f-2-deoxy-2-fluoro - glucose(f - fdg ) , an f - labeled glucose analog , is a gold standard for positron emission tomography ( pet ) in cancer diagnosis . however , f - fdg - pet has several limitations that can result in false positive or negative diagnoses , and its ability to predict tumor response to chemotherapy or radionuclide therapy is poor . for instance , f - fdg can not distinguish tumor tissue from inflammatory or normal brain tissues because each type of tissue has high glucose consumption . therefore , radiolabeled amino acids have been developed as an alternative to f - fdg in diagnostic imaging of cancer . their use in tumor detection is based on an increased uptake of amino acids in tumor cells , which is assumed to reflect enhanced amino acid transport , metabolism , and protein synthesis . among all radiolabeled amino acids , aromatic ones such as tyrosine and its derivatives are more suitable for use in cancer diagnostic imaging given their readily alterable chemistry and favorable biological characteristics . to date , tyrosine and its -methyl - substituted analog -methyl tyrosine ( amt ) have been successfully labeled with c , f , and i for pet imaging , as well as i for single - photon emission computed tomography ( spect ) imaging , respectively [ 27 ] . most of these radiolabeled tyrosine analogs show promise in preclinical and clinical research in diagnostic tumor imaging , especially for brain tumors . they have also been successfully applied to imaging breast cancer , which is the most common malignancy among women in the world . for instance , c - labeled tyrosine appears to be better than f - fdg for breast cancer imaging because of its lower uptake in noncancerous fibrocystic disease . however , most existing radiolabeled tyrosine derivatives require an on - site cyclotron to produce the radioisotope , which is inconvenient and costly . in addition to that , the radioisotopes have either a too short ( e.g. , 20 minutes for c ) or too long ( e.g. , 57.4 days for i ) half - life , making them impractical for clinical use . so far , f has the most appropriate half - life ( 110 minutes ) ; however , the radiosynthesis yield of f - labeled radiotracers is relatively low , apparently because of the electrophilic fluorination and tedious purification of f. therefore , a radiotracer with simpler chemistry and an affordable isotope that can be used clinically in most major medical facilities is needed . technetium-99  m ( tc ) is an ideal radioisotope for diagnostic imaging studies because of its favorable physical characteristics . it emits a 140  kev gamma ray in 89% abundance , which is commonly used in gamma and spect imaging . the half - life of tc is 6.02 hours , allowing serial images and , thus , overcoming a drawback of f . unlike most cyclotron - produced radionuclides , which use covalent chemistry for labeling the combinations of nitrogen , oxygen , and sulfur appear to be stable chelators for tc [ 1012 ] . in fact , this chelator could allow the same target ligand to be labeled with several different radioisotopes , such as gallium-68 , indium-111 , or rhenium-188 , for future diagnostic and therapeutic applications . here , we report the synthesis of tc - labeled tyrosine using 1,4,8,11-tetra - azacyclotetradecane ( n4 ) cyclam as a chelator and evaluate its potential in gamma imaging using in vitro and in vivo breast tumor models . all chemicals of analytical grade and solvents of high - performance liquid chromatography ( hplc ) grade were obtained from sigma - aldrich ( st . louis , mo , usa ) . nuclear magnetic resonance ( nmr ) was performed on a bruker 300-mhz spectrometer ( bruker biospin corporation , billerica , ma , usa ) , and mass spectra were performed on a waters q - tof ultima mass spectrometer ( waters , milford , ma , usa ) at the chemistry core facility at the university of texas md anderson cancer center ( houston , tx , usa ) . sodium pertechnetate ( natco4 ) was obtained from a mo / tc generator ( mallinckrodt , hazelwood , mo , usa ) . n - t - butoxycarbonyl - l - tyrosine methyl ester ( 25.00  g ; 0.085  mol ) was dissolved in acetone ( 300  ml ) . 1,3-dibromopropane ( 17.3  ml ; 0.169  mol ) and k2co3 ( 58.00  g ; 0.420  mol ) were added under a nitrogen atmosphere . was cooled and filtered , the solvent was removed under reduced pressure and the residue was dissolved in chloroform . the product was purified by column chromatography using a silica gel column eluted with hexane : ethyl acetate ( 2  :  1 , v / v ) . n4 cyclam ( 15.00  g ; 74.88  mmol ) was dissolved in 150  ml of anhydrous ethanol , and diethyl oxalate ( 10.94  g ; 74.88  mmol ) was added . the solvent was rotary evaporated , and the crude product was dissolved in minimum quantity of ethanol and filtered and recrystallized in 400  ml of acetone / ethanol to yield white crystals of compound 2 . compound 2 ( 0.63  g ; 2.46  mmol ) was dissolved in 20  ml of anhydrous dimethylformamide ; it was reacted with a solution of compound 1 ( 1.06  g ; 2.46  mmol ) in 40  ml of dimethylformamide under a nitrogen atmosphere . the mixture was refluxed at 75c for 18 hours and then cooled to room temperature . the crude compound was purified via silica gel column using chloroform : methanol solution ( 9  :  1 , v / v ) . ms ( m / z ) 591.31 [ m ] . to deprotect n - t - butoxycarbonyl group , compound 3 ( 0.60  g ; 1.00  mmol ) was dissolved in anhydrous dichloromethane ( 15  ml ) , and 2.5  ml of trifluoroacetic acid was added to it . the solution was stirred overnight at room temperature , and the solvents were then removed in vacuo . the crude compound was purified by chromatography over silica gel ( chloroform : methanol 9  :  1 , v / v ) giving an off - white solid . compound 4 ( 0.25  g ; 0.50  mmol ) was dissolved in 5  ml of water , and 2.5  ml of 10  n naoh was added to it . the solution was stirred and refluxed overnight at 90c , and the solvent was evaporated in vacuo . the crude compound was then dissolved in 5  ml of water , and the ph of the solution was neutralized to ph 7 by adding 5  m hcl . the final solution was lyophilized overnight to obtain white powder , which was then dissolved in 25  ml of anhydrous methanol , filtered , evaporated , and lyophilized to obtain off - white powder . n4-tyrosine ( 1  mg ) was dissolved in 0.2  ml of sterile water , and tin ( ii ) chloride ( 0.1  ml , 1  mg / ml ) was added . the required amount of natco4 was added to the n4-tyrosine solution at the room temperature . radiochemical purity was determined by radio - hplc using an c-18 reverse column ( waters ) , eluted with a 7  :  3 acetonitrile : water solution ( v / v ) at a flow rate of 0.5  ml / minute . to determine the lipophilicity , 20  l of tc - n4-tyrosine was added into an equal volume mixture of 1-octanol and sterile water in a centrifuge tube . the mixture was vortexed at room temperature for 1 minute and then centrifuged at 5,000  rpm for 5 minutes to allow phase separation . from each phase , 0.1  ml of the aliquot was taken out , and the radioactivity was measured by gamma counter ( cobra quantum , packard , mn , usa ) . the measurement was repeated 3 times , and care was taken to avoid cross - contamination between the phases . the partition coefficient value , expressed as log  p , was calculated using the following equation : 
 ( 1)log  p=  log(radioactivity  in  1octanol  layerradioactivity  in  sterile  water  layer ) 
  rat breast tumor cell line 13762 ( american type culture collection , rockville , md , usa ) was used for both in vitro studies and to create the animal model for in vivo evaluation . all cells were maintained in dulbecco 's modified eagle 's medium and nutrient mixture f-12 ham ( dmem / f12 ; gibco , grand island , ny ) at 37c in a humidified atmosphere containing 5% co2 . for in vitro cellular uptake analysis , cells were plated onto 6-well tissue culture plates ( 2  10 cells / well ) 48 hours before adding the radiotracers and incubated with tc - n4-tyrosine ( 0.05  mg / well , 8  ci / well ) , tc - n4 ( 0.025  mg / well , 8  ci / well ) , or f - fdg ( 8  ci / well ) for 04 hours . after incubation , cells were washed with ice - cold phosphate - buffered solution twice and detached using a treatment of 0.5  ml of trypsin for 5 minutes . cells were then collected , and the radioactivity of the cells was measured in triplicate with a gamma counter ( cobra quantum ) . radioactivity was expressed as mean  standard deviation ( sd ) percent of cellular uptake ( % uptake ) . all animal work was carried out in the small animal imaging facility at md anderson under a protocol approved by the institutional animal care and use committee . for blood clearance analysis , 3 normal female fischer 344 rats ( 150  25  g ; harlan sprague - dawley , indianapolis , in , usa ) were intravenously injected with 30  ci of tc - n4-tyrosine . blood samples ( n = 3/rat ) were drawn from each rat through the lateral tail vein by microliter pipette ( 10  l ) at several time points between 5 minutes and 24 hours after injection . the blood samples were measured for radioactivity by gamma counter , and the radioactivity was expressed as a percentage of the injected dose per gram of blood ( % id / g ) . tissue distribution studies of  tc - n4-tyrosine were conducted using normal female fischer 344 rats ( 150  25  g , n = 9 ) . the rats were divided into 3 groups , and each rat was injected intravenously with 25  0.5  ci of tc - n4-tyrosine . each group was examined at 1 of 3 time points ( 0.5 , 2 , or 4 hours after injection ) . at each time point , the rats were killed , and the selected tissues were excised , weighed , and measured for radioactivity by gamma counter . for each sample , radioactivity was expressed as mean percentage of the injected dose per gram of tissue wet weight ( % id / g ) . counts from a 1/10 diluted sample of the original injection were used as a reference . individual organ and whole - body time - activity curves were fitted to exponential functions using olinda / exm software ( version 1.1 ; vanderbilt university , nashville , tn , usa ) . these functions were then integrated analytically to determine the area under the curve to yield the residence time of tc - n4-tyrosine in each organ . it was assumed that the injected activity of tc - n4-tyrosine was uniformly distributed throughout the body immediately following injection . mass correction factors were used to account for the different ratios of organs to total body weight and different scaling of residence times between rats and humans . residence times were then used to calculate target organ absorbed radiation doses for a 70  kg standard man model using the olinda / exm software . cells from the breast tumor cell line 13762 , suspended in phosphate - buffered solution ( 10 cells/0.1  ml solution per rat ) , were injected subcutaneously into the right calf muscle of female fischer 344 rats ( n = 9 ) . planar scintigraphic imaging of tc - n4-tyrosine was performed 1214 days after inoculation when tumors reached approximately 1  cm in diameter . the breast tumor - bearing rats ( n = 3/time point ) were anesthetized and injected intravenously with tc - n4-tyrosine ( 0.3  mg / rat , 300  ci / rat ) , and images were acquired at 30 , 120 , and 240 minutes after administration of tracers . planar scintigraphic images were obtained using m - cam ( siemens medical solutions , hoffman estates , il , usa ) equipped with a low - energy high - resolution collimator . computer - outlined regions of interest ( rois in counts per pixel ) between tumor and muscle tissue were used to calculate tumor - to - muscle ( t / m ) ratios . percent of injected dose ( % i d ) in the tumor was also calculated from the reference standard , which was 1/10 of the original injection activity of tc - n4-tyrosine . to investigate whether tc - n4-tyrosine can differentiate tumor tissue from inflammatory tissue , a rat model bearing both a mammary tumor and tissue with turpentine oil - induced inflammation was created . rat breast tumor cells ( from cell line 13762 ; 10 cells/0.1  ml phosphate - buffered solution per rat ) were injected into the right calf muscles of 3 female fischer 344 rats . after the tumors reached 1  cm in diameter , turpentine oil ( 0.1  ml / rat ) was injected into the left calf muscles of the rats to induce inflammation . the anesthetized rats were injected intravenously with tc - n4-tyrosine 24 hours after the turpentine injection . planar scintigraphic images were acquired at 30 , 120 , and 240 minutes after the tc - n4-tyrosine injection , and rois of the tumor , inflamed tissue , and muscle were used to calculate the tumor - to - muscle ratios ( t / ms ) , inflammation - to - muscle ratios ( i / ms ) , and tumor - to - inflammation ratios ( t / is ) , respectively . micro - pet imaging of f - fdg was performed using an r4 micro - pet scanner ( concorde microsystems , knoxville , tn , usa ) . three breast tumor- and inflammation - bearing rats were injected intravenously with f - fdg ( 500  ci / rat ) , and dynamic pet scans with a spatial resolution of 2.2  mm were obtained at 30 , 60 , and 90 minutes after f - fdg injection . pet images were reconstructed by using the ordered subset expectation maximization ( osem ) algorithm . t / m , i / m , and t / i ratios were calculated using the regional radioactivity concentrations ( ci / cm ) that were estimated from the average pixels within rois drawn around the tumor , inflamed tissue , or muscle on transverse slices of the reconstructed image sets . as mentioned previously , radiolabeled amino acid analogues have shown great potentials in cancer imaging by targeting the increased amino acids uptake in tumor cells . in the present study , we report the synthesis of tc - labeled tyrosine using ( n4 ) cyclam as a chelator and evaluate its potential in breast cancer diagnosis using the breast tumor models in vitro and in vivo . the structure and purity of n4-tyrosine were confirmed by h- and c - nmr , mass spectra , and hplc . the h - nmr results ( d2o  , ppm ) were as follows : 7.18 ( d , 2h , phenyl ring ) , 6.95 ( d , 2h , phenyl ring ) , 4.073 ( t , 2h , o - ch2 ) , 3.43 ( t , 1h , chn ) , 2.283.00 ( m , 2oh , och2 and nch2- ) , 1.871.91(m , 2h , c - ch2-c ) , and 1.681.79 ( m , 4h , c - ch2-c ) . c- nmr results ( d2o  , ppm ) were as follows : 162.88 , 156.72 , 130.76 , 117.57 , 115.25 , 68.55 , 57.29 , 53.91 , 51.56 , 49.95 , 49.17 , 48.96 , 48.24 , 48.03 , 46.80 , 45.73 , 45.35 , 39.27 , 24.90 , 24.31 , and 23.87 . because the starting material , n - t - butoxycarbonyl - l - tyrosine methyl ester , was commercially available , the synthetic yield of n4-tyrosine in our study was much higher ( 38% ) than that of its -methyl derivative n4-amt ( 14% ) , which was reported in a previous study . for the radiolabeling , the top and middle panels of figure 2 show the ultraviolet absorbance of tc - n4-tyrosine at 210  nm and 274  nm , while the bottom panel indicates the peak from nai detector that only detects the radioactivity . since precursor n4-tyrosine has a unique ultraviolet absorbance at 274  nm , we selected 274  nm for one of the ultraviolet detectors . on the other hand , the 210  nm detector can detect almost every molecule ; therefore , we were able to detect any impurities in our compound . the retention time of three detectors matched very well , which indicated precursor n4-tyrosine was labeled with tc successfully with high radiochemical purity ( > 95% ) . because tc - n4-tyrosine is a kit product and labeled without any further purification , its radiochemical yield was assumed to be identical to its radiochemical purity . for the partition coefficient value , the log  p of tc - n4-tyrosine was 2.83  0.082 , which was lower than that of its -methyl derivative tc - n4-amt ( 2.02  0.168 ) . since the log  p value is positively correlated with lipophilicity of the compound , we found that the lipophilicity of the tyrosine - based radiotracer could be increased by adding a methyl group on its -carbon . the cellular uptake kinetics of tc - n4-tyrosine , tc - n4 , and f - fdg in rat breast tumor cells is shown in figure 3 . the uptake for tc - n4-tyrosine increased dramatically up to 240 minutes , but this was not true for the tc - n4 chelator , suggesting that tc - n4-tyrosine can enter tumor cells specifically and accumulate rapidly . in addition , the mean % uptake of tc - n4-tyrosine was much higher than that of f - fdg , which indicated that tc - n4-tyrosine had better imaging potential than f - fdg in this in vitro breast cancer model . the blood clearance curve for tc - n4-tyrosine in normal fischer 344 rats is shown in figure 4 . the plasma half - life of the distribution phase ( t1/2 ) was 9.31  0.759 minutes , and the plasma half - life of the elimination phase ( t1/2 ) was 90.14  1.901 minutes , indicating that tc - n4-tyrosine had relatively fast blood clearance in normal rats . tissue distribution of tc - n4-tyrosine in normal fischer 344 rats is shown in table 1 . low thyroid and stomach uptake of tc - n4-tyrosine was observed , suggesting that tc - n4-tyrosine has high stability in vivo . high uptake was found in the kidneys , which was consistent with the planar scintigraphic imaging findings in the breast tumor - bearing rats ( see figure 5 ) . this high kidney uptake may be a result of the low lipophilicity of tc - n4-tyrosine , or it may be related to the biological characteristics of tc - labeled tyrosine because similar results were also observed in the previously developed tyrosine - based radiotracers tc - n4-amt , tc - ec - amt , and tc - ec - tyrosine [ 14 , 15 ] . to estimate the maximum dose of tc - n4-tyrosine that could be safely administered to humans , individual organ absorbed radiation doses the kidneys received the highest absorbed dose ( 4.86e-02  msv / mbq or 1.80e-01  rem / mci ) , which made it the dose - limiting organ . other organs with relatively high absorbed radiation doses included the heart wall , spleen , and liver . according to the us food and drug administration regulations , human exposure to radiation from the use of  radioactive research drugs should be limited to 3  rem per single administration and 5  rem per year to the whole body , blood - forming organs ( red marrow , osteogenic cells , spleen ) , the lens of the eye , and gonads ( testes and uterus ) ; the limit for other organs is 5  rem per single administration and 15  rem annually . total rem of tc - n4-tyrosine absorbed by each organ was below these limits at the proposed injection of 15  mci per patient . the selected planar scintigraphic images of breast tumor - bearing rats at 30 , 120 , and 240 minutes after tc - n4-tyrosine injection are shown in figure 5 . the t / m ratios at 30 , 120 , and 240 minutes were 5.12 , 4.88 , and 5.20 , respectively . tumor % i d at these three time points were 1.82% , 1.87% , and 1.71% , respectively . the selected planar scintigraphic images of breast tumor- and inflammation - bearing rats at 30 , 120 , and 240 minutes after tc - n4-tyrosine injection , as well as micro - pet images at 30 , 60 , and 90 minutes after f - fdg injection , are shown in figure 6 . the t / m ratio of tc - n4-tyrosine was relatively higher than i / m ratio at each time point . although t / i ratios were greater than 1 at all time points for both tc - n4-tyrosine and f - dfg , indicating that the tumor had higher uptake than the inflammation site , the t / i ratios for tc - n4-tyrosine were lower than those for f - fdg . this suggests that tc - n4-tyrosine was inferior to f - fdg in differentiating tumor from inflammation in this breast tumor - bearing rat model . it may be worth testing tc - n4-tyrosine in another animal model using radiation to induce inflammation so that the model better mimics patients undergoing radiation therapy . in summary , n4-tyrosine was synthesized and labeled efficiently with tc with high radiochemical purity . although it appears to inferior f - fdg in distinguishing breast tumor tissue from chemical - induced inflammatory tissue , tc - n4-tyrosine has high tumor - to - muscle uptake ratios and can detect breast tumor tissue clearly by gamma camera . by taking the advantage of chelator n4 cyclam , we could label n4-tyrosine with gallium-68 for pet imaging and , furthermore , labeled with rhenium-188 for radionuclide therapy for breast cancer treatment in future .
the purpose of this study was to develop an efficient way to synthesize 99mtc - o-[3-(1,4,8,11-tetraazabicyclohexadecane)-propyl]-tyrosine ( 99mtc - n4-tyrosine ) , a novel amino acid - based radiotracer , and evaluate its potential in breast cancer gamma imaging . 
 precursor n4-tyrosine was synthesized using a 5-step procedure , and its total synthesis yield was 38% . 
 it was successfully labeled with 99mtc with high radiochemical purity ( > 95% ) . 
 cellular uptake of 99mtc - n4-tyrosine was much higher than that of 99mtc - n4 and the clinical gold standard 18f-2-deoxy-2-fluoro - glucose ( 18f - fdg ) in rat breast tumor cells in vitro . 
 tissue uptake and dosimetry estimation in normal rats revealed that 99mtc - n4-tyrosine could be safely administered to humans . 
 evaluation in breast tumor - bearing rats showed that although 99mtc - n4-tyrosine appeared to be inferior to 18f - fdg in distinguishing breast tumor tissue from chemical - induced inflammatory tissue , it had high tumor - to - muscle uptake ratios and could detect breast tumors clearly by planar scintigraphic imaging . 99mtc - n4-tyrosine could thus be a useful radiotracer for use in breast tumor diagnostic imaging .