1 INTRODUCTION
When it’s beyond the capability of accompanying vasculature to supply oxygen, the growth of tumors can result in hypoxia, which is a common characteristic of many solid tumors [1]. Tumor hypoxia can cause adverse effect in therapeutic oncology, such as resistance to conventional radiotherapy and chemotherapy [2-5]. To improve the therapeutic efficacy, it is important to develop hypoxia markers that can detect hypoxic regions within tumors effectively. As such, there are a lot of hypoxia markers for PET and SPECT imaging [6-9], and they are mainly divided into two classes: compounds with nitroimidazole and compounds without nitroimidazole.
99mTc-BnAO is a nonnitroaromatic 99mTc-labelled hypoxia marker and its hypoxic biological evaluation has been reported [10, 11]. 99mTc-BnAO is similar to BMS181321 (99mTc-[PnAO-1-(2-nitroimidazole)]), which also showed a significant hypoxic/normoxic differential. However, the mechanism for why 99mTc-BnAO was retained in hypoxic cells was unknown. Jia [12] proposed that the mechanism may relate to the interconversion between the penta-coordinated mono-oxo form and hexa-coordinated di-oxo form of the 99mTc-BnAO complex. Lately, some derivatives of 99mTc-BnAO have been extensively studied by Hsia [13] and Sun [14], such as HL-91-ET, OH-BnAO, EtO-BnAO and Et-BnAO. The biological results indicated that hypoxia uptake was weakly correlated with the lipophilicity of those radiotracers.
Athough the reduction potential of 3-nitro-1,2,4-triazole is more negative than that of 2-nitroimidazole, 3-nitro-1,2,4-triazole has been used as redox center in hypoxia markers [15-18]. In our previous work, 99mTc-labeled hydroxy-iminoamide complex containing nitrotriazole (NTPA) exhibited a good uptake in hypoxic S180 cells and a high tumor-to-muscle ratio [19]. Recently, two 99mTc-labeled PnAO complexes containing nitrotriazole were prepared; in vitro and in vivo results showed that both complexes displayed significant hypoxic/normoxic differential [20].
In 2011, Hsia [21] synthesized BnAO-NI, which contained two redox centers, 2-nitroimidazole, and 99mTc-BnAO. It was reported that 99mTc-BnAO-NI did not achieve a higher specific uptake in hypoxic tissue than 99mTc-BnAO, although 99mTc-BnAO-NI was more lipophilic than 99mTc-BnAO. In our preceding work, we found that 99mTc-labeled PnAO-NT (99mTc-[PnAO-1-(3-nitro-1,2,4-triazole)]) had a higher hypoxic cellular uptake than BMS181321 (99mTc-[PnAO-1-(2-nitroimidazole)]) with the same cell line [20, 22]. Hence, it is interesting and necessary to explore the effect of 3-nitro-1,2,4-triazole on the hypoxia uptake of 99mTc-BnAO derivatives.
In this work, 3,3,10,10-tetramethyl-1-(3-nitro-1,2,4-triazole)-4,9-diazadodecane-2,11-dionedioxime (BnAO-NT) was synthesized and labeled with 99mTc in a superior yield (Fig. 1). Some of its physicochemical properties such us stability, protein binding, electrical property and lipophilicity were investigated. The cellular uptakes of 99mTc-BnAO-NT and 99mTc-BnAO were performed using the S180 and H22 cell lines. The biodistribution of 99mTc-BnAO-NT in mice bearing a S180 tumor was also studied.
2 EXPERIMENTAL SECTION
2.1 General
3-nitro-1H-1,2,4-triazole was acquired from J&K (Beijing, China) and 1,4-diaminobutane and N,N-diisopropylethylamine (98%) were provided by Acros Organics (Geel, Belgium). All other reagents were of analytical grade without further purification. The 99Mo-99mTc generator was purchased from China Institute of Atom Energy (Beijing). Mass spectra were measured on a Bruker APEX IV FTMS (Faellanden, Switzerland), positive mode, ESI. NMR spectra were obtained on Bruker (400 MHz) spectrometers (Bruker, Faellanden, Switzerland). RP-HPLC analyses were performed on a reversed-phase column (Agilent HC-C18, 4.6×150 mm, size 5 microns), a Waters 1525 binary HPLC pumps and a Waters 2478 UV absorbance dual λ detector (Milford, MA USA). The elution was monitored with a Packard 500 TR flow scintillation radioactivity detector (Meriden, CT, USA). The radioactivity was detected by 2470 WIZARD2 Automatic Gamma Counter (PerkinElmer, MA, USA). Murine sarcoma S180, hepatocarcinoma H22 cell lines, and male Kunming mice were purchased from Department of Laboratory Animal Science, Peking University. Dulbecco’s Modified Eagle’s Medium (DMEM) was from Gibco BRL Life Technologies (Grand Island, NY, USA) and fetal bovine serum was from Hyclone (Logan, UT, USA). The JPSJ-605 dissolved oxygen meter was purchased from REX Instrument Factory of Shanghai Precision & Scientific Instrument Co., LTD (Shanghai, China). The 99mTc-BnAO kit was purchased from Beijing Xin branch of Star Medical Technology Co., LTD. (Beijing, China).
2.2 Synthesis of BnAO-NT
The synthesis route for the compound BnAO-NT is depicted in Scheme 1, which is similar to that reported in our previous work [20]. 1, 3-diaminopropane was replaced with 1, 4-diaminobutane. The compound 2 (3-chloro-3-methyl-2-butanoneoxime) was prepared by the addition of concentrated hydrochloric acid, isoamyl nitrite and 2-methylbut-2-ene. The substitution reaction of compound 2 with an excess of 1,4-diaminobutane produced the compound 3 (N-(4-aminobutyl)-3-amino-3-methyl-2-butanone-oxime). 1-bromo-3-methylbut-2-ene was added to a mixture of K2CO3 and 3-nitro-1-H-1,2,4-triazole in acetone solution to get the compound 4 (3-methyl-1-(3-nitro-1H-1,2,4-triazole-1-yl)-2-butene). Concentrated hydrochloric acid was added to the mixture of compound 4 and isoamyl nitrite to get the compound 5 (3-chloro-3-methyl-1-(3-nitro-1H-1,2,4-triazol-1-yl) butan-2-one oxime). BnAO-NT was then prepared by substitution reaction of compound 3 and 5 in dry acetonitrile. The crude product was purified by column chromatography (silica gel, CH2Cl2:CH3OH, 4:1) and recrystallized with CH2Cl2 to provide a white solid (yield 65%). HRMS (ESI): mass calculated for BnAO-NT (C16H30N8O4), 399.24628; m/z found, 399.24708 (M+H+). 1H NMR (DMSO-d6) δ11.41(s, 1H, C=N-OH), 11.17(s, 1H, C=N-OH) 8.84(s, 1H, triazole-H), 5.20(s, 2H, NCH2), 2.71(t, 2H, CH2NH), 2.31(t, 2H, CH2NH), 1.83(m, 4H, CH2CH2CH2CH2), 1.63(s, 3H, N=CCH3), 1.41(s, 6H, C(CH3)2), 1.27 (s, 6H, C(CH3)2) .
2.3 Radiolabeling
Na99mTcO4 was obtained from a 99Mo/99mTc generator. To get a high labeling yield, the effects of the dosage of stannous chloride dihydrate (SnCl2.2H2O), intermediate ligand (sodium tartrate solution), and ligand BnAO-NT were investigated. For comparison, the radiolabeling of ligand BnAO was also performed and analyzed using the same method.
The best radiolabeling method for 99mTc-BnAO-NT we screened as follows: 99mTcO4-(10 μL, 3~5 MBq), the ligand solution (20 μL, 2 mg/mL), phosphate buffer solution (100 μL, pH 7.4, 0.2 mol/L), and sodium tartrate solution (25 μL, 2 mg/mL) were mixed in a 2 mL centrifugal tube. Then the fresh SnCl2 solution (0.5 μL, 2 mg/mL) was added. The mixture was stirred in a water bath at a temperature of 75 oC for 15 min, and then filtrated by a 0.22 μm filter. As for 99mTc-BnAO, 99mTcO4- (10 μL, 3~5 MBq) was added to 99mTc-BnAO kit, and kept at room temperature for 15 min. 99mTc-BnAO-NT and 99mTc-BnAO were analyzed utilizing the radio high-performance liquid chromatography (radio-HPLC). Solvent systems for HPLC analysis: flow rate 1.0 mL/min; Phase A, 0.1 M ammonium acetate; Phase B, CH3CN. Gradient: 0-10 min 90%-30% A; 10-15 min 70%-20% A; 15-17min 20% A; 17-20 min 20%-70% A (Fig. 2).
2.4 Stability, Protein binding, Partition coefficient and Electrical property
2.4.1 Stability
In vitro stability was determined in phosphate buffer and mouse serum. The labeled complex 99mTc-BnAO-NT was dissolved in the phosphate buffer (0.1 M, pH 7.4) and incubated at room temperature after preparation. Certain aliquots of samples were taken at different time points (1, 2, 8 h) and the radiochemical purity was analyzed by HPLC.
The stability of the complex 99mTc-BnAO-NT in mouse serum was also determined for 8 h with a similar method as reported [23]. The labeled complex (0.1 mL) was dissolved in 1 mL mouse serum and incubated at 37 oC after preparation. Then certain samples were taken at different time points (1, 2, 8 h). Ethanol (200 μL) was added to each sample to precipitate the protein. The samples were centrifuged, filtrated by 0.22 μm filter, and finally the supernatant was tested by HPLC.
2.4.2 Protein binding
The serum protein binding experiment was investigated along with that of the stability in mouse serum [23]. The radioactivity of the full sample was A, and that of the supernatant was B. The percentage of protein-binding was calculated as (A-B)/A.
2.4.3 Octanol/Water Partition Coefficient (Po/w)
Octanol (1 mL), 99mTc-labeling solution (0.5 mL), and saline (0.5 mL) were combined in a 5 mL centrifugal tube. The tube was vigorously vortexed for 5 min and centrifuged at 3000 rpm for another 5 min. The radioactivities of a 100 μL solution of each phase were measured. The partition coefficient Po/w was the ratio of radioactivity of the octanol layer to that of the water layer. The process was repeated three times.
2.4.4 Paper electrophoresis
The experiment was taken by the Whatman 3 paper using the method as reported [24]. The labeling complex was put in the middle of the paper, and after the solvent was dried, the paper was placed in the phosphate buffer solution (pH 7.4, 0.05 M) under a voltage of 200 V. 1 h later, the paper was taken out, dried, cut into sections of 1 cm and counted for radioactivity. Therefore, the paper electrophoresis map was obtained.
2.5 In Vitro Study
In vitro cellular uptake experiments were performed according to the literature methods [20, 22]. The S180 or H22 cells were suspended in fresh DMEM medium with 10% (v/v) fetal bovine serum at a cell concentration of approximately 106 cells/mL. Aliquots of 20 mL were placed into glass vials, and then incubated at 37 oC with gentle magnetic stirring under normoxic (95% air with 5% carbon dioxide) or hypoxic (95% nitrogen with 5% carbon dioxide) conditions. In hypoxic condition, the oxygen concentration was 0.00 mg/L after 60 min equilibration, which was determined by the JPSJ-605 dissolved oxygen meter. The 99mTc-labeling complex was added to each glass vial at an activity of approximately 0.1 MBq/mL, and the concentration was roughly 1 μg/mL. More than 1 mL of the sample was removed every 30 mins. For each sample, five 200 μL aliquots were pipetted, and centrifuged at 1500 rpm for 5 min. 160 μL aliquot of supernatant was removed and measured. The radioactivity of the supernatant was marked as A, and that of the residue containing cells and medium was regarded as B. The cell uptakes were calculated as % uptake = [(B-A/4)/(A+B)]*100%. The cells viability was determined by the trypan blue exclusion assay for 4 h.
2.6 Biodistribution Study
All the animal experiments were performed in accordance with the national laws related to the conduct of animal experimentation. Hypodermic injection of approximately 106 cells into the left front leg of male Kunming mice was performed to establish the S180 tumor model. Tumors grew to diameters of 10 mm during 8-10 days. The complex 99mTc-BnAO-NT was administered by tail vein injection (about 0.1 MBq, 0.1 mL, 1.5 μg). Five mice were killed at 0.5, 1, 2, 4 and 8 h after injection. Various organs and tissues (blood, brain, muscle, tumor, heart, stomach, kidney, spleen, intestine, liver, and lung) were excised, washed, weighed and counted for radioactivity in a gamma counter. The percent of injected dose per gram (% ID/g) was calculated and the final results were expressed as mean±SD (standard deviation).
3 Results and Discussion
3.1 Radiolabeling
The labeling yields of the 99mTc-complexes were tested using the radio high-performance liquid chromatography. The retention times of 99mTc-sodium tartrate and 99mTcO4- were 1.8 and 2.8 min respectively, while the radioactivity signal of 99mTc-BnAO and 99mTc-BnAO-NT showed a distinct peak at 3.9 min and 6.6 min, respectively. The labeling yields of 99mTc-BnAO-NT and 99mTc-BnAO were both above 98%.
3.2 Stability, Protein binding, Partition coefficient and Electrical property
99mTc-BnAO-NT was observed to be stable in the prepared medium and in mouse serum as their radiochemical purities remained above 95% for up to 8 h. The protein binding ratios of 99mTc-BnAO-NT were 30.49%, 32.68% and 27.39% at 1, 2 and 8 h, respectively. The octanol/water partition coefficient of 99mTc-BnAO-NT was 0.26±0.01, compared with 0.089±0.007 for 99mTc-BnAO [21], which demonstrated that 99mTc-BnAO-NT was more lipophilic than 99mTc-BnAO.
The results of the paper electrophoresis of 99mTcO4-, 99mTc-BnAO, and 99mTc-BnAO-NT are presented in Fig. 3. As 99mTcO4- was negatively charged, it shifted toward the anode. The paper electrophoresis demonstrated that 99mTc-BnAO and 99mTc-BnAO-NT were neutral under physiological condition.
3.3 In Vitro Study
In vitro studies of 99mTc-BnAO-NT and 99mTc-BnAO were performed using the S180 and H22 cell lines. Hepatocarcinoma H22, liver cancer cells, is one of widely used tumor model [25, 26]. Cellular uptakes of 99mTc-BnAO-NT under normoxic and hypoxic conditions are plotted versus time in Fig. 4.
As for the S180 and H22 cell lines, the significant differences in cellular uptake between normoxic and hypoxic conditions was observed. The maximum hypoxic cellular uptakes of 99mTc-BnAO-NT under normoxic conditions were 27.11±0.73% and 14.85±0.83% for S180 and H22 cells respectively. The maximum hypoxic uptake in H22 cells was only half of that in S180 cells. In S180 cells, the initial hypoxic cellular uptake was 13.15±1.68% at 30 min, and then it reached up to 15.12±0.68%, 23.95±1.09 and 27.11±0.73% at 60, 90 and 180 min. However, normoxic cellular uptake was 6.16±2.38% at 30 min, and there was little significant increase over 4 h. Under hypoxic condition, the initial cellular uptake in H22 cells was 7.77±1.73% at 30 min, then it reached up to 11.64±0.82%, 12.51±1.44%, and 14.85±0.83% at 90, 150 and 210 min. Under normoxic conditions, the cellular uptakes of 99mTc-BnAO-NT fluctuated with time and there was no fixed trend.
99mTc-BnAO, the complex containing no nitrotriazole, showed a significant difference in cellular uptake between normoxic and hypoxic uptakes in KHT sarcoma cells [21]. In this work, an in vitro study of 99mTc-BnAO was also carried out using S180 and H22 cell lines. According to Fig. 5, the uptake of 99mTc-BnAO steadily increased with time under hypoxic condition, but fluctuated with time at the range about 5% under normoxic condition. At the time of 30 min, the uptake was only about 8.18±1.47% in hypoxic cells. Subsequently, there was a significant increase over 4 h. The uptakes were 11.89±1.12%, 18.18±1.07%, and 28.13±0.97% at 60, 90, and 120 min. Then there was a slight increase, and maximum uptake was about 30.79±0.44% at 180 min. Under normoxic conditions, the uptake was 6.01±0.62% at 30 min, and there was little significant increase over four hours. It was shown that 99mTc-BnAO was enriched in hypoxic S180 cells selectively. However, the result was not satisfactory like that of 99mTc-BnAO-NT in H22 cells. The difference between normoxic and hypoxic uptakes was not observed in the initial 150 min and the maximum hypoxic uptake was only 9.66±1.20% at 240 min.
The results exhibited that 99mTc-BnAO-NT and 99mTc-BnAO selectively accumulated in hypoxic S180 and H22 cells, although the results in hypoxic H22 cells were unsatisfactory. Some studies have explored the cell dependence of hypoxia markers. For example, the uptake and retention of 64Cu-ATSM in MDA468, FaDu, and R3327-AT cells was cell line dependent [27]. Hoigebazar also determined the uptake of 68Ga-labeled DOTA-nitroimidazole derivatives in the Hela, CHO, and CT-26 cell lines, and CT-26 showed the greatest radiotracer uptake [28]. However, the cell dependence of hypoxia markers in S180 and H22 cells has rarely been reported. According to Figs. 4 and 5, it could be seen that the cell lines dramatically affected the cellular uptake under hypoxic conditions, but weakly affected the cellular uptake under normoxic conditions. The difference between S180 and H22 cells suggested that the hypoxic cellular uptake of 99mTc-BnAO-NT and 99mTc-BnAO was cell dependent, and S180 cells may be better than H22 cells in the term of hypoxia evaluation.
For the uptake of 99mTc-BnAO and 99mTc-BnAO-NT in S180 cells, their maximum hypoxic cells were 30.79±0.44% and 27.11±0.73%, respectively. Obviously, the nitrotriazole may not play the key role in the cells’ uptake of 99mTc-BnAO-NT. The conclusion was similar to that of 99mTc-BnAO and 99mTc-BnAO-NI described by Hsia et al [21]. In their work, the cellular uptake of 99mTc-BnAO-NI in KHT cells was lower than that of 99mTc-BnAO.
3.4 Biodistribution study
During in vitro study, the radiotracer uptake was higher for S180 cells than H22 cells, thus the investigation of the biodistribution of 99mTc-BnAO-NT in Kunming male mice bearing a S180 tumor was carried out. The biodistribution results are listed in Table 1.
| Tissue | 0.5 h | 1 h | 2 h | 4 h | 8 h |
|---|---|---|---|---|---|
| Blood | 0.96±0.54 | 1.00±0.37 | 0.80±0.17 | 0.64±0.10 | 0.52±0.21 |
| Brain | 0.13±0.03 | 0.16±0.05 | 0.20±0.03 | 0.22±0.04 | 0.19±0.02 |
| Muscle | 0.47±0.11 | 0.71±0.28 | 1.03±0.15 | 0.42±0.15 | 0.32±0.08 |
| Tumor | 0.97±0.48 | 1.52±0.57 | 1.78±0.26 | 1.35±0.41 | 0.72±0.19 |
| Heart | 0.70±0.20 | 0.90±0.23 | 1.21±0.36 | 1.31±0.28 | 0.72±0.14 |
| Stomach | 0.67±0.32 | 1.14±0.38 | 2.00±0.48 | 1.70±0.50 | 1.01±0.14 |
| Spleen | 2.03±1.13 | 3.17±1.41 | 4.85±0.17 | 1.81±0.42 | 0.91±0.28 |
| Intestine | 1.42±0.55 | 3.54±0.17 | 3.23±0.79 | 2.88±0.65 | 1.85±0.37 |
| Kidney | 0.56±0.22 | 0.79±0.27 | 0.99±0.25 | 0.77±0.30 | 0.49±0.18 |
| Lung | 0.88±0.19 | 1.31±0.39 | 1.32±0.22 | 1.29±0.27 | 0.57±0.12 |
| Liver | 2.91±1.42 | 7.07±2.02 | 4.56±0.58 | 4.15±0.15 | 2.32±0.92 |
| T/B | 1.10±0.46 | 1.52±0.49 | 1.96±1.11 | 2.31±0.34 | 1.49±0.29 |
| T/M | 1.74±0.72 | 2.22±0.93 | 1.75±0.37 | 3.79±0.98 | 2.29±0.59 |
The radioactivities of complex 99mTc-BnAO-NT in the liver, spleen, intestine, and kidney were clearly higher than that in other tissues, which was probably related to its lipophilicity and this result suggested that the complex was cleared away through both the hepatobiliary pathway and urinary pathway. As we could see in Table 1, the tumor uptake increased in the first two hours, and then decreased in the subsequent four hours: 0.5 h after injection, the tumor uptake was 0.97±0.48% ID/g, and increased to 1.78±0.26 % ID/g at 2 h, then decreased to 0.72±0.19 % ID/g at 8 h. The complex may undergo an enrichment and then clearance process. The tumor-to-blood ratio was 2.31±0.34, and tumor-to-muscle ratio was 3.79±0.98 at 4 h. The results showed that 99mTc-BnAO-NT had high tumor specificity. The results were similar to that of 99mTc-BnAO[24] using the same S180 tumor. The maximum tumor uptake of 99mTc-BnAO was 1.60±0.35% ID/g at 0.5 h. The tumor-to-blood ratio was 2.40±1.04, and the tumor-to-muscle ratio was 3.94±1.25 at 4 h. The tumor specificity of 99mTc-BnAO-NT did not significantly increase after introducing the redox center 3-nitro-1,2,4-triazole.
4 Conclusion
This study demonstrated that 99mTc-BnAO-NT and 99mTc-BnAO could accumulate in the S180 and H22 cell lines under hypoxic conditions, especially in the S180 cells. When combined with 3-nitro-1,2,4-triazole, 99mTc-BnAO-NT was more lipophilic than 99mTc-BnAO. However, 99mTc-BnAO-NT did not exhibit higher cellular uptake in vitro and the biodistribution of 99mTc-BnAO and 99mTc-BnAO-NT were similar. Therefore, 3-nitro-1,2,4-triazole moiety did not have a significant influence on the hypoxic uptake of 99mTc-BnAO-NT. Both in vitro and in vivo studies hinted that 99mTc-BnAO-NT may be a hypoxia-sensitive radio probe for monitoring hypoxic regions in the sarcoma S180 tumor model.
Angiogenesis ischemic and neoplastic disorders
. Annu Rev Med, 2003, 54: 17-28. DOI: 10.1146/annurev.med.54.101601.152418Molecular imaging of hypoxia
. J Nucl Med, 2008, 49: 129S-148S. DOI: 10.2967/jnumed.107.045914Hypoxia and radiotherapy: opportunities for improved outcomes in cancer treatment
. Cancer Metastasis Rev, 2007, 26: 241-248. DOI: 10.1007/s10555-007-9056-0Hypoxia and aggressive tumor phenotype: implications for therapy and prognosis
. Oncologist, 2008, 13: 21-26. DOI: 10.1634/theoncologist.13-S3-21Hypoxia as a target for combined modality treatments
. Eur J Cancer, 2002, 38: 240-257. DOI: 10.1016/S0959-8049(01)00361-6Imaging hypoxia in tumors
. Semin Nucl Med, 2001, 31: 321-329. DOI: 10.1053/snuc.2001.26191Molecular imaging of hypoxia
. J Nucl Med, 2011, 52: 165-168. DOI: 10.2967/jnumed.110.075663Molecular imaging of hypoxia with radiolabelled agents
. Eur J Nucl Med Mol Imaging, 2009, 36: 1674-1686. DOI: 10.1007/s00259-009-1195-9Tumor hypoxia imaging
. Mol Imaging Biol, 2011, 13: 399-410. DOI: 10.1007/s11307-010-0420-zTc-99m-HL91 - "Hot spot" detection of ischemic myocardium in vivo by gamma camera imaging
. Circulation, 1998, 97: 2557-2566. DOI: 10.1161/01.CIR.97.25.2557Studies of 99mTc-BnAO (HL-91): a non-nitroaromatic compound for hypoxic cell detection
. Int J Radiat Oncol, 1998, 42: 737-740. DOI: 10.1016/S0360-3016(98)00301-0The interconversion mechanism between TcO3+ and TcO2+ core of 99mTc labeled amine-oxime (AO) complexes
. Theor Chem Acc, 2008, 121: 271-278. DOI: 10.1007/s00214-008-0474-zThe preparation and biological characterization of a new HL91-derivative for hypoxic imaging on stroke mice
. Appl Radiat Isotopes, 2010, 68: 1610-1615. DOI: 10.1016/j.apradiso.2010.03.008Preliminary studies of 99mTc-BnAO and its analogue: synthesis, radiolabeling and in vitro cell uptake
. Nucl Med Biol, 2010, 37: 117-123. DOI: 10.1016/j.nucmedbio.2009.09.003AK-2123 (Sanazol) as a radiation sensitizer in the treatment of stage III cervical cancer: Results of an IAEA multicentre randomised trial
. Radiother Oncol, 2007, 82: 24-29. DOI: 10.1016/j.radonc.2006.11.007Preparation and preliminary biological evaluation of a 177Lu labeled sanazole derivative for possible use in targeting tumor hypoxia
. Bioorg Med Chem, 2004, 12: 6077-6084. DOI: 10.1016/j.bmc.2004.09.007Pre-clinical evaluation of a 3-nitro-1,2,4-triazole analogue of [18F]FMISO as hypoxia-selective tracer for PET
. Nucl Med Biol, 2010, 37: 565-575. DOI: 10.1016/j.nucmedbio.2010.03.011Technetium-99m-cyclam AK 2123: a novel marker for tumor hypoxia
. Appl Radiat Isot, 2001, 54: 81-88. DOI: 10.1016/S0969-8043(00)00104-4Synthesis and preliminary biological evaluation of the 99mTc labeled nitrobenzoimidazole and nitrotriazole as tumor hypoxia markers
. Bioorg Med Chem Lett, 2006, 16: 1831-1833. DOI: 10.1016/j.bmcl.2006.01.001Synthesis, radiolabeling and biological evaluation of propylene amine oxime complex containing nitrotriazole as hypoxia markers
. Molecules, 2012, 17: 6808-6820. DOI: 10.3390/molecules17066808The biological characterization of 99mTc-BnAO-NI as a SPECT probe for imaging hypoxia in a sarcoma-bearing mouse model
. Appl Radiat Isot, 2011, 69: 649-655. DOI: 10.1016/j.apradiso.2010.12.009Effect of a second nitroimidazole redox centre on the uptake of a hypoxia marker: Synthesis and in vitro evaluation of 99mTc-labeled bisnitroimidazole propylene amine oxime complex
. Bioorg Med Chem Lett, 2012, 22: 172-177. DOI: 10.1016/j.bmcl.2011.11.042Synthesis and biological evaluation of novel 99mTcN-labeled bisnitroimidazole complexes containing monoamine-monoamide dithiol as potential tumor hypoxia markers
. J Rad Nucl Chem, 2014, 301: 831-838. DOI: 10.1007/s10967-014-3235-6Synthesis, radiolabeling and biodistribution of 99Tcm-BnAO ethylene glycol monoethyl ether derivate
. Isotopes, 2011, 24: 29-33.Synthesis and biological evaluation of a novel asymmetrical Tc-99m-nitrido complex of metronidazole derivative
. J Label Compd Radiopharm, 2007, 50: 1137-1142. DOI: 10.1002/jlcr.1292Saikosaponin-D enhances radiosensitivity of hepatoma cells under hypoxic conditions by inhibiting hypoxia inducible factor-1 alpha
. Cell Physiol Biochem, 2014, 33: 37-51. DOI: 10.1159/000356648Cell line-dependent differences in uptake and retention of the hypoxia-selective nuclear imaging agent Cu-ATSM
. Nucl Med Biol, 2005, 32: 623-630. DOI: 10.1016/j.nucmedbio.2005.05.003Synthesis of 68Ga-labeled DOTA-nitroimidazole derivatives and their feasibilities as hypoxia imaging PET tracers
. Bioorg Med Chem, 2011, 19: 2176-2181. DOI: 10.1016/j.bmc.2011.02.041