Development Of Two Novel ^99mTc-labeled Nitroimidazole Imaging Agents And Research On Targeting Hypoxia In Vivo And In Vitro

Background and Objective:Hypoxia is a common pathophysiological feature of malignant tumors.Hypoxia can promote tumor invasion and metastasis,and be more resistant to a variety of treatments such as radiotherapy and chemotherapy.The detection of hypoxia in tumors has been an important research hotspot.In the field of nuclear medicine,although many radiopharmaceuticals have been developed for hypoxic imaging,only a few such as ¹⁸FMISO,¹²³I-IAZA,^99mTc-HL91 are currently used in clinical practice,and they are also not very ideal because of chemical structure and biological characteristics.Newer radiopharmaceuticals with better pharmacokinetics and higher tumor/background ratios need further development.In recent years,improvements in detectors and other hardware and reconstruction algorithms have enabled the era of quantitative analysis of radionuclide single-photon imaging.In the near future,it is entirely possible to obtain tumor images with high resolution and clear tissue structure using the SPECT/CT all-in-one machine and guide the delineation of radiotherapy hypoxic biological target areas.Technetium(^99mTc)has many advantages and ^99mTc labeled hypoxic radiopharmaceuticals can complement the advantages of PET positron radiopharmaceuticals.This subject designed and synthesized a new 5-nitroimidazole-aspartic acid derivative,explored structural modification of the compound and ^99mTc labeling method,conducted elemental analysis verification and physical property quality control tests.Then we have conducted research on tumor hypoxia selectivity from three levels of molecular level,in vitro cells and mouse transplanted tumor models and compared with ^99mTc-BRU59-21,a representative of 2-nitroimidazoles which has excellent performance in current SPECT hypoxic drugs and has not been studied in China.This study would provide the basis of developing a new type of SPECT radiopharmaceutical for hypoxia with simple structure,easy preparation,high benefit-cost ratio,and good hypoxia selectivity.Methods:A new ligand 5-nitroimidazole aspartic acid derivative（3-amino-4-[2-（2-methyl-5-nitro-1H-imidazolyl）-ethylamino]-4-oxo-Butyric acid,codenamed NASn）and 2-nitroimidazole HMPAO derivatives（coded as BRU59-21）were designed and synthesised.The ^99mTc labeling method was explored and optimized.TLC method and HPLC method were used to control the quality of the labled agents and to test the stability of labled agents in vivo and in vitro,lipid-water partition coefficient,charge distribution,plasma protein binding rate,abnormal toxicity and other general properties.The optimal time for A549 adenocarcinoma cells to be cultured under hypoxic condition was determined by CCK-8 cell activity assay,Western Blot and RT-PCR detection of endogenous hypoxia marker HIF-1α.Through in vitro cell uptake experiments and in vivo biodistribution experiments in nude mice bearing A549adenocarcinoma,the aggregation of the two agents in hypoxic cells and tissues,and blood clearance in normal mice were studied,and the two agents were compared.Comparison the differences of the distribution of the two agents in nude mouse models bearing lung cancer A549 with different tumor weights,and SPECT imaging results.Finally,the immunohistochemical staining of the hypoxic marker PIMO or HIF-1αwith the tumor tissue sections and the autoradiography results of the same tumor tissue sections were compared.Qualitative and quantitative detection of the distribution of the above two labled agents in tumors were done to verify hypoxia selectivity of them.Results:（1）The NMR and MS analysis of the two unlabeled precursors showed the same structure as the design.NASn was radiolabelled with ^99mTc as[^99mTcN]-NASn through two-step method,and BRU59-21 was directly labeled with ^99mTc to form^99mTc-BRU59-21.The radioactive labeling conditions of NASn were optimized,and the radiochemical purity of both labeled products was>95%.（2）[^99mTcN]-NASn and^99mTc-BRU59-21 are both electrically neutral compounds,and their lipid-water partition coefficients are 1.7 and 1.38,which are both lipophilic.When placed at room temperature,human serum albumin at 37°C,and mouse liver homogenate at 37°C for 4 h,the radiochemical purity of the markers did not decrease significantly.The plasma protein binding rates of[^99mTcN]-NASn and ^99mTc-BRU59-21 were 68.8%and50.5%,respectively.Two labeled agents,74MBq,were injected into the experimental mice through tail vein for 7 days.All the mice survived and there were no obvious adverse reactions.（3）The survival rates of cultured A549 cells at different time under hypoxic condition were significantly different（P=0.009）,and the highest survival rate was（96.30±2.79）%in the group under hypoxic condition for 24h.Under normal and hypoxic conditions,A549 cells were cultured at different times（4h⁴8h）,and the expression of HIF-1a protein began to increase after 4h of culture under hypoxia,reaching a peak at 24h,and its expression decreased after 48h.There was a statistically significant difference in expression under two oxygen conditions（3.69±0.37 vs 1.01±0.04 at 24h,P=0.000）;HIF-1a mRNA expression began to increase significantly at12h,and peaked at 24h（3.27±0.32 vs 1.03±0.28,P=0.000）.In vitro cell uptake experiments showed that:10 minutes after adding the agents,the percentage of agent uptake in A549 cells in the hypoxic system gradually increased with time,and all were higher than the percentage of uptake in cells in the corresponding non-hypoxic phase.The peak time of ^99mTc-NASn uptake was 60min,and the peak time of ^99mTc-BRU59-21 cells was up to 120min[（28.51±2.36）%and（24.34±2.65）%respectively].The uptake values were all about 5 times as much as those under the normoxic state,and the difference was statistically significant（p<0.05）.（4）The blood clearance experiment showed that the blood clearance of the two agents in normal mice conformed as the two-compartment metabolism model,and the elimination phase half-lives were112.47min and 61.28min for ^99mTc-NASn and ^99mTc-BRU59-21,respectively.The distribution data of nude mice bearing lung adenocarcinoma A549 showed that the clearance of the two markers was slow after entering the blood,and were mainly excreted through the liver and intestines,and some were excreted by the kidneys.Tumor uptake gradually increased with time.NAsn uptake reached a peak（4.57±0.29）%ID/g at 120min,and tumor/muscle uptake ratio（T/M）was 6.80;the absolute uptake value of NAsn was better than BRU-5921,the latter reached a peak（2.66±0.22）%ID/g at 60 min,and the T/M was 3.54.BRU-5921 in blood was cleared faster,and the tumor-to-blood ratio（T/B）was higher than NASn before 60min.SPECT/CT imaging of nude mice bearing A549 adenocarcinoma was similar to the distribution in vivo.Imaging comparison of nude mice bearing A549 with two tumor volumes of 0.5cm or1.5cm.The tumor/background ratio（T/N）of two agents of the same tumor volume was statistically significant（1.5cm-5.17±0.68 vs 2.45±0.53,0.5cm-4.53±0.55 vs2.09±0.48,p<0.005）.Comparing the immunohistochemical results of HIF-1a with the same tumor tissue sections and imaging findings,the larger the tumor volume（1.5cm vs 0.5cm）,the higher the HIF-1a expression content（88.23%±4.78%vs 48.62%±3.48%）,and the higher the imaging T/N（NASn 5.17±0.68 vs 4.53±0.55;BRU59-21 2.45±0.53 vs 2.09±0.48）,indicating that both agents are both good hypoxia selectivity in the tumors.（5）There was a significant positive correlation between the uptake of these two radiolabeled agents and the immunohistochemical staining of PIMO:the correlation between tumor uptake of ^99mTc-NASn and PIMO positive in autoradiography（Pearson correlation r=0.861,P=0.000）.Relationship between ^99mTc-BRU59-21 uptake and PIMO positive（r=0.71,P=0.002）.The correlation between ^99mTc-NASn and PIMO is higher than the correlation between^99mTc-BRU59-21 and PIMO（χ2=8.38,P=0.001）.Conclusions:[^99mTcN]-NASn and ^99mTc-BRU59-21 are electrically neutral,lipophilic compounds,have few abnormal toxicity,and have good stability in vitro and in vivo.In vitro cell uptake and SPECT imaging,immunohistochemistry,autoradiography studies of tumor-bearing animal models showed that both of them have good hypoxia selectivity.[^99mTcN]-NASn’s relatively slow blood clearance mode and two hypoxic reducing groups in the structure may make tumor uptake and retention better,which is worth further development as a radiopharmaceutical for tumor hypoxia imaging.