Preparation,Biological Characteristics Evaluation,and Preliminary Imaging Studies Of ¹⁸⁸Re-Ibandronate

Objective:Ibandronate and ¹⁸⁸Re were selected for radiolabeling in our research to explore the best labeling conditions and biological characteristics of¹⁸⁸Re-ibandronate,and study the biodistribution and imaging characteristics of¹⁸⁸Re-ibandronate in normal animals and nude mice with bone metastasis,to explore its feasibility as a new type of bone-seeking radiopharmaceuticals with both imaging and therapeutic effects.Methods:We determined the effects of ibandronate,ascorbic acid,KRe O₄,Sn Cl₂,¹⁸⁸Re O₄^-activity,p H,temperature,and reaction time on the radiochemical purity（RCP）of ¹⁸⁸Re-ibandronate by the method of controlled variables.First,we sequentially mixed 0.1-1.8 mg,0-0.5 mg,0.02-0.4 mg,and 0-0.019 mg of ibandronate,ascorbic acid,Sn Cl₂,and KRe O₄,respectively.We then added the fresh eluted Na¹⁸⁸Re O₄ solution 18.5-444 MBq.Subsequently,we adjusted the p H value to 0.5-4.The reaction occurred at temperatures of room temperature（25±2）,60,and 95 for 10-60 min,respectively.After the reaction was completed,it was cooled to room temperature.The p H value of each tube was adjusted to 6-7.RCP of ¹⁸⁸Re-ibandronate was analyzed by thin layer chromatography.The stability of ¹⁸⁸Re-ibandronate incubated in human serum（37℃）and normal saline（room temperature,25±2℃）for 0.5 h,1 h,3 h,6 h,8h,and 24 h was studied.To study the plasma protein binding rate（plasma protein binding rate,PPB）of ¹⁸⁸Re-ibandronate with human plasma at 37℃for 2hours.Using n-Octanol as organic phase,the lipid-water distribution coefficient（lipid-water distribution coefficient,log P）of ¹⁸⁸Re-ibandronate was studied.Blood samples were collected at 5 min,10 min,15 min,30 min,1 h,2 h,3 h,4h,6 h,and 8 h in rats after injection of 3.7 MBq ¹⁸⁸Re-ibandronate.The percentage of the injected dose per gram of tissue（%ID/g）was calculated,and the blood clearance curve of the drug was draw and the pharmacokinetic parameters were calculated.Sixteen mice were selected for toxicity test.The mice were divided into four groups:high dose group(37 MBq ¹⁸⁸Re-ibandronate),medium dose group(18.5 MBq ¹⁸⁸Re-ibandronate),low dose group(3.7 MBq¹⁸⁸Re-ibandronate),and control group（normal saline）;and there are 4 mice in each group,half male and half female.The survival,activity,vomiting,food consumption,and stool of the mice were observed within 28 days,and the weight within 28 days was recorded.The mice were killed after 28 days,and the heart,liver,spleen,lung,kidney,stomach,small intestine,brain,bone marrow,and muscle were taken for pathological examination.The in vivo distribution of 48mice were studied.The mice randomly divided into 12 groups with 4 mice in each group,half male and half female.Six groups were injected with ¹⁸⁸Re-ibandronate 3.7 MBq,and the other 6 groups were injected with Na¹⁸⁸Re O₄ 3.7MBq;blood,heart,liver,spleen,lung,kidney,stomach,thyroid,small intestine,brain,femur,muscle,and gonadal tissue were taken at 1 h,3 h,6 h,8 h,24 h,and48 h after injection,and the%ID/g was calculated and the radioactivity uptake ratios of bone to heart,liver,blood,and muscle of the two groups were calculated respectively.Three New Zealand rabbits were injected with ¹⁸⁸Re-ibandronate74-100 MBq,and bone imaging was performed at 20 min,40 min,60 min,80min,100 min,120 min,3 h,4 h,6 h,8 h,24 h,and 48 h.The nude mice model of bone metastasis of prostate cancer PC-3 and breast cancer MDA-MB-231 was established by tibial bone marrow injection,and the bone metastasis model was judged by computed tomography.The in vivo distribution of 20 nude mice with bone metastasis were studied.They were randomly divided into 5 groups with 4mice in each group,and half of the two models.Blood,heart,liver,spleen,lung,kidney,stomach,thyroid,small intestine,brain,the healthy side tibia,the healthy side muscle,affected side tibia,and affected side soft tissue were taken to calculate the%ID/g at 1 h,3 h,6 h,24 h,and 48 h after injection of ¹⁸⁸Re-ibandronate 1.85 MBq.The radioactivity uptake ratios of the healthy side tibia and affected side tibia to heart,liver,blood,and the healthy side muscle were calculated,respectively;and the radioactivity uptake ratio of affected side tibia to the healthy side tibia and affected side soft tissue,affected side soft tissue to the healthy side muscle was calculated.3 PC-3 and 3 MDA-MB-231 nude mice with bone metastasis were used for imaging study.Bone imaging was performed in nude mice at 1 h,2 h,3 h,6 h,16 h,and 32 h after injection of ¹⁸⁸Re-ibandronate 9.3-13 MBq.Results:We have successfully prepared ¹⁸⁸Re-ibandronate which could be consistently prepared in>95%RCP.The optimum preparation conditions were as follows:ibandronate,0.8–1.2 mg;ascorbic acid,0.20–0.35 mg;Sn Cl₂,0.14–0.18 mg;KRe O₄,0.005 mg;and ¹⁸⁸Re O₄^-activity,18.5–296 MBq.They were reacted for 30min at 95 and p H=2.¹⁸⁸Re-ibandronate has good stability.Its radiochemical purity is more than 95%at room temperature（25±2）and in normal saline for 8 h,and is still more than 90%after 24 h.Its PPB was 79.8±0.71%and log P was-2.33±0.02.Its metabolic process accords with two-compartment model and blood clearance is fast.There was no obvious adverse reaction in mice after using ¹⁸⁸Re-ibandronate at different doses（high dose,37 MBq;middle dose,18.5 MBq;low dose,3.7 MBq）in 28 days;the high dose group inhibited the weight gain of mice to a certain extent,but there was no obvious damage to tissues and organs.The distribution of ¹⁸⁸Re-ibandronate in mice showed the skeletal uptake was high,with%ID/g of 10.394±3.849 at 6 h;since then,bone uptake has decreased,but it was still as high as 5.699±1.331%ID/g at 48 h.In addition,the radioactivity ratios of bone to heart,liver,blood,and muscle were high to 327.902,111.183,326.053,and 291.551,respectively.The distribution of Na¹⁸⁸Re O₄ in vivo showed that the high uptake of stomach and thyroid（the highest at 1 h,%ID/g was 22.747±5.673 and 20.247±2.489,respectively）,while the skeletal uptake of Na¹⁸⁸Re O₄ was low（the highest was 1.67±0.345%ID/g）.In the bone imaging of New Zealand rabbits showed that ¹⁸⁸Re-ibandronate had rapid blood clearance rate and low soft tissue uptake;and the whole-body bone imaging was clear,and the contrast between bone and background was high;at48 h,the whole-body bone still had significant uptake of ¹⁸⁸Re-ibandronate.The distribution of ¹⁸⁸Re-ibandronate in nude mice with bone metastasis showed that the uptake in the healthy side bone was high,and the uptake reached the maximum at 1 h（9.331±0.541%ID/g）;since then,bone uptake has decreased,but it can be maintained at a high level,and the%ID/g at 6 h was 7.662±2.934and 48 h was 4.737±0.863.The uptake of ¹⁸⁸Re-ibandronate in affected side bone reached the maximum at 6 h（8.417±1.820%ID/g）;since then,bone uptake has decreased,but it was still as high as 6.503±0.010%ID/g at 48 h.In addition,the radioactivity ratio of affected side bone to the heart,liver,blood,the healthy side muscle,and the affected side soft tissue was high,and the highest was 183.399,146.359,314.817,158.088,and 154.239,respectively.Bone imaging of the nude mice with bone metastasis showed obvious accumulation of affected side limb at each time point,and the uptake of affected side bone was significantly higher than that of the healthy side bone at 6-32 h.Conclusion:¹⁸⁸Re-ibandronate is an excellent bone-seeking radiopharmaceutical with high RCP,good stability,high plasma protein binding rate,good hydrophilicity,rapid blood clearance,safe,and low toxicity.It has good bone targeting ability and stays in bone for a long time,and its targeting ability for bone metastasis is higher than that of normal bone tissue;its non-target tissue is cleared quickly and the whole-body bone imaging is clear with high contrast.¹⁸⁸Re-ibandronate is a potential radiopharmaceutical for imaging and treatment of bone metastases.