Influence of Linker Molecules in Hexavalent RGD Peptides on Their Multivalent Interactions with Integrin αvß3.

Multivalent RGD peptides have been used as an excellent targeting vector to integrin αvß3-positive tumors. However, little attention has been paid to the influence of linker molecules in multivalent RGD peptides on their dissociation kinetics from tumor cells. In this study, we evaluated the dissociation kinetics of 99mTc-labeled hexavalent RGD peptides which have (CH2-CH2-O)n (n = 4, [99mTc][Tc(L1)6]+ and n = 12, [99mTc][Tc(L2)6]+) or (DPro-Gly)n (n = 1, [99mTc][Tc(L3)6]+; n = 6, [99mTc][Tc(L4)6]+; and n = 9, [99mTc][Tc(L5)6]+) as a linker molecule. The results showed that [99mTc][Tc(L4)6]+ and [99mTc][Tc(L5)6]+ displayed slower dissociation kinetics and [99mTc][Tc(L4)6]+ showed exceptionally high in vitro cellular uptake (203.1 ± 16.7% dose/mg protein) and the highest tumor to blood ratio (138.1 ± 26.3 at 4 h p.i.) in tumor bearing nude mice. These findings indicate that the use of appropriate length of (DPro-Gly)n would maximize the binding of multivalent RGD peptides to clustered integrin αvß3.

Phase I clinical study with different doses of 99mTc-TRODAT-1 in healthy adults.

OBJECTIVES: To study the pharmacokinetics, biodistribution, and injection doses of 99mTc-TRODAT-1 in healthy adults. METHODS: Thirty healthy individuals comprising 15 females and 15 males were randomly divided into three groups and the injection doses of 99mTc-TRODAT-1 of group 1, 2, and 3 were 370 MBq, 740 MBq, and 1110 MBq, respectively. Assessments of subjective symptoms and tests were performed before and after injection. Blood and urine collections and whole-body planar imaging were analyzed at various time points. Bilateral brain striatal SPECT images obtained at 3.5 h PI were assessed visually and semiquantitatively. RESULTS: No serious adverse events or deaths were observed in our study. The pharmacokinetic analysis showed that 99mTc-TRODAT-1 was eliminated rapidly from the circulation, with just about 4% of the injected dose remaining in blood at 1 h post-injection. The mean cumulative urinary excretion over 24 h was just 2.96 ± 0.96%ID. The time-activity curve demonstrated that the radioactivity was mainly in liver and abdomen. The highest absorbed dose was in the dose-limiting organ, liver (20.88 ± 4.45 × 10-3 mSv/MBq). The average effective dose was 5.22 ± 1.05 × 10-3 mSv/MBq. The clarity of striatal images assessed visually in group 1 was worse than that in group 2 and 3. The semiquantitative analysis showed that there were no differences in striatum/cerebellum between the three groups (group 1: 1.77 ± 0.11, group 2: 1.62 ± 0.14, and group 3: 1.75 ± 0.20; P = 0.088). CONCLUSIONS: 99mTc-TRODAT-1 was safe to use in humans and showed the status of dopaminergic neurons specifically and clearly. The injection dose we suggested was 740 MBq.

Preclinical pharmacokinetic, biodistribution, radiation dosimetry, and toxicity studies of 99mTc-HYNIC-(Ser)3-LTVPWY: A novel HER2-targeted peptide radiotracer.

Accurate assessment of the HER2 expression is an essential issue for predicting response to anti-HER2 therapy in breast cancer patients. The aim of this study was to evaluate 99mTc-HYNIC-(Ser)3-LTVPWY (99mTc-HYNIC-LY) peptide as a novel HER2-targeted radiolabeled peptide in healthy mice to examine the applicability of this imaging agent in a first-in-human clinical trial. To this end, pharmacokinetic and dosimetry studies were performed according to the ICH guideline M3 (R2) with 99mTc-HYNIC-LY. To estimate the radiation-absorbed doses in humans, the accumulated activity in each mouse organ was calculated based on biodistribution data. In addition, toxicology assessment was performed based on mortality events, body weights, and serum biochemical, hematological, and histopathological assays. The pharmacokinetic study showed rapid blood clearance. Based on the results of biodistribution study, the highest radioactivity was observed in the kidneys. The projected absorbed doses to the kidneys, liver, lungs, stomach, and spleen were obtained as 1.70E-02, 1.42E-02, 1.02E-02, 8.62E-03, and 8.34E-03 mSv/MBq, respectively. The results also revealed that serum biochemical and hematological parameters were in the normal range. No significant morphologic alterations were observed in the liver, kidneys, and spleen tissues. Consequently, the results were indicative of the suitability of 99mTc-HYNIC-LY peptide for advancement to a first-in-human clinical trial.

Preclinical Evaluation of Mesothelin-Specific Ligands for SPECT Imaging of Triple-Negative Breast Cancer.

Mesothelin is a cell-surface glycoprotein restricted to mesothelial cells overexpressed in several types of cancer, including triple-negative breast cancer not responding to trastuzumab or hormone-based therapies. Mesothelin-targeting therapies are currently being developed. However, the identification of patients potentially eligible for such a therapeutic strategy remains challenging. The objective of this study was to perform the radiolabeling and preclinical evaluation of 99mTc-A1 and 99mTc-C6, two antimesothelin single-domain antibody (sdAb)-derived imaging agents. Methods: A1 and C6 were radiolabeled with 99mTc and evaluated in vitro on recombinant protein and cells, as well as in vivo in xenograft mouse models of the triple-negative breast cancer cell lines HCC70 (mesothelin-positive) and MDA-MB-231 (mesothelin-negative). Results: Both 99mTc-A1 and 99mTc-C6 bound mesothelin with high affinity in vitro, with 99mTc-A1 affinity being 2.4-fold higher than that of 99mTc-C6 (dissociation constant, 43.9 ± 4.0 vs. 107 ± 16 nM, P < 0.05). 99mTc-A1 and 99mTc-C6 remained stable in vivo in murine blood (>80% at 2 h) and ex vivo in human blood (>90% at 6 h). In vivo 99mTc-A1 uptake (percentage injected dose) in HCC70 tumors was 5-fold higher than in MDA-MB-231 tumors and 1.5-fold higher than that of 99mTc-C6 (2.34% ± 0.36% vs. 0.48% ± 0.18% and 1.56% ± 0.43%, respectively, P < 0.01) and resulted in elevated tumor-to-background ratios. In vivo competition experiments demonstrated the specificity of 99mTc-A1 uptake in HCC70 tumors. Conclusion: Mesothelin-positive tumors were successfully identified by SPECT using 99mTc-A1 and 99mTc-C6. Considering its superior characteristics, 99mTc-A1 was selected as the most suitable tool for further clinical translation.

Development of a 99mTc-Labeled CXCR4 Antagonist Derivative as a New Tumor Radiotracer.

Due to high expression of CXCR4 (CXC chemokine receptor type 4) receptors in many tumors and metastasis, synthesis and labeling of CXCR4 receptor-targeted analogs as tumor imaging agents have been encouraged. Herein, CXCR4 receptor-targeted peptide antagonist was prepared and thereafter its labeling with 99mTc by a bifunctional chelating agent and tricine coligand was developed. Radiotracer purity, stability, and tumor cell binding were assessed. Bioevaluation of radiotracer was performed in mice bearing xenograft tumor. More than 95% labeling yield and stability up to 24 hours were observed. Radiotracer-related tumor accumulation was 3.61 ± 0.15% ID/g at 1 hour postinjection. High stability and specific tumor uptake are important characteristics of the radiotracer that could nominate this as a targeted imaging agent in the future.

Synthesis, optimization and biological evaluation of 99mTc-digoxin as possible cardiac imaging agent.

Heart imaging radiopharmaceuticals could improve the diagnostic value of routine heart scanning for detecting cardiac disorders. The aim of the study was to prepare high radiochemical purity 99mTc-Digoxin in a yield of about 98%. The optimal conditions for labelling were as follows: 100µg of Digoxin, 2µg of SnCl2•2H2O, room temperature (25±1°C), reaction retention time of 30 min at pH 7. Under these conditions, the radiochemical yield of 99mTc-Digoxin reaches 98%. In vivo bio distribution was performed in normal Swiss Albino mice at different time intervals after administration of 99mTc-Digoxin.Scintigraphic study of 99mTc-Digoxin was performed in rabbits. The heart uptake of 99mTc-Digoxin was sufficiently high and thus may be a potential myocardial imaging radiopharmaceutical applicable in cardiology.

Optimization of SPECT Measurement of Myocardial Blood Flow with Corrections for Attenuation, Motion, and Blood Binding Compared with PET.

Myocardial blood flow (MBF) and myocardial flow reserve (MFR) measured with PET have clinical value. SPECT cameras with solid-state detectors can obtain dynamic images for measurement of MBF and MFR. In this study, SPECT measurements of MBF made using 99mTc-tetrofosmin were compared with PET in the same patients. Methods: Thirty-one patients underwent PET MBF rest-stress studies performed with 82Rb or 13N-ammonia within 1 mo of their SPECT study. Dynamic rest-stress measurements were made using a SPECT camera. Kinetic parameters were calculated using a 1-tissue-compartment model and converted to MBF and MFR. Processing with and without corrections for attenuation (+AC and -AC), patient body motion (+MC and -MC), and binding of the tracer to red blood cells (+BB and -BB) was evaluated. Results: Both +BB and +MC improved the accuracy and precision of global SPECT MBF compared with PET MBF, resulting in an average difference of 0.06 ± 0.37 mL/min/g. Global MBF and detection of abnormal MFR were not significantly improved with +AC. Global SPECT MFR with +MC and +BB had an area under the receiver-operating curve of 0.90 (+AC) to 0.95 (-AC) for detecting abnormal PET MFR less than 2.0. Regional analysis produced similar results with an area under the receiver-operating curve of 0.84 (+AC) to 0.87 (-AC). Conclusion: Solid-state SPECT provides global MBF and MFR measurements that differ from PET by 2% ± 32% (MBF) and 2% ± 28% (MFR).

Development of a freeze-dried kit formulation for the preparation of 99mTc-NTP 15-5, a radiotracer for scintigraphic imaging of proteoglycans.

The cartilage-targeting strategy is based on the strong affinity of quaternary ammonium (QA) functions for cartilage proteoglycans. We use a bifunctional agent containing QA moiety and a polyazamacrocycle structure able to complex technetium-99m. (99m)Tc-NTP 15-5 was selected for its high stability and its high affinity for proteoglycans in vivo. Labeling conditions of NTP 15-5 were optimized, and a lyophilized kit was developed for radiolabeling of (99m)Tc-NTP 15-5 (radiochemical yields 94.6±1.8%). (99m)Tc-NTP 15-5 was stable and resulted in favorable biological evaluations.

Preparation of 99mTc-TRODAT-1 with high labeling yield in boiling water bath: a new formulation.

A new formulation for preparation of (99m)Tc-labeled tropane derivative, (99m)Tc-TRODAT-1, which is useful as a potential CNS dopamine transporter imaging agent, was evaluated and characterized. Preparation of (99m)Tc-TRODAT-1 was attained previously by a formulation in which vial has to be autoclaved at 121 °C for 30 min. It is highly desirable to further improve the preparation method by developing a simplified one vial formulation which will be labeled in boiling water bath (95 °C) for 15 min and a high labeling yield will be achieved. A formulation contained 10 µg of TRODAT-1, 20 µg tricine, 40 µg SnCl2 and 20mg manitol was prepared. Labeling was performed at 95 °C for 15 min and radiochemical analysis involved ITLC and HPLC methods. The stability of radioconjugate was checked in the presence of human serum at 37 °C up to 24h. (99m)Tc-TRODAT-1 was prepared with a radiochemical purity of more than 95% and specific activity of 64.3 MBq/nmol. Biodistribution studies of this new formulation in rats revealed similar regional brain distribution as compared with those obtained with the previous preparation in which brain uptake was high in striatum and striatum to cerebellum ratio was high. Requiring no autoclave facility for labeling, this new formulation will significantly improve the using feasibility of this radiopharmaceutical in clinic.

[(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.

Application of (99m)Technetium-HYNIC(tricine/TPPTS)-Aca-Bombesin(7-14) SPECT/CT in prostate cancer patients: a first-in-man study.

RATIONALE: The peptide bombesin (BBN) and its derivatives exhibit high binding affinity for the gastrin-releasing peptide receptor (GRPR), which is highly expressed in prostate cancer. We used the BBN-based radiopharmaceutical (99m)Technetium-HYNIC(tricine/TPPTS)-Aca-Bombesin(7-14) ((99m)Tc-HABBN) to perform a first-in-man clinical pilot study to evaluate the feasibility of (99m)Tc-HABBN SPECT/CT for detection of prostate cancer in patients. METHODS: Eight patients with biopsy-proven prostate cancer who were scheduled for either radical prostatectomy or external beam radiotherapy underwent (99m)Tc-HABBN scintigraphy and SPECT/CT prior to treatment. Serial blood samples were taken to assess blood radioactivity and to determine in vivo metabolic stability. Clinical parameters were measured and reported side effects, if present, were recorded. Prostate cancer specimens of all patients were immunohistochemically stained for GRPR. RESULTS: (99m)Tc-HABBN was synthesized with high radiochemical yield, purity and specific activity. There were no significant changes in clinical parameters, and there were no adverse or subjective side effects. Low metabolic stability was observed, as less than 20% of (99m)Tc-HABBN was intact after 30 min. Immunohistochemical staining for GRPR was observed in the prostate cancer specimens in all patients. (99m)Tc-HABBN scintigraphy and SPECT/CT did not detect prostate cancer in patients with proven disease. CONCLUSIONS: (99m)Tc-HABBN SPECT/CT for visualization of prostate cancer is safe but hampered by an unexpected low in vivo metabolic stability in man. The difference between the excellent in vitro stability of (99m)Tc-HABBN in human serum samples determined in our previous study regarding (99m)Tc-HABBN and the low in vivo metabolic stability determined in this study, is striking. This issue warrants further study of peptide-based radiopharmaceuticals.

A comparison of single plasma sample methods to estimate renal clearance using 99mTc-ethylenedicysteine and 99mTc-mercaptoacetyltriglycine.

Several single sample methods for determination of (99m)Tc-mercaptoacetyltriglycine (MAG3) clearance are being used clinically. Kabasakal et al. proposed a similar formula for (99m)Tc-ethylenedicysteine (EC). This study was performed to compare his method with Bubeck et al. formula for (99m)Tc-MAG3 already in use. Twenty-eight subjects divided in two groups were registered which included 22 patients with various renal diseases (group-I) and six normal volunteers (group II). All subjects were studied twice using both the radiopharmaceuticals. The images and renogram parameters, that is TMAX and T1/2 of both the agents, were similar in all the subjects. The clearance of the (99m)Tc-EC was however considerably higher than (99m)Tc-MAG3 in both the groups (mean ± SEM =279 ± 14 ml min(-1)/1.73 m(2) versus 177 ± 15 ml min(-1)/1.73 m(2) in group-I and 377 ± 11.90 ml min(-1)/1.73 m(2) versus 238 ± 8.23 ml min(-1)/1.73 m(2) in group II). This difference was more pronounced in cases with reduced renal functions. Among the Effective Renal Plasma Flow (ERPF) values determined from EC and MAG3 clearances in six normal volunteers, four cases only in MAG3 had ERPF below the lower limit. This study has demonstrated superiority of single sample method for (99 m)Tc-EC clearance over its analogous method for (99m)Tc-MAG3.

Chronic intestinal ischaemia: measurement of the total splanchnic blood flow.

A redundant collateral network between the intestinal arteries is present at all times. In case of ischaemia in the gastrointestinal tract, the collateral blood supply can develop further, thus accommodating the demand for oxygen even in the presence of significant stenosis or occlusion of the intestinal arteries without clinical symptoms of intestinal ischaemia. Symptoms of ischemia develop when the genuine and collateral blood supply no longer can accommodate the need for oxygen. Atherosclerosis is the most common cause of obliteration in the intestinal arteries. In chronic intestinal ischaemia (CII), the fasting splanchnic blood flow (SBF) is sufficient, but the postprandial increase in SBF is inadequate and abdominal pain will therefore develop in relation to food intake causing the patient to eat smaller meals at larger intervals with a resulting weight loss. Traditionally, the CII-diagnosis has exclusively been based upon morphology (angiography) of the intestinal arteries; however, substantial discrepancies between CII-symptoms and the presence of atherosclerosis/stenosis in the intestinal arteries have been described repeatedly in the literature impeding the diagnosis of CII. This PhD thesis explores a method to determine the total SBF and its potential use as a diagnostic tool in patients suspected to suffer from CII. The SBF can be measured using a continuous infusion of a tracer and catheterisation of a hepatic vein and an artery. By measuring the SBF before and after a standard meal it is possible to assess the ability or inability to enhance the SBF and thereby diagnosing CII. In Study I, measurement of SBF was tested against angiography in a group of patients suspected to suffer from CII due to pain and weight loss. A very good agreement between the postprandial increase in SBF and angiography was found. The method was validated against a well-established method independent of the hepatic extraction of tracer using pAH in a porcine model (study II). An excellent agreement was found between the two methods for the measurement of SBF. In the same set-up metabolism and recirculation in the intestines of the 99mTechnetium labelled tracer was rejected based on the consistency between the portal and arterial contents of tracer. Based on this study we concluded that an arterial blood sample can be used instead of a portal blood sample, making the method applicable to patients. In study III, 20 healthy volunteers and 29 patients with weight loss and abdominal pain but normal morphology of the intestinal arteries were investigated. A reference value for the meal induced SBF-increase and the relation to bodyweight was established designating that bodyweight should be taken into account when diagnosing CII based on measurement of SBF. The clinical method for measuring the SBF based on hepatic 99mTc-MBF extraction is a robust method. It allows determination of the postprandial increase in SBF providing knowledge about the circulatory physiology in intestines in patients with weight loss and abdominal pain with or without intestinal arterial stenosis. Future studies within this field could include measurement of the SBF before and after revascularisation in order to quantify the effect of revascularisation or investigate whether arterial blood sampling could be avoided or the amount of blood samples (and thus the time spend) could be reduced. The three studies were presented at eleven national and international congresses and Helle Damgaard Zacho has been awarded three prizes for the presentations.

Design, synthesis, and biological evaluation of catecholamine vehicle for studying dopaminergic system.

Catecholamine mimetic EDTA-bis(tyramide) was synthesized and characterized by various spectroscopic techniques (NMR, mass spectroscopy) and λem 310 nm for the excitation at 270 nm. Molecular docking studies were performed with human serum albumin (PDB 1E78), showing binding pattern with amino acid residues Arg218, Arg222, and Lys444, identifies the ligand-human serum albumin interaction for the transportation affinity of the ligand at the specific site of the target. Subsequently, binding study with human serum albumin at λex  = 350 nm found to be 5.847 × 10(4)  m(-1) shows effective quenching effect. Additionally, to go more insight, acetylcholinesterase binding affinity was investigated, which shows 90% binding affinity for the 10 mm concentration. IC50 value was found 18.60 µm for MAO-B inhibition. Finally, EDTA-bis(tyramide) labeled with (99m) Tc to investigate its in vivo radiopharmaceutical efficiency having 97% binding affinity with 98% radiochemical purity. In vivo studies were carried out for (99m) Tc-EDTA-bis(tyramide) included blood kinetics showed a quick wash out from the circulation via renal route, and biodistribution revealed that maximum %ID/g was found in kidney at 1 h, and its scintigraphy image shows 3.96% brain uptake with respect to whole body.

Preliminary studies of acetylcholinesterase activity in the rat brain using N-phenylferrocenecarboxamide labelled by the technetium-99m.

There is currently great interest in developing radiolabeled substrates for acetylcholinesterase that would be useful in the in vivo imaging of patients with Alzheimer's disease. The reduction of acetylcholinesterase (AChE) activity in the brain has been measured in dementia disorders such as Alzheimer's disease and dementia with Lewy bodies using (11)C and (18)F-labeled acetylcholine analogues. Our aim was to develop a new 99mTc-labeled acetylcholine analogue: N-phenylferrocenecarboxamide labelled with technetium-99m (99mTc-TPCC) to study acetylcholinesterase activity. In vivo and in vitro studies demonstrated that the labelled compound was a substrate for acetylcholinesterase. The hydrolytic rate of this substrate was measured and the specificity was evaluated using the inhibitor BW 284 C51. In rat experiments, the 99mTc-TPCC showed desirable properties for studying the acetylcholinesterase in the rat brain: high hydrolytic rate and a moderate specificity of the substrate for acetylcholinesterase.

99mTc(CO)3(NTA) and 131I-OIH: comparable plasma clearances in patients with chronic kidney disease.

UNLABELLED: The pharmacokinetics of the tricarbonyl core radiopharmaceutical (99m)Tc(CO)3-nitrilotriacetic acid ((99m)Tc(CO)3(NTA)) in rats and subjects with normal renal function are comparable to those of (131)I-o-iodohippuran ((131)I-OIH), the radiopharmaceutical gold standard for the measurement of effective renal plasma flow. Our objective was to compare the pharmacokinetics of these 2 tracers in subjects with renal failure. METHODS: (99m)Tc(CO)3(NTA) was prepared with commercially available NTA and a commercially available labeling kit and isolated by reversed-phase high-performance liquid chromatography. Approximately 74 MBq (2.0 mCi) of (99m)Tc(CO)3(NTA) were coinjected with approximately 11.1 MBq (300 µCi) of (131)I-OIH in 8 subjects with stage 3-4 renal failure; simultaneous images were obtained for 24 min, followed by an anterior image over the gallbladder and abdomen. Plasma clearances were determined from 10 blood samples obtained 3-180 min after injection using the single-injection, 2-compartment model. Plasma protein binding, red cell uptake, and percentage injected dose in the urine at 30 and 180 min were determined. RESULTS: There was no difference in the plasma clearances of (99m)Tc(CO)3(NTA) and (131)I-OIH (177 ± 63 vs. 171 ± 66 mL/min/1.73 m(2), respectively) (P = 0.41). The plasma protein binding and red cell uptake of (99m)Tc(CO)3(NTA) were 35% ± 7% and 6% ± 3%, respectively; both values were significantly lower than the plasma protein binding (71% ± 5%) and red cell uptake (16% ± 2%) of (131)I-OIH (P < 0.001). There was no significant difference in the percentage injected dose in the urine at 30 min (P = 0.24) and at 3 h (P = 0.82); for comparison, the percentage dose in the urine at 3 h was 77% ± 9% for (99m)Tc(CO)3(NTA) and 78% ± 11% for (131)I-OIH. Image quality with (99m)Tc(CO)3(NTA) was excellent and no activity was identified in the gallbladder or intestine. CONCLUSION: Results in patients with renal failure show the clearance and rate of urine excretion of (99m)Tc(CO)3(NTA) to be equivalent to that of (131)I-OIH.

(99m)Tc-daunorubicin a potential brain imaging and theranostic agent: synthesis, quality control, characterization, biodistribution and scintigraphy.

Daunorubicin is a chemotherapeutic antibiotic of the anthracycline family used for the treatment of many type of cancers when doxorubicin or other less effective drugs cannot be used. The aim of the present study was labeling of Daunorubicin with (99m)Tc, quality control, characterization, and biodistribution of radiolabeled Daunorubicin. Labeling efficiency was determined by ascending paper chromatography. All the experiments were performed at room temperature (25°C±2°C). More than 96% labeling efficiency with (99m)Tc was achieved at pH 5-6, 2-4 µg stannous chloride and 300 µg of ligand in few minutes. The characterization of the compound was performed by using HPLC, electrophoresis and shake flask assay. Electrophoresis indicates that Tc-99m-Daunorubicin is neutral, HPLC confirms the single specie of the labeled compound, while shake flask assay confirms high lipophilicity. The biodistribution studies of (99m)Tc-Daunorubicin were performed in rats. Significantly higher accumulation of (99m)Tc-Daunorubicin was seen in brain of normal rats. Scintigraphy was also indicating higher accumulation of (99m)Tc-Daunorubicin in brain of normal rabbits.

Synthesis and biological evaluation of a new triazole-oxotechnetium complex.

A new triazole oxotechnetium chelating agent was synthesized via a 'Click-to-Chelate' strategy. In vivo evaluation of the corresponding (99m)Tc complex shows that the tracer exhibits very interesting properties for molecular imaging.

[Comparative study of the biodistribution of (99m)Tc-HYNIC-Lys3-Bombesin obtained with the EDDA/tricine and NA/tricine as coligands]. / Estudio comparativo de la biodistribución de (99m)Tc-HYNIC-Lys3-bombesina obtenido con los coligandos EDDA/tricina y AN/tricina.

The aim of present investigation was to evaluate biodistribution in healthy animals and in tumor models of the radiopharmaceuticals (99m)Tc-EDDA/tricine-HYNIC-Lys3-Bombesin (HYNIC-Lys3-BN) and (99m)Tc-NA/tricine-HYNIC-Lys3-BN. Biodistribution and pharmacokinetics were carried out over 24 hours. To do so, 24 healthy Wistar rats were used and were administered 37.0 ± 0.8 MBq/rat of each radiopharmaceutical. For the tumor model study, 20 CD-1 nude mice were used and prostate tumors (PC3) were implanted in all the mice. Ten days later, tumor volumes were calculated and 40.00 ± 0.04 MBq/mice of each radiopharmaceutical were injected. Both showed high radiochemical purity: 98.08 ± 0.25% for EDDA/tricine product and 95.1 ± 0.3% for the conjugate with NA/tricine. Uptake of the radiopharmaceutical with NA/tricine was significantly higher in organs of the reticulo-endothelial system of healthy Wistar rats during 24h, specifically in the liver and spleen. Both labeled compounds showed no significant differences between their blood elimination half lives. Average of tumor growth was 0.93 ± 0.02 cm(3) and affinity for tumors showed a growing and specific binding of both radiopharmaceuticals, although it was significantly higher for the EDDA/tricine conjugate. This outcome made it possible to corroborate the direct relationship between the density of gastrin releasing peptide and its receptors (GRPr) and the variation of the accumulation of the radiopharmaceuticals in the tumor. Use of EDDA/tricine as coligand is more appropriate than NA/tricine for labeling of HYNIC-Lys3-BN with (99m)Tc.

(99m)Tc-labeled rituximab for imaging B lymphocyte infiltration in inflammatory autoimmune disease patients.

PURPOSE: The rationale of the present study was to radiolabel rituximab with 99m-technetium and to image B lymphocytes infiltration in the affected tissues of patients with chronic inflammatory autoimmune diseases, in particular, the candidates to be treated with unlabelled rituximab, in order to provide a rationale for 'evidence-based' therapy. PROCEDURES: Rituximab was labelled with (99m)Tc via 2-ME reduction method. In vitro quality controls of (99m)Tc-rituximab included stability assay, cysteine challenge, SDS-PAGE, immunoreactive fraction assay and competitive binding assay on CD20+ve Burkitt lymphoma-derived cells. For the human pilot study, 350-370 MBq (100 µg) of (99m)Tc-rituximab were injected in 20 patients with different chronic inflammatory autoimmune diseases. Whole body anteroposterior planar scintigraphic images were acquired 6 and 20 h p.i. RESULTS: Rituximab was labelled to a high labelling efficiency (>98%) and specific activity (3515-3700 MBq/mg) with retained biochemical integrity, stability and biological activity. Scintigraphy with (99m)Tc-rituximab in patients showed a rapid and persistent spleen uptake, and the kidney appeared to be a prominent source for the excretion of radioactivity. Inflamed joints showed a variable degree of uptake at 6 h p.i. in patients with rheumatoid arthritis indicating patient variability; similarly, the salivary and lacrimal glands showed variable uptake in patients with Sjögren's syndrome, Behçet's disease and sarcoidosis. Inflammatory disease with particular characteristics showed specific uptake in inflammatory lesions, such as, dermatopolymyositis patients showed moderate to high skin uptake, a sarcoidosis patient showed moderate lung uptake, a Behçet's disease patient showed high oral mucosa uptake and a polychondritis patient showed moderate uptake in neck cartilages. In one patient with systemic lupus erythematosus, we did not find any non-physiological uptake. CONCLUSION: Rituximab can be efficiently labelled with (99m)Tc with high labelling efficiency. The results suggest that this technique might be used to assess B lymphocyte infiltration in affected organs in patients with autoimmune diseases; this may provide a rationale for anti-CD20 therapies.