Study Of Tumor Proliferation Imaging Agents

Tumors are classified on the basis of the tissue of origin, cell type, whetherbegign or malignant, degree of differentiation, anatomic site and function. Because ofthis diversity, no single imaging technique is capable of detecting all tumors. Whilethe radiological techniques （X-ray, CT, MRI and Ultrasound） can provide information,regarding location and size of the tumor with better resolution than nuclear imagingtechniques, these lack the specificity and sometimes can not even distinguish residual,viable disease from fibrosis. Positron emission tomography （PET） and single photonemission computed tomography （SPECT） techniques, on the other hand, have thepotential to provide functional status of the tumor tissue （metabolism and receptorexpression） and offer higher specificity.In clinical oncology,2-[¹⁸F]fluoro-2-deoxy-d-glucose (¹⁸F-FDG), a glucosederivative, has been widely used for tumor imaging with positron emissiontomography （PET） in recent years. However, high¹⁸F-FDG uptake is a non-specificmeasure of overall glucose utilities, ranging from inflammation caused bymacropages to tumor cell.Yet, the assay of tumor proliferation and DNA synthesis aremore attractive for tumor imaging. Therefore, many studies have been focus on thedevelopment of a variety of DNA precursors.In principle, the ideal tracer to image cell proliferation should rapidly andspecifically be incorporated into tumor DNA so that tracer retention reflects DNAsynthesis and, hence, tumor cell proliferation. The labeled thymidine analog targetsspecifically the proliferative activity of malignant lesions. So far several usefulligands such as11C-labeled nucleoside thymidine and3’-deoxy-3’-[¹⁸F]fluorothymidine (¹⁸F-FLT) have been demonstrated their good imaging features. However,the short half-life of, complicated radiochemical synthesis, and the lowerradiochemical yield have become main obstacles for its production. Most nuclearmedicine procedures are performed with [^99mTc] technetium-basedradiopharmaceuticals, and therefore it is important to develop a technetium labeledthymidine analog so as to provide the ideal characteristics needed for routine clinicalstudies.Our study was about radiolabeled thymidine analogs as tumor imaging agents,which involves two parts. Part one: The study of¹⁸F labeled thymidine analog¹⁸F-FLT. Part two: The study of^99mTc labeled thymidine analogs. Part one: The study of¹⁸F labeled thymidine analog¹⁸F-FLT1, Preparation of the labeling precursors of¹⁸F-FLTThere are several labeling precursors of¹⁸F-FLT, among which [5’-O-（4,4’-dimethoxytriphenylmethyl）-2’-deoxy-3’-O-（4-nitrobenzesulfonyl）-β-D-threopentofuranosyl] thymine （Nosyl-FLT） and3-N-t-butoxycarbonyl-（5’-O-（4,4’-dimethoxytriphenylmethyl）-2’-deoxy-3’-O-（4-nitrobenzesulfonyl）-β-D-threopentofuranosyl） thymine （N-BOC-FLT）with high label yield. The labeling precursors Nosyl-FLT and N-BOC-FLT weresynthesized from the raw material thymidine and the reaction conditions wereoptimized. Fist, thymidine was protected at the5’-O-position with dimethoxytrityl.Second, the configuration of hydroxide in3’-position was overturn and then esterifiedwith4-nitrobenzensulfonyl to obtain Nosyl-FLT, Nosyl-FLT was protected at the3-Nposition with t-butoxycarbonyl to obtain N-BOC-FLT. Nosyl-FLT, N-BOC-FLT andall intermediates were verified with IR,1HNMR and MS.2, Determination of thymidine derivatives as labeling precursors of¹⁸F-FLT（1） Determination of Nosyl-FLT as labeling precursor of¹⁸F-FLT byHPLC-ELSDAn HPLC-ELSD method was established for the determining the concentrationof Nosyl-FLT, which was one of labeling precursors of¹⁸F-FLT. A C8column （5μm,4.6×250mm） was used with the mobile phase of methanol/water/trifluoroacetate（90/10/0.1, v/v/v） at a flow rate of0.5ml/min. The content of Nosyl-FLT in samplewas measured by external standard method. The resolution of Nosyl-FLT andimpurity was good and the calibration curve was linear in the concentration range of10-100μg/ml. The limit of detection was5ng and RSDs were less than1.25%.（2） Determination of N-BOC-FLT as labeling precursor of¹⁸F-FLT by HPLC-UVA simple and rapid method was established for determining the concentration ofN-BOC-FLT. The content of N-BOC-FLT was determined by reversed phase HPLCusing a C18column（5μm,4.6×250mm） with the mobile phase of acetonitrile/water（85/15, v/v） at a flow rate of0.8ml/min and235nm as detect wavelength. The contentof N-BOC-FLT in sample was measured by external standard method. The retentiontime of the main peak was6.6min and that of impurity was4.5min. The resolution ofN-BOC-FLT and impurity was>1.5. The calibration curve was linear in theconcentration range of2-10μg/mL. Detection limit of N-BOC-FLT was2.5ng（S/N≥3）. 3,¹⁸F labeling and preliminary biological evaluation of¹⁸F-FLTNucleophilic substitution of fluororeplacement reaction was proceeding withN-BOC-FLT as labeling precursor at120°C. Radiochemical purity was determinedwith TLC and HPLC. Biodistribution was performed with mice. The results showedthat radiochemical yield was35.2%±5.8（n=2, decay corrected） and finalradiochemical purity was above97%which was stable within6h. Biodistribution of¹⁸F-FLT in mice suggested that the uptakes of¹⁸F-FLT in kidney, spleen and intestinewere higher than that of¹⁸F-FLT in heart, liver and lung at60min postinjection.MicroPET image of tumor in tumor-bearing nude mice was clear.4, Conclusions（1） In this study, an economical and simple method of preparation of¹⁸F-FLTlabeling precursors, Nosyl-FLT and N-BOC-FLT, was established through theoptimization of reaction conditions.（2） The method is simple and reliable to detect Nosyl-FLT content and relatedimpurity content by high performance liquid chromatography with evaporative lightscattering detector.（3） The method is simple and reliable to detect N-BOC-FLT content by highperformance liquid chromatography with UV detector, which provides an effectivemeans of quality control of the kit.（4） In this study,¹⁸F-FLT was obtain by labeling precursor N-BOC-FLT, the finalradiochemical yield was35.2%, radiochemical purity was above97.8%, in vitrostability, and a higher uptake at the tumor site, all these suggested¹⁸F-FLT can beused for further clinical studies.Part two: The study of^99mTc labeled thymidine analogs1, Preparation and preliminary biological evaluation of^99mTc-ANMdUThe labeling precursor5-{2-sulfanylethyl-[2-（2-sulfanylethylamino）acetyl]amino}-methyl-2’-deoxyuridine （ANMdU） was synthesizedin a six-step reaction sequencefrom the raw material thymidine. Using SnCl2as reducing agent, and in the presenceof sodium glucoheptonate, ethylene diamine tetraacetic aciddisodium salt（EDTA-2Na）, the reaction mixture was added to100°C and kept for30min to obtain^99mTc-ANMdU. The radiochemical purity of the^99mTc-ANMdU was over95%.Partition coefficients （lgP） were0.92and0.70at pH7.0and pH7.4of thephosphate buffer saline, respectively. Biodistribution of^99mTc-ANMdU intumor-bearing mice showed that the high initial uptake of^99mTc-ANMdU in vivo and the clearance was rapid. The highest uptake of^99mTc-ANMdU wasin kidney, whichmeans that the clearance of^99mTc-ANMdU was mainly through the renal pathway. At1h p.i.,the ratios of the percentage administered activity per gram of tissue （%ID/g）of tumor-to-muscle, tumor-to-bone and tumor-to-blood were1.58±0.17，1.95±0.31and1.17±0.08, respectively.2, Preparation and preliminary biological evaluation of^99mTc-NHTThe labeling precursor N3-{N’-[2-sulfanyl-ethylamino)acetyl]-2-aminoethylsulfanyl-1-hexanamide}-thymidine （NHT） was synthesizedin a five-step reactionsequence from the raw material thymidine. Using SnCl2as reducing agent, and in thepresence of sodium glucoheptonate, ethylene diamine tetraacetic acid disodiumsalt （EDTA-2Na）, the reaction mixture was added to100°C and kept for30min toobtain^99mTc-NHT. The radiochemical purity of the^99mTc-NHT was above95%.Partition coefficients （lgP） were0.76and0.56at pH7.0and pH7.4of the phosphatebuffer saline, respectively. Biodistribution of^99mTc-NHT in tumor-bearing miceshowed that the high uptake of^99mTc-NHT in liver and kidney, which means that theclearance of^99mTc-NHT was mainly through the hepatobiliary pathway and the renalpathway. At2h p.i., the ratios of%ID/g of tumor-to-muscle, tumor-to-bone andtumor-to-blood were4.41±0.32,2.45±0.24and1.51±0.18, respectively.3, Preparation and preliminary biological evaluation of two novel^99mTc-labelledlong chain thymidine analogs.Two kinds of novel long chain thymidine derivatives,N-thymidine-yl-N’-methyl-N’-{N’’-[2-sulfanyl-（ethylamino）acetyl]-2-amino-ethylsulfanyl-1-hexanamide}–ethanediamine （TMHEA） and N-thymidine-yl-N’-methyl-N’-{N’’-[2-sulfanyl-（ethylamino） acetyl]-2-amino-ethylsulfanyl-1-hexanamide}–hexanediamine （TMHHA） were prepared and successfullylabeled with^99mTc in high labeling yields. The in vitro stability and in vivobiodistribution of^99mTc-TMHEA and^99mTc-TMHHA were investigated and compared.The biodistribution studies indicate that the radiotracer^99mTc-TMHEA has selectiveuptake in the tumor. At2h p.i. of^99mTc-TMHEA, the ratios of%ID/g oftumor-to-muscle, tumor-to-bone and tumor-to-blood were2.84±0.76,1.27±0.20and1.87±0.02, respectively. However, the ratios of%ID/g of tumor-to-muscle,tumor-to-bone and tumor-to-blood of^99mTc-TMHHA were2.42±0.33,0.90±0.16and1.28±0.22respectively at2h p.i. 4，ConclusionsBiodistribution of^99mTc-ANMdU in mice showed that the high initial uptake of^99mTc-ANMdU in vivo and the clearance was rapid, though the ratios of%ID/g oftumor-to-muscle, tumor-to-bone and tumor-to-blood were not high. The clearance of^99mTc-NHT in mice was shower than that of^99mTc-ANMdU. However, ratiosof%ID/g of tumor-to-muscle, tumor-to-bone and tumor-to-blood of^99mTc-NHT were4.41±0.32,2.45±0.24and1.51±0.18respectively, which were higher than those of^99mTc-ANMdU. The linker chain between the thymidine and N2S2ligand of^99mTc-NHT is longer than that of^99mTc-ANMdU, which means that extension oflinker chain maybe increased tumor uptake. However, ratios of%ID/g oftumor-to-muscle, tumor-to-bone and tumor-to-blood of long chain thymidinederivatives^99mTc-TMHEA and^99mTc-TMHHA were lower than those of^99mTc-NHT.This indicates that limitless extension of the carbon chain is not always beneficial toimprove the uptake ratio of tumor-to-muscle, tumor-to-bone and tumor-to-blood.Among four^99mTc-labelled thymidine analogs (^99mTc-ANMdU,^99mTc-NHT,^99mTc-TMHEA and^99mTc-TMHHA), ratios of%ID/g of tumor-to-muscle,tumor-to-bone and tumor-to-blood of^99mTc-NHT were higest. These indicated that^99mTc-NHT is a probably potentialSPECT tumor imaging agent and further study isneeded.