Imaging of Clot by 99mTc-HMPAO Labeled Platelet in Animal Model Induced Thrombosis.

99mTc-HMPAO labeled platelet (LP) imaging may integrate thrombosis imaging into routine clinical procedures. In the current study, we assessed the feasibility of the use of 99mTc-HMPAO LP for imaging of small clots in an animal model. Thrombosis was induced by application of FeCl3 solution in the distal part of the inferior vena cava (IVC) of a 6100 g anesthetized rabbit and in a male Wistar rat weighing 420 g. Twenty minutes later, 178 MBq 99mTc-HMPAO LP was injected. 99mTc-HMPAO LP preparation was done as defined and standardized in a previous report. Whole body and SPECT imaging were done 60, 90, and 120 min after tracer injection. Then, the clotted part of the vein was extracted and then its activity and pathologic evaluations were compared with the proximal part of the IVC at a similar volume. A 17 × 6 mm clot was clearly detected with both planar and SPECT imaging. The count to pixel ratio (CPR) of the clotted part of the vein was 35, 40, and 40 compared to the non-clotted vein (i.e. 19, 18, and 21) at 60, 90, and 120 min, respectively. After clot extraction, the CPR decreased to 14. The clot activity was 0.44 MBq compared to 0.01 MBq of the normal control vein. Also, clot induction was pathologically proven. 99mTc-HMPAO LP preparation is logistically possible in clinical nuclear medicine and the ability of imaging small size clots encourages future trials for real clinical thrombotic scenarios.

Preclinical evaluation of new GnRH-I receptor radionuclide therapy with 177 Lu-peptide tracer.

The purpose of this study was to develop preclinical evaluation of a novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor targeting peptide for prostate cancer therapy. The new antiproliferative agent of GnRH-I analogue was developed on the basis of the D-Trp6 -GnRH-I scaffold, and in vivo pharmacokinetics and receptor binding affinity were enhanced by the substitution of Gly-NHNH2 for Gly-NH2 at position 10 in D-Trp6 -GnRH-I. To evaluate 177 Lu-DOTA-triptorelin-hydrazide as radionuclide therapy of tumor, the quality control tests and preclinical stage assessment were carried out. Solid-phase method was used to synthesize new peptide. Characterization and purity of peptide were done by mass spectroscopy and high-performance liquid chromatography (HPLC). In order to be utilized in targeted therapy, the new GnRH-I agonist was coupled with pSCN-Bn-DOTA. The precipitate crude of DOTA-triptorelin-hydrazide was then purified via preparative HPLC. At optimal conditions of time, temperature, ligand amount, and lutetium content, DOTA-triptorelin-hydrazide was labeled with 177 Lu (specific activity not less than 925 GBq/mg). Investigation of the in vivo biodistribution and in vitro studies for 177 Lu-DOTA-TRPHYD was performed in three different ways, and the binding of radiopeptide to GnRH receptors was expressed on the human cell lines using 125 I-labeled D-TRP6 GnRH-I as a tracer, respectively. Synthesized novel GnRH-I was obtained with purity greater than 98%. Paper chromatography was found to be the most suitable with Rf of the complex and observed radiochemical purity of RTLC and HPLC greater than 97%. For in vivo studies, 177 Lu-DOTA-triptorelin-hydrazide showed promising results with fast clearance from the blood and resulted in good T/NT ratios at 1, 4, and 24 hours postinjection and satisfactory biodistribution with no significant activity seen in normal tissue. The values of internalization efficiency and receptor affinity of new radiopeptide binding were IC50 = 0.47 ± 0.06 vs 0.13 ± 0.01 nM for triptorelin and cellular uptake: 3.4 ± 0.7% at 1 hour and 6.8 ± 1.17% at 4 hours of the internal reference. The results showed a good stability and radiochemical purity of the obtained radioconjugate. For in vivo and in vitro studies, new radiopeptide showed a high uptake of 177 Lu conjugate in tumor and rapid clearance from the blood stream almost entirely via the renal/urinary pathway and binding to the GnRH receptors with high specificity and affinity, respectively.

Development of Dendrimer Encapsulated Radio-Ytterbium and Biodistributionin Tumor Bearing Mice.

The aim of this study is preparation of dendrimer encapsulated ytterbium-175 radio-nanoparticles and investigation of the compound chemical characteristic before and after the neutron irradiation and also study the in vivo biodistribution for targeted radiopharmaceutical dose delivery to solid tumors. For preparation of dendrimer-metal nanocomposite, a dendrimer compound containing an average of 55 Yb+3 ions per dendrimer was prepared. The synthesized encapsulated ytterbium irradiated by neutron for 2 h at 3×1011 n.cm [Formula: see text] neutron flux. The resulting mixture was injected into 2 separate groups of tumor bearing mice. One group were injected intravenously and the other group were injected directly in tumor and were excised, weighed and counted at certain times to study the biodistribution and to compare the tumor treatment and the leakage of the radiopharmaceutical to non-target organs. The formation of dendrimer-Yb3+complex was confirmed by UV-vis spectrometer. High-resolution transmission electron microscopy (HRTEM) and Dynamic Light Scattering (DLS) results showed a particle size of less than 10 nm. The specific activity and radio-ytterbium purity of the irradiated nano-composite were as follows: 7 MBq/mg and >95%. The measured radiochemical purity by Instant Thin Layer Chromatography (ITLC) was more than 99%. In intravenous injection the complex showed rapid up take in liver, spleen, and lung, while accumulation in other organs was insignificant. In tumor direct injection the average size of the tumor mass in mice was reduced by 30%.

Preparation and Biological Study of (68)Ga-DOTA-alendronate.

OBJECTIVES: In line with previous research on the development of conjugated bisphosphonate ligands as new bone-avid agents, in this study, DOTA-conjugated alendronate (DOTA-ALN) was synthesized and evaluated after labeling with gallium-68 ((68)Ga). METHODS: DOTA-ALN was synthesized and characterized, followed by (68)Ga-DOTA-ALN preparation, using DOTA-ALN and (68)GaCl3 (pH: 4-5) at 92-95° C for 10 min. Stability tests, hydroxyapatite assay, partition coefficient calculation, biodistribution studies, and imaging were performed on the developed agent in normal rats. RESULTS: The complex was prepared with high radiochemical purity (>99% as depicted by radio thin-layer chromatography; specific activity: 310-320 GBq/mmol) after solid phase purification and was stabilized for up to 90 min with a log P value of -2.91. Maximum ligand binding (65%) was observed in the presence of 50 mg of hydroxyapatite; a major portion of the activity was excreted through the kidneys. With the exception of excretory organs, gastrointestinal tract organs, including the liver, intestine, and colon, showed significant uptake; however, the bone uptake was low (<1%) at 30 min after the injection. The data were also confirmed by sequential imaging at 30-90 min following the intravenous injection. CONCLUSION: The high solubility and anionic properties of the complex led to major renal excretion and low hydroxyapatite uptake; therefore, the complex failed to demonstrate bone imaging behaviors.

Development of a (68)Ga-peptide tracer for PET GnRH1-imaging.

OBJECTIVE: Total synthesis, quality control and preclinical evaluation of [(68)Ga]-DOTA-triptorelin ([(68)Ga]-DOTA-TRP) is reported as a possible PET radiotracer for GnRH receptor imaging. METHODS: DOTA-TRP was totally synthesized in two steps and after characterization went through radiolabelling optimization studies followed by tracer stability. The biodistribution of the tracer in normal male rats and 4T1 tumour-bearing mice was performed in 120 min after i.v. injection. RESULTS: The peptide and the conjugates were synthesized with >95 % chemical purity. [(68)Ga]-DOTA-TRP complex was prepared in high radiochemical purity (>99 %, ITLC, HPLC) and specific activity of 1400-2100 MBq/nM at 95 °C using 40-60 µg of the peptide in 5-7 min followed by solid phase purification. The IC50 [nM] DOTA-TRP was comparable to the intact peptide, 0.11 ± 0.01 and 0.22 ± 0.05, respectively. The biodistribution of the tracer demonstrated kidney, stomach, and testes significant uptake, all in accordance with GnRH receptor ligands. Significant tumour uptake was observed in 4T1 tumour-bearing female mice 30-120 min post-injection with tumour:blood and tumour:muscle ratios of 28 and >50 in 60 min, respectively. Kidney is rapidly washed from the tracer. [(68)Ga]-DOTA-TRP can be proposed as a possible tracer for GnRH-R imaging studies.

Nano Liposomes Labeled with (99m)Tc-HMPAO, a Novel Agent for Blood Pool Imaging.

In-vitro labeling of RBC with (99m)Tc is an intricate procedure and there is always a need for an alternate blood pool imaging agent. The aim of this study was to prepare an effective nano sized liposome (NLs) similar to human RBC for blood pool scintigraphy. This study formulates PEG-NLs and non-PEG-NLs using film method plus high pressure homogenization technique. Biodistribution studies were performed on BALB/C mice 1, 4 and 24 h after tail vein injection of labeled NLs with (99m)Tc hexamethylpropylene-amine-oxime ((99m)Tc-HMPAO). Planar images were acquired using a 256 × 256 matrix following(99m) Tc-HMPAO-NLs injection into ear vein of rabbits 1, 2 and 24 h later. SPECT images were obtained 15 minutes after the injection (64 slices, 30 second/projection). The mean diameter, zeta potential and polydispersity index (PDI) of the PEG-NLs and the NLs were (80.88 ± 0.594 nm, -12.5 ± 0.56 mv, 0.158 ± 0.025) and (94.14 ± 0.114 nm, -35.5 ± 0.67 mv and 0.198 ± 0.007), respectively. (99m)Tc-HMPAO-PEG-NLs showed a significant circulation tracer activity (7.74 ± 1.63%ID/g at 1 h and 4.9 ± 0.77 %ID/g at 4 h), with low liver accumulation (12.07 ± 3.66 %ID/g at 1 h and 14.85 ± 1.3 %ID/g at 4 h). Heart to liver, spleen and background ROIs (region of interests) for (99m) Tc-HMPAO-PEG-NLs were 1.25, 4 and 4.28 respectively at 2 h which changed to 1.06, 1.75 and 2.51 respectively at 24 h. The (99m)Tc-HMPAO-PEG-NLs with a prolonged blood circulation time could be an excellent RBC alternative for scintigraphy and gastrointestinal bleeding.

Development of (153) Sm-folate-polyethyleneimine-conjugated chitosan nanoparticles for targeted therapy.

The aim of this study was to develop biocompatible, water-soluble (153) Sm-labeled chitosan nanoparticles (NPs) containing folate and polyethyleneimine functionalities i.e. chitosan-graft-PEI-folate (CHI-DTPA-g-PEI-FA), suitable for targeted therapy. The physicochemical properties of the obtained NPs were characterized by dynamic light-scattering analysis for their mean size, size distribution, and zeta potential; scanning electron microscopy for surface morphology; and (1) H-NMR, FT-IR analyses for molecular dispersity of folate in the NPs. NPs were spherical with mean diameter below 250 nm, polydispersity of below 0.15, and positive zeta potential values. The NP complex ((153) Sm-CHI-DTPA-g-PEI-FA) was stable at 25 °C (6-8 h, >90% radiochemical purity, instant thin layer chromatography (ITLC)). Binding studies using fluorescent NPs for internalization also demonstrated significant uptake in MCF-7 cells. MCF-7 cell internalization was significantly greater for 4T1. In blocking studies, both MCF-7 and 4T1 cell lines demonstrated specific folate receptor (FR) binding (decreasing 45%). In vivo biodistribution studies indicated major excretion of NPs metabolites and/or free (153) Sm through the kidneys. The preliminary imaging studies in 4T1 tumor-bearing mice showed minor uptake up to 96 h. The present folic acid that functionalized chitosan NP is a candidate material for folate receptor therapy.

Activity estimation in radioimmunotherapy using magnetic nanoparticles.

OBJECTIVE: Estimation of activity accumulated in tumor and organs is very important in predicting the response of radiopharmaceuticals treatment. In this study, we synthesized (177)Lutetium ((177)Lu)-trastuzumab-iron oxide nanoparticles as a double radiopharmaceutical agent for treatment and better estimation of organ activity in a new way by magnetic resonance imaging (MRI). METHODS: (177)Lu-trastuzumab-iron oxide nanoparticles were synthesized and all the quality control tests such as labeling yield, nanoparticle size determination, stability in buffer and blood serum up to 4 d, immunoreactivity and biodistribution in normal mice were determined. In mice bearing breast tumor, liver and tumor activities were calculated with three methods: single photon emission computed tomography (SPECT), MRI and organ extraction, which were compared with each other. RESULTS: The good results of quality control tests (labeling yield: 61%±2%, mean nanoparticle hydrodynamic size: 41±15 nm, stability in buffer: 86%±5%, stability in blood serum: 80%±3%, immunoreactivity: 80%±2%) indicated that (177)Lu-trastuzumab-iron oxide nanoparticles could be used as a double radiopharmaceutical agent in mice bearing tumor. Results showed that (177)Lu-trastuzumab-iron oxide nanoparticles with MRI had the ability to measure organ activities more accurate than SPECT. CONCLUSIONS: Co-conjugating radiopharmaceutical to MRI contrast agents such as iron oxide nanoparticles may be a good way for better dosimetry in nuclear medicine treatment.

Synthesis and preliminary evaluation of a new (99m)tc labeled substance p analogue as a potential tumor imaging agent.

Neurokinin 1 receptors (NK1R) are overexpressed on several types of important human cancer cells. Substance P (SP) is the most specific endogenous ligand known for NK1Rs. Accordingly,a new SP analogue was synthesized and evaluated for detection of NK1R positive tumors.[6-hydrazinopyridine-3-carboxylic acid (HYNIC)-Tyr(8)-Met(O)(11)-SP] was synthesized and radiolabeled with (99m)Tc using ethylenediamine-N,N'-diacetic acid (EDDA)and Tricine as coligands. Common physicochemical properties of radioconjugate were studied and in-vitro cell line biological tests were accomplished to determine the receptor mediated characteristics. In-vivo biodistribution in normal and tumor bearingnude mice was also assessed. The cold peptide was prepared in high purity (>99%) and radiolabeled with (99m)Tc at high specific activities (84-112GBq/µmol) with an acceptable labeling yield (>95%). The radioconjugate was stable in-vitro in the presence of human serum and showed 44% protein binding to human serumalbumin. In-vitro cell line studies on U373MG cells showed an acceptable uptake up to 4.91 ± 0.22% with the ratio of 60.21 ± 1.19% for its specific fraction and increasing specific internalization during 4 h. Receptor binding assays on U373MG cells indicated a mean Kd of 2.46 ± 0.43 nM and Bmax of 128925 ± 8145 sites/cell. In-vivo investigations determined the specific tumor uptake in 3.36 percent of injected dose per gram (%ID/g) for U373MG cells and noticeable accumulations of activity in the intestines and lung. Predominant renal excretion pathway was demonstrated. Therefore, this new radiolabeled peptide could be a promising radiotracer for detection of NK1R positive primary or secondary tumors.

Preparation and radiolabeling of a lyophilized (kit) formulation of DOTA-rituximab with 9°Y and ¹¹¹In for domestic radioimmunotherapy and radioscintigraphy of non-Hodgkin's lymphoma.

BACKGROUND: On the basis of results of our previous investigations on 90Y-DTPA-rituximab and in order to fulfil national demands to radioimmunoconjugates for radioscintigraphy and radioimmunotherapy of Non-Hodgkin's Lymphoma (NHL), preparation and radiolabeling of a lyophilized formulation (kit) of DOTA-rituximab with 111In and 90Y was investigated. METHODS: 111In and 90Y with high radiochemical and radionuclide purity were prepared by 112Cd (p,2n)111In nuclear reaction and a locally developed 90Sr/90Y generator, respectively. DOTA-rituximab immunoconjugates were prepared by the reaction of solutions of p-SCN-Bz-DOTA and rituximab in carbonate buffer (pH = 9.5) and the number of DOTA per molecule of conjugates were determined by transchelation reaction between DOTA and arsenaso yttrium(III) complex. DOTA-rituximab immunoconjugates were labeled with 111In and 90Y and radioimmunoconjugates were checked for radiochemical purity by chromatography methods and for immunoreactivity by cell-binding assay using Raji cell line. The stability of radiolabeled conjugate with the approximate number of 7 DOTA molecules per one rituximab molecule which was prepared in moderate yield and showed moderate immunoreactivity, compared to two other prepared radioimmunoconjugates, was determined at different time intervals and against EDTA and human serum by chromatography methods and reducing SDS-polyacrylamide gel electrophoresis, respectively. The biodistribution of the selected radioimmunoconjugate in rats was determined by measurement of the radioactivity of different organs after sacrificing the animals by ether asphyxiation. RESULTS: The radioimmunoconjugate with approximate DOTA/rituximab molar ratio of 7 showed stability after 24 h at room temperature, after 96 h at 4°C, as the lyophilized formulation after six months storage and against EDTA and human serum. This radioimmunoconjugate had a biodistribution profile similar to that of 90Y-ibritumomab, which is approved by FDA for radioimmunotherapy of NHL, and showed low brain and lung uptakes and low yttrium deposition into bone. CONCLUSION: Findings of this study suggest that further investigations may result in a lyophilized (kit) formulation of DOTA-rituximab which could be easily radiolabeled with 90Y and 111In in order to be used for radioimmunotherapy and radioscintigraphy of B-cell lymphoma in Iran.

Synthesis, Radiolabeling and Biological Evaluation of (99m)Tc-labeled Deoxyglucose Derivatives for Molecular Imaging.

Two deoxyglucose (DG) derivatives, (α,ß)-2-deoxy-2-amino(ethylcarbamate)-D-glucose (ECB-DG) and (α,ß)-2-deoxy-2-amino(1,2-dihydroxypropyl)-D-glucose (DHP-DG), were synthesized and radiolabeled successfully with [(99m)Tc(H2O)3(CO)3](+) complex. [(99m)Tc]-ECB-DG and [(99m)Tc]-DHP-DG complexes were prepared (96% and 93% radiochemical purities respectively) by using 46 mCi of Na(99m)TcO4 in 1 mL saline. Radio-HPLC analysis of [(99m)Tc]- ECB-DG at pH = 7.4, revealed that labeling with (99m)Tc leads to formation of one radiochemical species with tR = 381 second. Three radiochemical species, Na(99m)TcO4, [(99m)Tc]-DHP-DG and [(99m)Tc(H2O)3(CO)3](+) complexes with tR = 342 sec, tR = 567 sec and tR = 1586 sec respectively, were obtained when [(99m)Tc]-DHP-DG complex evaluated by HPLC. Biodistribution of two complexes were studied on normal mice at 10, 30 and 60 min post-injections. Compared to the (18)F-FDG, [(99m)Tc]-ECB-DG displayed a 2.8-fold reduction in brain uptake (1.7 ± 0.2 versus 0.61% ± 0.09) ,whereas [(99m)Tc]-DHP-DG just showed 1.9-fold reduction in heart uptake (2.2 ± 0.05 towards 1.16±0.10) at 1 h post-injection. On the basis of our results, it seems that ECB-DG and DHP-DG analogues could be used as brain and heart imaging agent respectively.

Toxicity of trastuzumab labeled 177Lu on MCF7 and SKBr3 cell lines.

In this study, we labeled trastuzumab with (177)Lu to synthesize a new radiopharmaceutical for therapy of breast cancer and at the first stage investigated its therapeutic effects on SKBr3 and MCF7 breast cancer cell lines. Trastuzumab-(177)Lu showed very good in-vitro characteristics such as high radiochemical purity (91+/-0.9%), good stability in PBS buffer (86+/-2.3%) and blood serum (81+/-2.7%) up to 96 h, appropriate immunoreactivity (85.4+/-1.1%) and high cytotoxicity in HER2 expression cells. 5 fold increase in toxicity of trastuzumab-(177)Lu was observed when compared with unlabeled trastuzumab on SKBr3 cells.