitted much better by a two-compartment model with a weight coefficient of 1/C2. The distribution half life was 0.52 min and the elimination half life was 37.79 min.4.The data from the distribution studies of ¹⁸⁸Re-RGD-4CK in C57 mice bearing melanoma xenografts indicated fast blood clearance and predominantly urinary excretion. Little accumulation of tissues was observed, e.g. brains, muscles, bones. Increasing activity was observed in tumors 3.0 h post injection. The peak value of tumor-to-normal tissue ratio (T/NT) for tumor over muscle and tumor over bone was 3.76. 5.SPECT imaging of rabbits indicated that T-A curves of the ROI of all the tissues were descending over time. 1.0 min after the tracer was injected, kidney image emerged. 5 min after injection, bladder image emerged. Heart image and liver image were attenuated. Radioactivity distribution of lung was asystematic, which was lower that liver. Radioactivty accumulation in bladder was gradually increased. 20 min after injection, tissue image was attenuated. Gall bladder and stomach images were not detected.Conclusions:1.¹⁸⁸Re-labeling of RGD-4CK via prethinning offers a convenient means of labeling peptides. ¹⁸⁸Re-RGD-4CK, with a considerably high RCP(>95%), has favorable physical and radiochemical characteristics, as well as a perfect distribution feature and an ideal tracer kinetics behavior in vivo. The method can also be applied for the formulation of a freeze-dried kit.2.¹⁸⁸Re-RGD-4CK, administered into C57 mice bearing B16 melanoma xenografts, is selectively accumulated in the tumors. Receptor-mediated specificity of the tumor accumulation of the radiolabeled RGD peptide for theα_vβ₃ integrin is demonstrated. The high tumor/muscle ratio provides a reasonable basis for anti-cancer target therapy studies in animals.