Preparation And Preliminary Biological Characteristics Evaluation Of ¹⁷⁷Lu Labeled Ibandronate

Objective:The purpose of this study was to radiolabel ibandronate?IBA,a third-generation bisphosphonate?with ¹⁷⁷Lu,investigate optimal labeling conditions and the physical and biological properties of this new bone-seeking radiopharmaceutical,analysis its ability to bind to inorganic substances of bone tissue,evaluate the characteristics of imaging in model animals,and explore its feasibility of becoming a new radiopharmaceuticals for bone imaging and targeted therapy.Methods:Excess ibandronic acid and appropriate ¹⁷⁷LuCl₃ in solution were combined to obtain an optimal ratio for radiolabeling purposes,varying the amount of ligand,the activity of ¹⁷⁷Lu³⁺,pH,temperature,and time to establish the ideal reactivity conditions.Radiochemical yield of the labeled compound was then assessed by thin-layer paper chromatography?TLC?.The stability of ¹⁷⁷Lu-IBA in saline and fresh human serum were determined in vitro.After intravenous injection of the labeled compound,blood samples were taken at specific time points to determine the in vivo stability.The anesthetized mice were injected intravenously with ¹⁷⁷Lu-IBA.Blood samples were collected at different time points for weighing,measurement and counting.Afterwards,the pharmacokinetic parameters of ¹⁷⁷Lu-IBA were plotted and calculated.The plasma protein binding rate of the labeled complexes were determined by the trichloroacetic acid method.The lipid-water partition of ¹⁷⁷Lu-IBA was calculated by using n-octanol as the organic phase and phosphate buffer as the inorganic phase.The hydroxyapatite-binding assay of ¹⁷⁷Lu-IBA to simulated bone matrix hydroxyapatite was determined in vitro.The biodistribution of¹⁷⁷Lu-IBA was investigated in 20 KM mice?Randomly divided into five groups,four in each group?.After tail intravenous injection of a 100?L volumes of¹⁷⁷Lu-IBA?3.7 MBq?for each mouse,four mice were killed by neck fracture at1 h,4 h,24 h,48 h,and 6 d respectively.The tissues and organs?ie,heart,liver,spleen,lung,kidney,intestine,stomach,muscle,femur,and blood?were excised to determine percentages of injected doses?%ID/g?in various samples of mouse tissues and the ratio of bone tissue to liver,kidney and muscle by gamma counter and microbalance.For comparison,biodistribution study of¹⁷⁷LuCl₃ was performed the same way.The toxicity study of ¹⁷⁷Lu-IBA was explored in sixteen normal Kunming mice?Randomly divided into four groups,four in each group,half male and half female?.Four groups were injected intravenously with normal saline,low,medium and high doses of ¹⁷⁷Lu-IBA respectively.The diet,body weight and activity.After four weeks,the mice were then sacrificed by cervical dislocation.The tissue of brain,heart,liver,spleen,lung,kidney,stomach,small intestine,muscle,bone tissue and gonadal were taken for pathological examination.For imaging studies,Three healthy New Zealand white rabbits were used.Bone imaging was performed three hours after injection of ^99mTc-MDP in the ear vein.After three days,¹⁷⁷Lu-IBA was injected intravenously,anesthesia was fixed,and bone imaging was performed at 2 h,12 h,24 h,48 h,and 6 d after injection.Establish a left tibia bone metastasis model of breast cancer?MDA-MB-231?and prostate cancer?PC-3?tumor-bearing mice.After micro-CT scan confirmed bone tissue destruction,injected each mouse intravenously with 11.1-14.8 MBq of¹⁷⁷Lu-IBA and fixed them after anesthesia and scanned them with gamma camera at specific time points.Establishing SD rat bone defect model,^99mTc-MDP bone imaging was performed one week after modeling,and¹⁷⁷Lu-IBA imaging was followed three days later.Results:Under the condition that the activity of ¹⁷⁷Lu³⁺was fixed at 3.7 MBq and the amount of IBA was200?g,adjusting pH to 5.0 and allowing reactions at 90°C for 30 min.High radiochemical purity?>97%?was obtained.The stabilities of ¹⁷⁷Lu-IBA was measured at saline and fresh human serum,showing that in vitro labeling efficiencies of the two labeled complexes were>90%at 72 h,and the stability at 37°C was better than normal temperature.Radiochemical purity of¹⁷⁷Lu-IBA remained stable within 2 h in vivo without significant metabolism.The ¹⁷⁷Lu-IBA was quickly cleared from the blood with an elimination half-life of 29.5 min.The plasma protein binding rates of ¹⁷⁷Lu-IBA was 62.3±2.16%.The Lipid-water partition lgP was-2.22±0.30.The hydroxyapatite binding rate was to 97%of ¹⁷⁷Lu-IBA.This distribution study in mice showed that¹⁷⁷Lu-IBA had a high bone accumulation,and the highest uptake value at 48 h?5.280±0.566?%ID/g,and the bone/muscle?T/NT?up to 523.At 6 days,the uptake of bone tissue was still higher than other tissues.¹⁷⁷Lu-IBA cleared rapidly in other non-target tissues,and almost completely cleared from blood after 24 hours.¹⁷⁷Lu-IBA was mainly metabolized by the kidneys.Compared with ¹⁷⁷LuCl₃,¹⁷⁷Lu-IBA has faster blood clearance,higher bone uptake,and significantly reduced liver intake.In the toxicity experiment of ¹⁷⁷Lu-IBA,the mice showed no obvious vomiting,diarrhea,decreased diet,decreased activity or other macroscopic changes after injection.No weight loss was observed in all groups,and there was no significant difference in body weight between groups.No serious tissue inflammation were observed in pathological sections.¹⁷⁷Lu-IBA imaging showed high contrast between bone systems and soft tissue,clear image quality and long-term retention.The image quality was so clear that it could be analyzed after 6 d.and the best imaging quality was obtained at24h-48h,which was similar to the common imaging agent ^99mTc-MDP.The rat bone defect model showed obvious concentration of ¹⁷⁷Lu-IBA at the lesion.Conclusions:Bisphosphonates can be easily labeled with ¹⁷⁷Lu.Its radiochemical purity ensured stability in vitro and in vivo.¹⁷⁷Lu-IBA is readily soluble in water,showing that there is almost no effect on the brain.¹⁷⁷Lu-IBA showed higher uptake in bone system and excellent T/NT ratios.Bone images are also clear,with obvious accumulation in bone lesion.These findings demonstrate superior bone-targeting characteristics of ¹⁷⁷Lu-IBA,suggesting that ¹⁷⁷Lu-IBA is a potential suitable radiopharmaceutical for bone imaging and bone targeted therapy.