[Radioimmunoimaging of lymphoma in mice with a two-step pretargeting strategy using biotinyled CD45 monoclonal antibody and (188)Re-avidin].

OBJECTIVE: To establish a two-step pretargeting approach to lymphoma radioimmunoimaging in mice using biotinynaled CD45 monoclonal antibody (McAb) and (188)Re-avidin in a tumor-bearing mouse model. METHODS: Six Nod-Scid mice bearing lymphoma cell xenograft were randomized to receive either an intravenous injection of 50 µg/200 µL biotinyled CD45 McAb followed 24 h later by an intraperitoneal injection of 3.7 MBq (50 µg/100 µL) (188)Re-avidin (two-step pretargeting group), or a single intravenous injection of 3.7 MBq (100 µg/100 µL) (188)Re-CD45 McAb (control group). SPECT was performed at 0.5, 1, 6 and 23 h post-injection to characterize (188)Re isotope biodistribution. At 24 h pos-injection, the mice were sacrificed for measurement of radioactivity uptake in the tumor and normal tissues and calculation of the tumor-to-non-tumor (T/NT) ratios. RESULTS: SPECT showed that the two-step pretargeting method resulted in a low radioactivity in the blood pool during the imaging and a concentrated radioactivity in the liver and spleen. The transplanted tumor began to be displayed at 1 h post-injection and was clearly displayed at 1-6 h; the images were clear even at 23 h. With the two-step pretargeting method, the radioactive uptake at 24 h post-injection were (1.34∓0.52)%, (6.77∓2.32)%, and (2.81∓1.25)% in the tumor, kidney and liver, respectively, with low radioactivity levels in other organs and high tumor/blood and tumor/muscle ratios (4.28∓0.82 and 8.00∓0.88, respectively). In the control group, SPECT revealed intense radioactivity in the liver, spleen, and kidneys with obscure display of the tumor; at 20 h, the radioactivity in the blood pool remained high but that in the tumor was low, and the tumor/blood and tumor/muscle ratios at 24 h were only 0.58∓0.06 and 3.21∓0.24, respectively. CONCLUSION: Compared with (188)Re-CD45 McAb, the two-step pretargeting approach exhibits a good specificity in targeting lymphoma with an increased T/NT ratio in mice and allows early tumor display at 1 h post-injection.

[Preparation of (99m)Tc-EDTA-MN and Its Bioimaging in Mouse].

BACKGROUND AND OBJECTIVE: Hypoxia is an important biological characteristics of solid tumor, it is not sensitive to radiotherapy and chemotherapy for which is the presence of hypoxic cell, thus increasing their resistance to conventional radiotherapy and chemotherapy, therefore, the detection of hypoxia degree of tumor tissue is of great significance. The hypoxia imaging of nuclear medicine can reflect the degree of tissue hypoxia, which can selectively retained on the hypoxic cells or tissues, including nitroimidazole and non nitroimidazole; the nitroimidazole is widely and deeply researched as hypoxic celles developer in China and abroad at present. The research about application of radionuclide labelled technique has clinical application value to develop the hypoxia imaging agent EDTA-MN complexes which was labeled. To study the feasibility of (99m)Tc by direct labeling method, the radiochemical properties evaluation of (99m)Tc-EDTA-MN, and observe the distribution characteristics of (99m)Tc radiolabeled EDTA-MN in the xenograft lung cancer nude mice bearing non-small cell lung cancer cell (A549), and provide experimental evidence for its further research and application. METHODS: The radiolabeling of EDTA-MN with (99m)Tc was performed with direct labeling method, respectively, on the reaction dosage (10 mg, 5 mg, 2 mg), stannous chloride dosage (8 mg/mL, 4 mg/mL, 2 mg/mL), mark system pH (2, 4, 5, 6) one by one test, using orthogonal design analysis, to find the optimal labeling conditions. Labelling rate, radiochemical purity, lipid-water partition coefficient and in vitro stability in normal saline (NS) were determined by TLC and HPLC, and the preliminary study on the distribution of (99m)Tc-EDTA-MN in nude mice. RESULTS: The labeling rate of 99mTc-EDTA-MN with the best labeling conditions was (84.11±2.83)%, and the radiochemical purity was higher than 90% by HPLC purification, without any notable decomposition at room temperature over a period of 12 h. The partition coefficient was lgP=-3.05, indicated that this complex was hydrophilic. At 3 h post-injection, the imaging of (99m)Tc-EDTA-MN in nude mice bearing non-small cell lung cancer cell showed that more radioactive gathered in bladder at 0.5 h, the transplanted tumor was clearly imaged at 1 h post-injection, during whole imaging radioactive in other tissues and organs was low. The radioactivity of tumor uptake by using of ROI technology were (88.14±11.59), (123.17±9.06), (98.08±14.40) and (79.87±10.57) at 0.5, 1, 2, 3 h post-injection, and the ratio of T/NT of tumor and liver area were (1.95±0.19), (3.58±0.78), (3.95±0.39) and (5.01±0.28), respectively. (99m)Tc-EDTA-MN could be quickly cleared from the blood in mice primarily through the kidneys, and the radioactivity in other tissues and organs remained low. CONCLUSIONS: (99m)Tc-EDTA-MN can be easily prepared and labeled compound with high labeling rate and stability, it appears to be suitable for further experiments requirement in vivo and in vitro application.

[Radiolabelling of a lung cancer-targeting small molecule polypeptide with (131)I and its radioactivity distribution in normal rabbits].

OBJECTIVE: To establish a labeling method for a specific lung cancer-targeting small molecule peptide cNGQGEQc with ¹³¹I and observe the radioactivity distribution of the labeled peptide in rabbits using single-photon emission computed tomography (SPECT). METHODS: Chloramine-T method was used for ¹³¹I labeling of the tyrosine amino group on cNGQGEQc, and the labeling efficiency and radiochemical purity of ¹³¹I-cNGQGEQc were determined with paper chromatography. The stability of ¹³¹I-cNGQGEQc in saline and human serum was assessed after incubation in water bath at 37 degrees celsius; for 24 h. The octanol-water partition coefficient lg P (the radioactivity counting ratio of ¹³¹I-cNGQGEQc dissolved in 100 µl octanol or in 100 µl saline) was calculated. SPECT was performed in 3 male New Zealand white rabbits after intravenous injection of ¹³¹I-cNGQGEQc to observe the dynamic distribution of the peptide with the time-radioactivity curve (T-A curve) of the region of interest (ROI). RESULTS: With a labeling efficiency of 90%, ¹³¹I-cNGQGEQc showed a radiochemical purity of was 95% after purification with HPLC. The radiochemical purity of ¹³¹I-cNGQGEQc was (93.12 ± 1.18)% and (88.34 ± 5.43)% after intubation in saline and human serum for 24 h, respectively. The octanol-water partition coefficient lg P of ¹³¹I-cNGQGEQc was -1.75, suggesting its hydrosolubility. In rabbits with intravenous injection of ¹³¹I-cNGQGEQc, SPECT visualized the kidneys at 1 min after the injection; the imaging of the heart and liver became attenuated at 5 min when the bladder was visualized with an increasing radioactivity. The radioactivity of the soft tissues began to fade at 30 min. No gallbladder visualization was detected, and the radioactivity of the abdomen remained low. No obvious radioactivity concentration was observed in the thyroid and stomach. The T-A curves of the ROI of all the tissues and organs descended over time. CONCLUSION: Radiolabeling of cNGQGEQc with ¹³¹I is simple and highly efficient. ¹³¹I-cNGQGEQc has good stability in vitro and good distribution characteristics for in vivo imaging, and is cleared mainly by renal excretion due to its hydrosolubility. These results provide experimental basis for further studies of diagnosis and therapy of lung cancer with targeting polypeptide.

[(99m)Tc radiolabeling of a novel polypeptide molecular probe for lung cancer and its biodistribution in animals].

OBJECTIVE: To develop a method for (99m)Tc radiolabeling of a small molecular peptide targeting lung carcinoma and observe the biokinetics and biodistribution of the labeled peptide in normal mice and rabbits. METHODS: MAG3-peptide (cNGQGEQc) was labeled with (99m)Tc and the labeling rate was determined with paper chromatography. In vitro stability test, cysteine challenge test and serum incubation test were performed for radiochemical evaluation of the labeled peptide. Blood (99m)Tc-peptide clearance in rabbits was evaluated by determining blood radioactive concentrations at different time points after injection of 37 MBq (99m)Tc-peptide, and its dynamic distribution was investigated by SPECT imaging. The percent injected dose per gram of tissue was calculated for each organ of mice injected intravenously with 7.4 MBq (99m)Tc-peptide based on gamma counter readings. RESULTS: The labeling rate of (99m)Tc-peptide exceeded 90%, and the radiochemical purity was 91% after placing for 12 h at room temperature and 85% after incubation at 37 degrees celsius; with human serum. The cysteine replacement rate was less than 7%, and the binding rate of (99m)Tc-peptide with serum proteins was below 5%. SPECT imaging showed that the labeled peptide could be quickly cleared from the blood in normal animals primarily through the kidneys, and the radioactivity in other tissues and organs remained low. CONCLUSION: (99m)Tc-peptide can be easily prepared with a high labeling yield. With good stability both in vitro and in vivo, (99m)Tc-peptide can be quickly cleared from the blood and excreted though the kidney with ideal biodistribution and biokinetics in vivo.

[IgG radiolabelling with (99m)Tc by tricarbonyl method and its biodistribution in mice].

OBJECTIVE: To synthesize the complex fac-[99(m)Tc(CO)3(H2O)3](+) for labeling IgG and investigate the in vitro stability of 99(m)Tc(CO)3(H2O)3-IgG and its biodistribution in mice. METHODS: fac-[99(m)Tc(CO)3(H2O)3](+) was synthesized and its radiochemical purity determined using polyamide membrane chromatography. IgG was directly labeled with fac-[99(m)Tc(CO)3(H2O)3](+) and the labeling ratio was determined using chromatography. The stability of 99(m)Tc(CO)3(H2O)3-IgG in human serum albumin and normal saline was evaluated. 99(m)Tc(CO)3(H2O)3-IgG was injected via the tail vein into 9 mice at the dose of 3.7×104 Bq/100 µl, and SPECT image was obtained at 2, 4 and 12 h after the injection. The mice were sacrificed at these time points to measure the radioactivity and calculate the %ID/g in each organ. RESULTS: Fac-[99(m)Tc(CO)3(H2O)3](+) had a radiochemical purity of 82.48% and remained stable in vitro at room temperature within 4 h. The labeling ratio of 99(m)Tc(CO)3(H2O)3-IgG was 57.04% with a radiochemical purity exceeding 90%. In the solution of human serum albumin, the labeled IgG maintained a stable radiochemical purity, but in normal saline, its radiochemical purity was lowered to 20% at 24 h. After injection in mice, the labeled IgG was deposited mainly in the liver, spleen, kidneys, and the blood pool showed a sustained radioactivity. CONCLUSION: 99(m)Tc(CO)3(H2O)3-IgG prepared in this study has good stability in vitro and in vivo in 24 h and shows a biodistribution pattern similar to that of IgG protein in vivo. The intermediate fac-[99(m)Tc(CO)3(H2O)3](+) can meet the experimental requirement for labeling monoclonal antibodies and polypeptides.