| Part One Radiochemical synthesis of18F-ML-10Objective:To develop the automated production method of18F-ML-10.Materials and methods:A new automated synthesis of18F-ML-10was developed, using one-pot two-step strategy and an upgraded Explora FDG4module integrated with a semi-preparative HPLC system. Firstly, the nucleophilic [18F]radiofluorination of precursor MML-95with activated18F ion was carried out at130℃for15min to yield the labeled intermediate. Secondly, the alkaline hydrolysis of the labeled intermediate was run at120℃for10min to produce18F-ML-10after addition of NaOH, and18F-ML-10was purified by HPLC. Quality controls were performed.Results:This automated production of18F-ML-10is of fast, reliable and multi-run features, being completed within70min with radiochemical yield of15%-20%(without decaycorrection). The quality control of18F-ML-10was identical with the radiopharmaceutical requirements, especially the radiochemical purity of greater than95%and high chemical purity and specific activity own to HPLC purification.Conclusions:18F-ML-10was prepared on a widely available automated module and passed the quality control. Part Two Experimenton18F-ML-10in detecting apoptosis of pancreatic cancer after chemotherapy in vitroObjective:To evaluate the detecting effectiveness of18F-ML-10on tumor apoptosis after chemotherapy in vitro.Materials and methods:Human pancreatic cancer SW1990cells were cultivated and plated in24-well culture dishes. The study consisted of3experiments:(1) to investigate the cellular uptake of18F-ML-10and the apoptotic ratio of SW1990cells at different time after chemotherapy by Gemcitabine;(2) to investigate the cellular uptake of18F-ML-10and the apoptotic ratio of SW1990cel ls after chemotherapy with different dosage of Gemcitabine:and(3)to investigate the cel lular uptake of18F-ML-10at different time after mixed with SW1990cel ls after chemotherapy.SPSS17.0was used for statistical analysis.Comparison among groups was conducted by one-way analysis of variance.The correlat ion between cellular uptake of18F-ML-10and the apoptotic ratio of SW1990cells was analyzed by linear correlation and regression.Results:(1) After the in vitro treatment of SWl990cells with Gemcitabine,the cellular uptake of18F-ML-10were(0.08±0.01)％,(0.43±0.06)％,(0.59±0.08)％,(0.97±0.10)％and(0.86±0.06)％in control group and groups at2,4,24and48h after chemotherapy, respectively.The corresponding apoptotic ratios were(5.65±0.62)％,(7.93±0.73)％,(11.58±0.90)％,(18.10±1.40)％and(13.48±0.51)％, respectively.8F-ML-10uptakes and apoptotic ratio were significantly different among groups(F=105.4and118.6,repectively, all P<0.001).(2)The cellular uptake of18F-ML-10were(0.09±O.02)％,(0.31±O.07)％,(0.63±0.08)％,(0.94±O.07)％and(0.51±0.06)％for different dosages of Gemcitabine at the1evel of0,1,10,100and1000ug/ml,respectively.The corresponding apoptotic ratios were (5.38±0.51)％,(6.65±0.39)％,(11.58±0.67)％,(18.05±1.13)％and(9.95±0.81)％,respectively..18F-ML-10uptakes and apoptotic ratios were significantly different among groups(F=98.2and179.1, repectively,all P<0.001).(3)Significant positive correlation was found between the cellular uptake of18F-ML-10and apoptotic ratio.(apoptotic ratio=13.99×uptake rate of18F-ML-10+3.31,r=0.97,P <0.001).(4)the cellular uptake of18F-ML-10were(0.30±0.05)％,(0.45±0.05)％,(0.97±0.08)％,(0.93±0.09)％,(0.78±0.10)％and(0.75±0.09)％at the binding time of15,30,60,90and120min, respectively.The cellular uptake of18F-ML-10culminated at60min,and subsequently decreased slowly.Conclusions:The cellular uptake of18F-ML-10can reflect the degree of SW1990cells apoptosis induced by chemotherapy in vitro.The cellular uptake of18F-ML-10peaked at60min after incubating SW1990cells with18F-ML-10. Part Three Biodistribution of18F-ML-10in normal animalsObjective:To evaluate the biodistribution and PET imaging of18F-ML-10in normal small animals to provide the basis for further studies on the detection of chemotherapy-induced apoptosis of tumor.Materials and methods:The study consisted of2experiments.(1) biodistribution of18F-ML-10:18F-ML-10was administrated intravenously to KM mice (5per group). At15,30,60,90,120, and180min after administration, mice were sacrificed by humane euthanasia, and the radioactivity in various tissues and organs was counted. Radioactivity values were presented as the percentage injected dose pergram (%ID/g) of tissue.(2)18F-ML-10microPET/CT imaging of normal mice and rats:mice and rats were injected with18F-ML-10via tail vein. The microPET/CT scans were performed at60min and120min after injection, respectively. The in vivo distribution and excretion of18F-ML-10was observed.Results:Biodistribution and PET imaging studies revealed clearance of18F-ML-10from blood predominantly through the kidneys, with a small part of18F-ML-10excreting through liver to gallbladder.18F-ML-10cleared from blood slowly. Retention of18F-ML-10were (2.855±0.250)%ID/g,(1.412±0.274)%ID/g,(0.733±0.074)%ID/g,(0.422±0.059)%ID/g, and (0.314±0.043)%ID/g in kidney, and (1.214±0.048)%ID/g,(1.156±0.086)%ID/g,(0.504±0.063)%ID/g,(0.361±0.049)%ID/g and (0.243±0.028)%ID/g in blood, at15,30,60,90,120, and180min after administration, respectively.18F-ML-10showed no accumulationin healthy tissues.Conclusions:18F-ML-10was eliminated from blood mainly by urinary system and no significant tracer metabolismin vivo was observed. The biodistribution profile of18F-ML-10is suitable as a baseline setting for the imaging of apoptosis-related foci of disease. Part Four Evaluation of18F-ML-10microPET/CT in detecting apoptosis of tumor after chemotherapyObjective:To evaluate the feasibility of18F-ML-10microPET/CT in the detection of apoptosis after a single dose of chemotherapy on mice bearing with pancreatic carcinoma to provide an experimental basis for clinical study in the future.Materials and methods:SW1990pancreatic carcinoma bearing Balb/c nude mice were randomly divided into five groups:control group and groups at6h,24h,48h and72h after chemotherapy. Gemcitabine (120mg/Kg) was administrated intraperitoneally to the mice in the treatment groups and physiological saline in the same volume was administrated intraperitoneally to the mice in the control groups24h before microPET/CT imaging. PET/CT scans were carried out60min after the injection of18F-ML-10. The maximum values of%ID/g of tumor were obtained by ROI analysis. Tumors were extracted immediately after imaging. TUNEL staining was performed, and the apoptosis index (AI) was calculated according to TUNEL staining to determine the degree of apoptosis in tumor tissues. Comparison among groups was conducted by one-way analysis of variance. The correlation between tumor uptake of18F-ML-10and AI of tumor was analyzed.Results:Uptakes of18F-ML-10were visualized by microPET/CT in local tumor at group24h,48h and72h after chemotherapy. The tumors of group6h and control group failed to uptake18F-ML-10. The%ID/g for tumor uptake of18F-ML-10were0.30±0.03,0.33±0.03,0.65±0.07,0.67±0.07and0.87±0.08, AI of tumor were1.40±0.55,1.60±0.55,5.00±1.22,5.20±1.30and7.80±1.30in control group, group6h,24h,48h and72h, respectively. The differences of both%ID/g and AI among these groups were significant (F=88.97and33.18, respectively, all P<0.001). The tumor uptake of18F-ML-10were significantly correlated with the AI (r=0.89, P<0.001)Conclusions:18F-ML-10microPET/CT can reflect apoptosis induced by chemotherapy in pancreatic carcinoma bearing nude mice. The optimal time window for the detection of apoptosis with18F-ML-10microPET/CT could be72h after chemotherapy. Part Fiver Evaluation of18F-ML-10microPET/CT in detecting apoptosis of tumor after radiotherapyObjective:To evaluate the feasibility of18F-ML-10microPET/CT in the detection of apoptosis on mice bearing with pancreatic carcinoma after radiotherapy.Materials and methods:Balb/c nude mice bearing with two SW1990pancreatic tumors at right and left flank were randomly divided into2groups: radiotherapy dose of5Gy and10Gy. The tumor at right flank received radiation and anther tumor at left flank was outside the field of radiation.18F-ML-10microPET/CT was performed24h after radiotherapy. The maximum values of%ID/g of tumor were obtained by ROI analysis. Tumors were extracted immediately after imaging. TUNEL staining was performed, and the apoptosis index (AI) was calculated according to TUNEL staining. Comparison among groups was conducted by t-test. The correlation between tumor uptake of18F-ML-10and AI of tumor was analyzed.Results:Uptakes of18F-ML-10were visualized by microPET/CT in local tumor at right and left flank in group10Gy, with more accumulation of the radiotracer in the right tumor. No significant accumulation of the radiotracer was observed in the two-lateral tumors at group5Gy. The%ID/g for uptake of18F-ML-10in right tumors at group10Gy were significantly higher than in left tumor (0.85±0.07VS0.72±0.04, t=3.39, P<0.02), while AIs were not different significantly (7.60±1.34VS6.40±1.14, t=1.52, P>0.05). The difference of%ID/g and AI between two lateral tumors at group5Gy was not significant, with0.40±0.06VS0.38±0.05and1.40±0.55VS1.20±0.45(t=0.33and0.63, respectively, all P>0.05). The tumor uptake of18F-ML-10were significantly correlated with AI (r=0.92, P<0.001).Conclusions:18F-ML-10microPET/CT can reflect apoptosis induced by radiotherapy in pancreatic carcinoma bearing nude mice. |
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