¹⁸⁸Re-MAG₃-QKRKRKKSRYKS: Preparation Characterization And Studies In Rabbits And Mice

Background and objectives:Tumor growth and metastasis is dependant on angiogenesis.Among many involved molecules in the process,vascular endothelial growth factor(VEGF) and vascular endothelial growth factor receptors(VEGFRs) play a pivotal role.VEGF binds to the extracellular binding domain of VEGFR,then activates tyrosine kinase to promote angiogenesis.VEGFR2(also named KDR/flk-1) is the main receptor of VEGF to induce angiogenesis.Physiologically,KDR is mainly expressed on endothelial cell.In some pathological conditions,for example,in many kinds of tumors KDR is upregulated.Therefore,KDR is considered to be a promising target for tumor imaging.VEGF comprises 7 exons and 6 introns.According to previous studies,it has been demonstrated that the peptide QKRKRKKSRYKS can inhibit the binding of VEGF to HUVEC and VEGF-induced angiogenesis.Thus,QKRKRKKSRYKS could be a good candidate to substitute intact VEGF for tumors imaging and treatment,Gene transfection is a common technique to improve the expression of protein.We constructed truncated KDR gene,which retains the binding activity to VEGF while deleting the intracellular tyrosine domain.Adenovirus,carrying the truncated KDR,could increase the expression of KDR in the tumors in vivo and induce the accumulation of labeled peptide. Anti-tumor effects are exerted through truncated KDR,peptide and evenβ-emitter at the same time,which may provide a new way for tumor therapy.In this study,we were aimed to investigate the following aspects:how to label QKRKRKKSRYKS with Rhenium-188,the biodistribution in mice and SPECT image of tumor with the labeled peptide,gene transfection of truncated KDR and effect of KDR gene transfection toward imaging.All the work was expected to found some background for future radioactive tumor therapy research. Methods:1.Labeling of QKRKRKKSRYKS with the introduction of bifunctional chelating agent NHS-MAG₃:we improved the conventional method,synthesizing the peptide GGGQKRKRKKSRYKS and modifying the peptide chain directly.Orthogonal design was also applied to detect the optimized labeling condition.2.Purification and identification of ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS:HPLC and Sep Pak C18 column were used to purify and identify ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS.The specific activity,radiochemical purity,stability and in vitro binding activity were also testified.3.Pharmacokinetic study of ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS:Five male Japanese rabbits were used for study.Purified ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS(37MBq per rabbit, 20μg) was administrated into the ear vein.0.15 mL blood sample was collected from the contralateral ear vein at different time intervals(1.5,3.0,5.0,10,30,60,90,120,180 and 300 min).The blood samples were subject to gamma counter,and after attenuation correction the results were formatted to radioactive concentrations(KBq/L).Compartment model and pharmacokinetic parameters were analyzed by software DAS2.0.4.Biodistribution and planar imaging of ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS in tumor bearing nude mice:15 tumor-bearing nude mice were used in the experiment.Studies were performed when the tumor was approximately 1cm³ in volume.Purified ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS(i.v.,0.1mL,18.5 MBq/mice) was injected via the tail vein of the mice.At 30,60,and 120 min after injection,mice were sacrificed and the blood, heart,liver,spleen,lungs,kidneys,intestines,brain,thyroid,tumor tissues and contralateral muscles were excised,weighed and counted for radioactivity by a gamma counter.The biodistribution were calculated as percentage of the injected radioactive dose per gram of tissue wet weigh(%ID/g).The ratio of%ID/g between tumor and the other tissues(T/NT ratio) was also calculated.As the tumor was 1.3cm³ in volume,18.5 MBq ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS was injected via the caudal vein.1 h and 3 h later,the mice were intraperitoneally anesthetized with pentobarbital and placed in a prone position. Posterior planar image was performed using a SPECT.5.Effects of truncated KDR gene transfection on SPECT imaging in tumor-bearing nude mice:the tumor-bearing nude mice were transfected with the truncated KDR adenovirus.At 48 h,the expression of the truncated KDR protein in tumor tissues was observed under fluorescence microscope.After injection of ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS,the effects of SPECT imaging was observed and evaluated.Results:1.Peptide synthesis and coupling with MAG₃:S-acetyl-MAG₃-QKRKRKKSRYKS₃ was synthesized in a comparatively easy way.The molecular weight was 1880.9 measured by chromatography,which was in accordance with the theoretical 1879.4.2.The optimized labeling conditions determined were as follows:30μL of 0.25 mol/L ammonium acetate buffer(pH 5.2),20μg of the preliminarily treated peptide,37 MBq ¹⁸⁸Re eluant,10μL of 55μg/μL potassium sodium tartrate,20μL of 5μg/μl SnCl₂ (prepared with 20 mmol/L HCl immediately before use,containing 1μg/μL ascorbic acid). The total volume was adjusted to 110μL with redistilled water,and pH was adjusted to 4.6. The reaction was performed under nitrogen atmosphere and 100℃in water bath for 60 min. Under these conditions,the labeling rate was 73.1%±5.45%.3.¹⁸⁸Re-MAG₃-QKRKRKKSRYKS was stable at room temperature.Specific activity was 2.594 TBq/mmol.Radiochemical purity was more than 95%after Sep Pak C18 purification.The radiochemical purity was still more than 90%after exposed 24 hours.In vitro competitive binding assay also demonstrated that the labeled peptide kept a good affinity to KDR.4.¹⁸⁸Re-MAG₃-QKRKRKKSRYKS pharmacokinetics in rabbits was in line with a three compartment model.5.At 1h after ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS injection,a significant radioactivity accumulation was viewed at the tumor site in the right anterior limb,and the image was clear.The%ID/g in tumor tissue was(1.846±0.511);while the tumor/contrlateral muscle was(2.43±0.30).Besides,there is also apparent accumulation in liver,intestine,kidneys and bladder.At 3 h after injection,the image of tumors disappeared.6.At 1h after ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS injection,the radioactivity accumulation at the tumor site in the transfection group was significantly higher than that in the non-transfection group.60 minutes after injection of ¹⁸⁸Re-MAG₃-QKRKRKKSRYKS via the caudal vein of mice,the%ID/g of tumor and the tumor/contralateral muscle tissues ratio were(1.98±0.38) and(2.53±0.33) in the control group,but was(3.08±0.84) and (3.61±0.59) in the transfection group,respectively.Conclusions:1.In the present study,we successfully labeled QKRKRKKSRYKS with ¹⁸⁸Re,and optimized the labeling conditions.The labeled peptide kept a good stability and binding affinity to KDR.2.There was a significant accumulation in tumor tissue and a high T/NT ratio.Truncated KDR gene transfection improved the uptake of labeled peptide in tumor tissue and the T/NT value.3.This study provides some basis for future radioactive tumor therapy investigation.