| Objective:In this study,NOTA-ICG and RESCA-ICG were designed and synthesized.The quality control analysis,fluorescence characteristics and biological distribution of the two precursors were performed.The two precursors were labeled with[18F]AlF,and the quality control analysis,octanol–water partition coefficient test,abnormal toxicity experiments,in vivo and in vitro stability test,and pharmacokinetic experiments were carried out.Rat models of liver cirrhosis and hepatic ischemia-reperfusion injury were established.The biological distribution of[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG in normal rats,rats with liver uptake inhibition,cirrhotic rats and hepatic ischemic reperfusion injuried rats was discussed.In this study,the characteristics of PET/CT imaging and liver clearance rates of the two PET radiopharmaceuticals were investigated in normal rats,cirrhotic rats and hepatic ischemia-reperfusion injured rats,and global and segmental liver functional reserve were evaluated.Methods:（1）The NOTA-ICG and RESCA-ICG were synthesized from NOTA-Bis（t Bu）ester and（±）-H3RESCA-TFP,respectively.Quality control analysis of NOTA-ICG and RESCA-ICG was carried out by HPLC and MS.The two precursors were dissolved by DCM and PBS respectively,and their excitation and emission spectra were determined.Normal rats were injected with the two precursors respectively via tail vein（n=5/group）,so as to explore the distribution of the two precursors in organs and tissues in vivo at different times.（2）NOTA-ICG was radiolabelled with[18F]AlF at 90℃and RESCA-ICG was radiolabelled with[18F]AlF at room temperature.Quality control analysis of[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG was carried out by HPLC and radio-TLC.The octanol-water partition coefficient of the two radiopharmaceuticals in PBS and n-octanol was calculated by anautomatic gamma counter.[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG were incubated in PBS and 5%BSA solution respectively,and their in vitro stability was determined by HPLC within 120 minutes.Two radiopharmaceuticals were injected into rats via tail vein,and the rats were sacrificed at different times.The liver tissue was ground and centrifuged,and the supernatant was taken.The in vivo stability of[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG was determined by radio-TLC.The mice were injected with high dose of two radiopharmaceuticals in abnormal toxicity group and the same amount of normal saline in control group（n=5/group）through tail vein.After 14days,the body weight,blood routine and blood biochemistry of all the mice were measured,and the main organs were stained with HE.The results of abnormal toxicity group and control group were compared.Rats were injected with[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG.The vein blood was collected at 2,5,10,15,30,60,90 and 120minutes after injection,and radioactivity was counted by an automatic gamma counter.The concentration-time curves of radioactive drugs were drawn to screen out the optimal metabolic model and obtain pharmacokinetic parameters.（3）The rats were injected subcutaneously with 50%CCl4 solution alternately in the abdomen 15 times for 9 weeks,using gradient increasing concentration of ethanol solution as the only water source.Then five cirrhotic rats were randomly selected for blood biochemical examination and HE staining of liver,kidney and intestine to evaluate the cirrhosis model.After 12 hours of fasting,the rats were underwent laparotomy and the blood flow into the right lobe of the liver was blocked by animal experimental vascular clamps.After 60 minutes,the vascular clamp was released and the abdominal opening was closed.Tail vein blood of rats was collected for blood biochemical examination to evaluate the hepatic ischemia-reperfusion injury model.Two radiopharmaceuticals were injected into normal rats,cirrhotic rats,hepatic ischemia-reperfusion injured rats,and hepatic uptake inhibited rats（high-dose precursors were injected 5 minutes before the injection of radiopharmaceutical）.The rats were sacrificed 5,15,30,60 and 90 minutes（n=5/group）after injection,and the radioactivity of the main tissues and organs was counted by an automatic gamma counter,and the%ID/g of main tissues and organs was calculated and compared.Dynamic micro-PET/CT scanning was performed in normal rats,cirrhotic rats and hepatic ischemia-reperfusion injured rats by injecting[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG through tail vein.The collection time were 5400 seconds.After PET/CT reconstruction,liver and kidney regions were delineated by time attenuation correction,SUVmax values at different time points were obtained,and liver clearance rates at 15 minutes,30 minutes,60 minutes and 90 minutes were calculated.The clearance rates of[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG in liver were analyzed and compared.Results:（1）The purity of NOTA-ICG and RESCA-ICG was 96.63%and 98.19%,respectively.Both precursors were confirmed by MS analysis.The wavelength peak of excitation light of NOTA-ICG was at 781.0 nm-787.0 nm,and the wavelength peak of emission light was at 806.0 nm-822.5 nm.The wavelength peak of excitation light of RESCA-ICG was at 782.5 nm-786.0 nm,and the wavelength peak of emission light was at808.0 nm-823.0 nm.The fluorescence values of NOTA-ICG and RESCA-ICG in liver were17.15±0.78×106 ph/s/cm2/sr and 15.32±1.07×106 ph/s/cm2/sr after 5 minutes,and then gradually decreased.The change trend of fluorescence value in intestinal tract was opposite to that in liver.Other tissues and organs,such as the kidney and brain,took up little of the two precursors.（2）The radiochemical purity of[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG were both greater than 98%.The radiochemical yields of[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG were 19.2±0.78%and 35.46±1.51%,respectively.The octanol-water partition coefficient of the two radiopharmaceuticals were-1.15±0.08 and-1.03±0.08,respectively.These two radiopharmaceuticals had good in vitro and in vivo stability and safety.The[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG met the two-compartment pharmacokinetic model,and the half-life of drug clearance in Beta phase was 21.31±2.56 min and 41.73±5.98 min,respectively.（3）Liver cirrhosis model and liver ischemia-reperfusion injury model were established successfully,and the rats of liver cirrhosis model were accompanied by renal function injury.[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG were rapidly taken by the liver in normal rats,and then gradually discharged into the intestine,without metabolism through the kidney or entering the brain tissue.Compared with normal group,the distribution of two radiopharmaceuticals in hepatic uptake inhibited group was reduced（P<0.05）,and the distribution of radiopharmaceuticals in blood and other organs with rich blood supply was increased（P<0.05）.In liver cirrhosis group,the distribution of two radiopharmaceuticals was increased at different time points in liver（P<0.05）,but the distribution was decreased in intestinal tract（P<0.05）.The distribution of the two radiopharmaceuticals in ischemia-reperfusion injuried liver was greater than that in the normal liver at different time points（P<0.05）.In micro-PET/CT imaging,the liver clearance rates of[18F]AlF-NOTA-ICG at 15,30 and 60min in normal liver were higher than those in liver with cirrhosis（P<0.05）,and the normal liver clearance rates at 15 and 30 min were higher than those in liver with ischemia-reperfusion injury（P<0.05）.The liver clearance rates of[18F]AlF-RESCA-ICG at30,60 and 90 min in normal liver were higher than those in liver with cirrhosis and ischemia-reperfusion injury（P<0.05）.[18F]AlF-NOTA-ICG had higher liver clearance rates than[18F]AlF-RESCA-ICG at different time points in different animal models.Conclusions:The NOTA-ICG and RESCA-ICG are successfully synthesized.[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG are radiolabelled by[18F]AlF.These two kinds of radiopharmaceuticals have high radiochemical purity,good stability,good hydrophilicity and no toxicity.[18F]AlF-NOTA-ICG and[18F]AlF-RESCA-ICG are mainly rapidly absorbed by the liver and discharged into the intestine,but not metabolized by the kidney.Both radiopharmaceuticals can be used to assess global and segmental liver functional reserve by liver clearance rates in micro-PET/CT imaging.[18F]AlF-RESCA-ICG has good radiochemical yield and good clinical transformation potential. |
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