White blood cell labeling with Technetium-99m (99mTc) using red blood cell extracellular vesicles-mimetics.

BACKGROUND: Extracellular vesicles, have gained increasing attention for their application in drug delivery. Here, we developed a novel method for radiolabeling WBCs with 99mTc using RBC-derived extracellular vesicles -mimetics (EVMs), and monitored in vivo inflammation tracking of 99mTc-WBC using gamma camera in acute inflammation mouse model. METHODS: Engineered EVMs from RBCs were produced by a one-step extrusion method. RBC-EVMs were analyzed by NTA and TEM. Cells were labeled with 99mTc by using 99mTc-RBC-EVMs. Inflammation mice model was prepared and confirmed by 18F-FDG PET/CT. 99mTc-WBCs were injected in mice, and their biodistribution was analyzed by gamma camera. FINDING: The radiochemical purity of 99mTc-RBC-EVMs was 100%. The 99mTc-labeling did't affect the size and morphology. The 99mTc in the cytoplasm of RBC-EVMs was successfully confirmed by high angle annular dark field STEM (scanning transmission electron microscope). Cells were successfully labeled with 99mTc using 99mTc-RBC-EVMs, and the counts per minute was increased in dose- and time-dependent manners. The 18F-FDG PET/CT images confirmed establishment of acute inflammation (left mouse foot). 99mTc-WBCs showed higher uptake in the inflamed foot than non-inflamed foot. INTERPRETATION: This novel method for radiolabeling WBCs using RBC-EVMs. 99mTc labeling may be a feasible method to monitor the in vivo biodistribution of cells.

Renal Handling of 99mTc-Labeled Antibody Fab Fragments with a Linkage Cleavable by Enzymes on Brush Border Membrane.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m (99mTc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG3)-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG3-GFK-suc-TFP, was designed, synthesized, and evaluated. 99mTc-labeled Fab was obtained by reacting MAG3-GFK-Fab conjugate with 99mTc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 (111In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [99mTc]Tc-MAG3-GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [99mTc]Tc-MAG3-Gly. However, [99mTc]Tc-MAG3-GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [99mTc]Tc-MAG3-Gly to the intestine. The retrospective comparison of [99mTc]Tc-MAG3-GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [99mTc]Tc-MAG3-Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG3-GFK-suc-TFP provided 99mTc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the post-labeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.

A novel delivery system for supraglottic atomization allows increased lung deposition rates of pulmonary surfactant in newborn piglets.

BACKGROUND: Earlier attempts to deliver effective lung doses of surfactant by aerosolization were unsuccessful, mostly because of technical shortcomings. We aimed at quantifying the lung deposition of poractant alfa with a new supraglottic delivery system for surfactant atomization in an experimental neonatal model. METHODS: The method involved six sedated 1-day-old piglets lying in the lateral decubitus, spontaneously breathing on nasal-mask continuous positive airway pressure (nCPAP). A pharyngeal cannula housing a multi-channel air-blasting atomization catheter was placed through the mouth with its tip above the glottis entrance. In all, 200 mg kg-1 of a 99mTc-surfactant mixture was atomized through the catheter synchronously with inspiration. Six intubated control piglets received an equal amount of intratracheally instilled 99mTc-surfactant mixture. The percentage of the 99mTc-surfactant mixture deposited in the lungs was estimated by scintigraphy. RESULTS: Median (range) deposition in the lungs was 40% (24-68%) after atomization and 87% (55-95%) after instillation (p < 0.001). Overall, almost 80% of the deposited surfactant was in the dependent lung. Effective atomization time (atomizer on) was 28 (17-52) min, yielding an output rate of 0.1-0.2 mL min-1. CONCLUSIONS: Without endotracheal intubation, in spontaneously breathing newborn piglets, this new supraglottic atomizer delivery system attained a median lung deposition of 40% of the nominal dose of surfactant.

Single-Photon Emission Computed Tomography Imaging Using Formyl Peptide Receptor 1 Ligand Can Diagnose Aortic Aneurysms in a Mouse Model.

BACKGROUND: Our previous studies showed that neutrophil infiltration and activation plays an important role in the pathogenesis of abdominal aortic aneurysms (AAA). However, there is a lack of noninvasive, inflammatory cell-specific molecular imaging methods to provide early diagnosis of AAA formation. Formyl peptide receptor 1 (FPR1) is rapidly upregulated on neutrophils during inflammation. Therefore, it is hypothesized that the use of cinnamoyl-F-(D)L-F-(D)L-F-K (cFLFLF), a PEGylated peptide ligand that binds FPR1 on activated neutrophils, would permit accurate and noninvasive diagnosis of AAA via single-photon emission computed tomography (SPECT) imaging. MATERIALS AND METHODS: Male C57BL/6 (wild-type) mice were treated with topical elastase (0.4 U/mL type 1 porcine pancreatic elastase) or heat-inactivated elastase (control), and aortic diameter was measured by video micrometry. Comparative histology was performed on Day 14 to assess neutrophil infiltration in aortic tissue. We performed near-infrared fluorescence imaging using c-FLFLF-Cy7 probe on Days 7 and 14 postelastase treatment and measured fluorescence intensity ex vivo in excised aortic tissue. A separate group of animals were injected with 99mTc-c-FLFLF 2 h before SPECT imaging on Day 14 using a SPECT/computed tomography/positron emission tomography trimodal scanner. Coexpression of neutrophils with c-FLFLF was also performed on aortic tissue by immunostaining on Day 14. RESULTS: Aortic diameter was significantly increased in the elastase group compared with controls on Days 7 and 14. Simultaneously, a marked increase in neutrophil infiltration and elastin degradation as well as decrease in smooth muscle integrity were observed in aortic tissue after elastase treatment compared with controls. Moreover, a significant increase in fluorescence intensity of c-FLFLF-Cy7 imaging probe was also observed in elastase-treated mice on Day 7 (approximately twofold increase) and Day 14 (approximately 2.5-fold increase) compared with respective controls. SPECT imaging demonstrated a multifold increase in signal intensity for 99mTc-cFLFLF radiolabel probe in mice with AAA compared with controls on Day 14. Immunostaining of aortic tissue with c-FLFLF-Cy5 demonstrated a marked increase in coexpression with neutrophils in AAA compared with controls. CONCLUSIONS: cFLFLF, a novel FPR1 ligand, enables quantifiable, noninvasive diagnosis and progression of AAAs. Clinical application of this inflammatory, cell-specific molecular probe using SPECT imaging may permit early diagnosis of AAA formation, enabling targeted therapeutic interventions and preventing impending aortic rupture.

Beta Cell Imaging-From Pre-Clinical Validation to First in Man Testing.

There are presently no reliable ways to quantify human pancreatic beta cell mass (BCM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. Furthermore, the lack of beta cell imaging hampers the evaluation of the impact of new drugs aiming to prevent beta cell loss or to restore BCM in diabetes. We presently discuss the potential value of BCM determination as a cornerstone for individualized therapies in diabetes, describe the presently available probes for human BCM evaluation, and discuss our approach for the discovery of novel beta cell biomarkers, based on the determination of specific splice variants present in human beta cells. This has already led to the identification of DPP6 and FXYD2ga as two promising targets for human BCM imaging, and is followed by a discussion of potential safety issues, the role for radiochemistry in the improvement of BCM imaging, and concludes with an overview of the different steps from pre-clinical validation to a first-in-man trial for novel tracers.

Imaging Hydrogen Sulfide in Hypoxic Tissue with [99mTc]Tc-Gluconate.

Hydrogen sulfide (H2S) is the third gasotransmitter and is generated endogenously in hypoxic or inflammatory tissues and various cancers. We have recently demonstrated that endogenous H2S can be imaged with [99mTc]Tc-gluconate. In the present study, we detected H2S generated in hypoxic tissue, both in vitro and in vivo, using [99mTc]Tc-gluconate. In vitro uptake of [99mTc]Tc-gluconate was measured under hypoxic and normoxic conditions, using the colon carcinoma cell line CT26, and was higher in hypoxic cells than that in normoxic cells. An acute hindlimb ischemia-reperfusion model was established in BALB/c mice by exposing the animals to 3 h of ischemia and 3 h of reperfusion prior to in vivo imaging. [99mTc]Tc-gluconate (12.5 MBq) was intravenously injected through the tail vein, and uptake in the lower limb was analyzed by single-photon emission computed tomography/computed tomography (SPECT/CT). SPECT/CT images showed five times higher uptake in the ischemic limb than that in the normal limb. The standard uptake value (SUVmean) of the ischemic limb was 0.39 ± 0.03, while that of the normal limb was 0.07 ± 0.01. [99mTc]Tc-gluconate is a novel imaging agent that can be used both in vitro and in vivo for the detection of endogenous H2S generated in hypoxic tissue.

SPECT/CT imaging of apoptosis in aortic aneurysm with radiolabeled duramycin.

The objective of this research was to estimate whether a [99mTc]duramycin probe can be used for apoptosis imaging in patients with aortic aneurysm (AA). Vascular smooth muscle cell (SMC) apoptosis has an important influence on AA development. Thus, non-invasive imaging of SMC apoptosis may be able to evaluate AA progress and risk stratification. SMCs were treated with hydrogen peroxide (H2O2; 200 µΜ) or culture medium as a control. Apoptosis was measured using flow cytometry and [99mTc]duramycin to detect the binding efficiency to apoptotic SMCs. C57/BL6 mice were administered angiotensin-II and beta-aminopropionitrile (BAPN) subcutaneously to establish an AA model, or saline for controls. Aortic specimens underwent pathological evaluation and their aortic diameters were measured after 6 weeks. Micro-SPECT/CT scanning of [99mTc]duramycin and 18F-FDG PET detection were performed. SMCs treated with H2O2 showed more apoptosis compared with the control group (67.2 ± 3.8% vs. 16.1 ± 0.6%, P < 0.01). The experimental group showed a high rate of AA formation (70%) compared with no AA formation in the control group. The average aorta diameter was higher and [99mTc]duramycin uptake at the AA site was higher in the experimental group compared with the control group. Compared with the normal aorta in the control group, AA in experiment group had more severe medial degeneration, elastic fiber reduction and fracture, and collagen degeneration. TUNEL staining verified the higher apoptosis rate at the AA site in experiment group compared with the control group (63.9 ± 3.7% in ascending AA, 66.4 ± 4.0% in thoracic AA, vs. 3.5 ± 0.3% in normal aorta, P < 0.01). [99mTc]Duramycin may be an effective probe to evaluate apoptosis in AA.

Non-cardiac uptake of technetium-99m pyrophosphate in transthyretin cardiac amyloidosis.

BACKGROUND: Technetium-based bone scintigraphy is rapidly becoming the most common non-invasive imaging tool in the diagnosis of Transthyretin cardiac amyloidosis (ATTR). Skeletal muscle uptake has been described with technetium-99m-3,3-diphosphono-1,2-propanodicarboxylic acid (TcDPD), and may account for masking of bony uptake. We sought to investigate skeletal muscle uptake of technetium-99m-pyrophosphate (TcPYP) in patients with ATTR. METHODS AND RESULTS: This was a retrospective analysis of 57 patients diagnosed with ATTR who underwent TcPYP scintigraphy. Cardiac uptake was assessed on whole-body planar imaging using a semiquantitative scale (grades 0 to 3) and on single-photon emission computed tomography (SPECT) with CT attenuation correction using total myocardial counts per voxel after a 3-hour incubation. Skeletal muscle (psoas and biceps), vertebral body, LV myocardium, and blood pool mean counts were calculated. In the cohort (age 78 ± 9 years, 77% male, and 30% hereditary ATTR), there was no visualized tracer uptake in skeletal muscle or soft tissue on qualitative SPECT assessment. Total and blood pool-corrected uptake in the muscle groups were significantly less than myocardium and bone (P < 0.001). Blood pool-corrected muscle uptake was not associated with semiquantitative grade 3 vs 2 uptake (psoas P = 0.66, biceps P = 0.13) or presence of hereditary ATTR (psoas P = 0.43, biceps P = 0.69). As bony uptake decreased, there was no corresponding increase in skeletal muscle uptake. CONCLUSIONS: In patients with ATTR cardiac amyloidosis, skeletal muscle uptake of TcPYP is minimal when assessed by qualitative and quantitative metrics, and is not significantly different in patients with grade 2 vs 3 semiquantitative uptake. The properties of this tracer may be different than TcDPD with respect to non-cardiac uptake.

Coordination-Mediated Synthesis of Purification-Free Bivalent 99mTc-Labeled Probes for in Vivo Imaging of Saturable System.

In the synthesis of technetium-99m (99mTc) labeled target-specific ligands, the presence of a large excess of unlabeled ligands over 99mTc in the injectate hinders target accumulation of 99mTc-labeled ligands by competing for target molecules. To circumvent the problem, we recently developed a concept of the metal coordination-mediated multivalency, and proved the concept with a 99mTc-labeled trivalent compound [99mTc(CO)3(CN-RGD)3]+. In this study, D-penicillamine (Pen) was selected as a chelating molecule and a cyclic RGDfK peptide was conjugated to Pen via a hexanoic linkage (Pen-Ahx-c(RGDfK)). 99mTc complexation reaction, and the stability, integrin αvß3 binding affinity, and biodistribution of the 99mTc-labeled probe were investigated to evaluate the applicability of the concept to bivalent probes. 99mTc-[Pen-Ahx-c(RGDfK)]2 was obtained over 95% radiochemical yields under low Pen-Ahx-c(RGDfK) concentration (50 µM). 99mTc-[Pen-Ahx-c(RGDfK)]2 showed approximately 10-times higher integrin αvß3 binding affinity than the monovalent compounds, Pen-Ahx-c(RGDfK) and c(RGDyV). In biodistribution studies, the tumor accumulation of 99mTc-[Pen-Ahx-c(RGDfK)]2 was decreased to 77% and 43% of HPLC-purified (Pen-Ahx-c(RGDfK)-free) 99mTc-[Pen-Ahx-c(RGDfK)]2 by the presence of 5 nmol of unlabeled Pen-Ahx-c(RGDfK) and Re-[Pen-Ahx-c(RGDfK)]2, respectively. 99mTc-[Pen-Ahx-c(RGDfK)]2 provided tumor image without removing unlabeled ligand, while a 99mTc-labeled monovalent probe prepared from a monovalent ligand could not. These findings indicate the availability of the design concept to prepare 99mTc-labeled bivalent probes with a variety of 99mTc core and other metallic radionuclides of clinical relevance.

A new method for studying cryptococcosis in a murine model using 99mTc-Cryptococcus gattii.

Cryptococcus gattii is one of the etiologic agents of cryptococcosis, a systemic mycosis that occurs in healthy and immunosuppressed humans and animals worldwide. Primary pulmonary infection caused by C. gattii is usually followed by fungal dissemination to the central nervous system, resulting in high mortality rates. In this context, animal models of cryptococcosis are useful in the study of fungal pathogenesis and host response against the pathogen, and for testing novel therapeutic options. The most frequently applied method to study fungal dissemination from the lungs to other organs is by culturing tissues, which is not accurate for the detection and quantification of fungal load at early stages of the infection. To overcome this problem, the purpose of this study was to develop a new method for the quantification of Cryptococcus dissemination. One C. gattii strain was efficiently radiolabeled with technetium-99m (99mTc), without affecting viability of the cells. Further, the 99mTc-C. gattii (111 MBq) strain was used to infect mice by intratracheal and intravenous route for biodistribution studies. 99mTc-C. gattii was successfully used in detection of the yeast in the brain of mice 6 hours postinoculation, while the detection using colony forming units was possible only 24 hours postinfection. Our results provided an alternative method that could be applied in further investigations regarding the efficacy of antifungals, fungal virulence, and host-pathogen interactions.

Evaluation of 99mTc-sulfadiazine as Bacillus microorganisms infection imaging agent using animal model.

Bacterial infection is one of the vital sources of morbidity and mortality. The development of single photon emission computed tomography (SPECT) radiotracer agents using antibiotics, for targeting in-vivo bacteria, helps in antibiotic dose calibration, targeted infection therapy and reduction in mortality rate. The aim of this study was to appraised 99mTc-labeling sulfadiazine as a radiopharmaceutical for bacillus infections imaging. Radiolabeling of sulfadiazine with technetium-99m was carried out by subsequent addition of 1.5 mL aqueous solution of sulfadiazine (1mg/mL), 120µg stannous tartrate, gentistic acid as stabilizing agent and 185 MBq normal saline solution of 99mTcO4-1 (pertechnetate) at pH = 5. The reaction mixture was incubated for 40 min at room temperature with light stirring. The quality control analysis (ITLC-SG and paper chromatography analysis) revealed ~ 98% labeling yield. Biodistribution and scintigraphic study was carried using bacillus bacterial infection induced New Zealand white rabbits. Due to the ease of 99mTc-sulfadiazine conjugation method, high labeling efficiency, shelf stability (>95% up to 6h), blood serum stability (~90% up to 6h) and high uptake in the infected muscle (T/NT =2.21 at 1H), 99mTc-SDZ could be used as radiopharmaceutical of choice for further pre-clinical and clinical studies.

Molecular imaging of fibrosis using a novel collagen-binding peptide labelled with 99mTc on SPECT/CT.

Fibrosis, closely related to chronic various diseases, is a pathological process characterised by the accumulation of collagen (largely collagen type I). Non-invasive methods are necessary for the diagnosis and follow-up of fibrosis. This study aimed to develop a collagen-targeted probe for the molecular imaging of fibrosis. We identified CPKESCNLFVLKD (CBP1495) as an original collagen-binding peptide using isothermal titration calorimetry and enzyme-linked immunosorbent assay. CBP1495 effectively bound to collagen type I (K d = 861 nM) and (GPO)9 (K d = 633 nM), a collagen mimetic peptide. Western blot and histochemistry validated CBP1495 targeting collagen in vitro and ex vivo. (Gly-(D)-Ala-Gly-Gly) was introduced to CBP1495 for coupling 99mTc. Labelling efficiency of 99mTc-CBP1495 was 95.06 ± 1.08 %. The physico-chemical properties, tracer kinetics and biodistribution of 99mTc-CBP1495 were carried out, and showed that the peptide stably chelated 99mTc in vitro and in vivo. SPECT/CT imaging with 99mTc-CBP1495 was performed in rat fibrosis models, and revealed that 99mTc-CBP1495 significantly accumulated in fibrotic lungs or livers of rats. Finally, 99mTc-CBP1495 uptake and hydroxyproline (Hyp), a specific amino acid of collagen, were quantitatively analysed. The results demonstrated that 99mTc-CBP1495 uptake was positvely correlated with Hyp content in lungs (P < 0.0001, r 2 = 0.8266) or livers (P < 0.0001, r 2 = 0.7581). Therefore, CBP1495 is a novel collagen-binding peptide, and 99mTc-labelled CBP1495 may be a promising radiotracer for the molecular imaging of fibrosis.

SPECT-CT Comparison of Lung Deposition using a System combining a Vibrating-mesh Nebulizer with a Valved Holding Chamber and a Conventional Jet Nebulizer: a Randomized Cross-over Study.

PURPOSE: To compare in vivo the total and regional pulmonary deposition of aerosol particles generated by a new system combining a vibrating-mesh nebulizer with a specific valved holding chamber and constant-output jet nebulizer connected to a corrugated tube. METHODS: Cross-over study comparing aerosol delivery to the lungs using two nebulizers in 6 healthy male subjects: a vibrating-mesh nebulizer combined with a valved holding chamber (Aerogen Ultra®, Aerogen Ltd., Galway, Ireland) and a jet nebulizer connected to a corrugated tube (Opti-Mist Plus Nebulizer®, ConvaTec, Bridgewater, NJ). Nebulizers were filled with diethylenetriaminepentaacetic acid labelled with technetium-99 m (99mTc-DTPA, 2 mCi/4 mL). Pulmonary deposition of 99mTc-DTPA was measured by single-photon emission computed tomography combined with a low dose CT-scan (SPECT-CT). RESULTS: Pulmonary aerosol deposition from SPECT-CT analysis was six times increased with the vibrating-mesh nebulizer as compared to the jet nebulizer (34.1 ± 6.0% versus 5.2 ± 1.1%, p < 0.001). However, aerosol penetration expressed as the three-dimensional normalized ratio of the outer and the inner regions of the lungs was similar between both nebulizers. CONCLUSIONS: This study demonstrated the high superiority of the new system combining a vibrating-mesh nebulizer with a valved holding chamber to deliver nebulized particles into the lungs as comparted to a constant-output jet nebulizer with a corrugated tube.

Single-Photon Emission Computed Tomography/Computed Tomography Imaging in a Rabbit Model of Emphysema Reveals Ongoing Apoptosis In Vivo.

Evaluation of lung disease is limited by the inability to visualize ongoing pathological processes. Molecular imaging that targets cellular processes related to disease pathogenesis has the potential to assess disease activity over time to allow intervention before lung destruction. Because apoptosis is a critical component of lung damage in emphysema, a functional imaging approach was taken to determine if targeting apoptosis in a smoke exposure model would allow the quantification of early lung damage in vivo. Rabbits were exposed to cigarette smoke for 4 or 16 weeks and underwent single-photon emission computed tomography/computed tomography scanning using technetium-99m-rhAnnexin V-128. Imaging results were correlated with ex vivo tissue analysis to validate the presence of lung destruction and apoptosis. Lung computed tomography scans of long-term smoke-exposed rabbits exhibit anatomical similarities to human emphysema, with increased lung volumes compared with controls. Morphometry on lung tissue confirmed increased mean linear intercept and destructive index at 16 weeks of smoke exposure and compliance measurements documented physiological changes of emphysema. Tissue and lavage analysis displayed the hallmarks of smoke exposure, including increased tissue cellularity and protease activity. Technetium-99m-rhAnnexin V-128 single-photon emission computed tomography signal was increased after smoke exposure at 4 and 16 weeks, with confirmation of increased apoptosis through terminal deoxynucleotidyl transferase dUTP nick end labeling staining and increased tissue neutral sphingomyelinase activity in the tissue. These studies not only describe a novel emphysema model for use with future therapeutic applications, but, most importantly, also characterize a promising imaging modality that identifies ongoing destructive cellular processes within the lung.

Evaluation of GX1 and RGD-GX1 peptides as new radiotracers for angiogenesis evaluation in experimental glioma models.

Gliomas are the most common type among all central nervous system tumors. The aggressiveness of gliomas is correlated with the level of angiogenesis and is often associated with prognosis. The aim of this study is to evaluate the novel GX1 peptide and the heterodimer RGD-GX1 radiolabeled with technetium-99m, for angiogenesis detection in glioma models. Radiolabeling and radiochemical controls were assessed for both radioconjugates. In vitro binding studies in glioma tumor cells were performed, as well as biodistribution in SCID mice bearing tumor cells, in order to evaluate the biological behavior and tumor uptake of the radiocomplexes. Blocking and imaging studies were also conducted. MicroSPECT/CT images were acquired in animals with experimentally implanted intracranial tumor. Open field activity was performed to evaluate behavior, as well as perfusion and histology analysis. The radiochemical purity of both radiotracers was greater than 96 %. In vitro binding studies revealed rather similar binding profi le for each molecule. The highest binding was for RGD-GX1 peptide at 120 min in U87MG cells (1.14 ± 0.35 %). Tumor uptake was also favorable for RGD-GX1 peptide in U87MG cells, reaching 2.96 ± 0.70 % at 1 h p.i. with 47 % of blocking. Imaging studies also indicated better visualization for RGD-GX1 peptide in U87MG cells. Behavior evaluation pointed brain damage and histology studies confirmed actual tumor in the uptake site. The results with the angiogenesis seeking molecule (99m)Tc-HYNIC-E-[c(RGDfk)-c(GX1)] were successful, and better than with (99m)Tc-HYNIC-PEG4-c(GX1). Future studies targeting angiogenesis in other glioma and nonglioma tumor models are recommended.

Development and Pharmacological Evaluation of New Bone-Targeted (99m)Tc-Radiolabeled Bisphosphonates.

A novel bisphosphonate, 1-(3-aminopropylamino)ethane-1,1-diyldiphosphonic acid (3), was coupled to the tridentate chelators di-2-picolylamine, 2-picolylamine-N-acetic acid, iminodiacetic acid, 3-((2-aminoethyl)thio)-3-(1H-imidazol-4-yl)propanoic acid, and 2-((2-carboxyethyl)thio)-3-(1H-imidazol-4-yl)propanoic acid to form ligands 6, 9, 11, 15, and 19, respectively. Organometallic complexes of the general formula [Re/(99m)Tc(CO)3(κ(3)-L)] were synthesized, where L denotes ligand 6, 9, 11, 15, or 19. The rhenium complexes were prepared at the macroscopic level and characterized by spectroscopic methods. The technetium-99m organometallic complexes were synthesized in high yield and were identified by comparative reversed-phase HPLC with their Re analogues. The (99m)Tc tracers were stable in vitro and exhibited binding to hydroxyapatite. In biodistribution studies, all of the (99m)Tc complexes exhibited high bone uptake superior to that of 25, which is the directly (99m)Tc-labeled bisphosphonate 3, and comparable to that of (99m)Tc-methylene diphosphonate ((99m)Tc-MDP). The tracers [(99m)Tc(CO)3(6)] (26), [(99m)Tc(CO)3(9)] (27), [(99m)Tc(CO)3(11)] (28), and [(99m)Tc(CO)3(15)] (29) exhibited higher bone/blood ratios than (99m)Tc-MDP. 26 had the highest bone uptake at 1 h p.i. The new bisphosphonates showed no substantial growth inhibitory capacity in PC-3, Saos-2, and MCF-7 established cancer cell lines at low concentrations. Incubation of 26 with the same cancer cell lines indicated a rapid and saturated uptake. The promising properties of 26-29 indicate their potential for use as bone-imaging agents.

The Use of Technetium-99m for Intravital Tracing of Transplanted Multipotent Stromal Cells.

We studied the possibility of in vivo tracing of multipotent mesenchymal stromal cells labeled with a radiophermaceutic preparation based on metastable isotope Technetium-99m and injected to rats with modeled traumatic brain injury. Accumulation of labeled cells occurred primarily in the liver and lungs. The cells distribution in internal organs greatly varied depending on the administration route. Cell injection into the carotid artery led to their significant accumulation in the damaged brain hemisphere, while intravenous injection was followed by diffuse cell distribution in all brain structures. Scintigraphy data were confirmed by magnetic resonance imaging and histological staining of cells. Visualization of stem cells labeled with Technetium-99m-based preparation by scintigraphy is an objective and highly informative method allowing real-time in vivo cell tracing in the body.

Tc-99m-labeled RGD-conjugated alpha-melanocyte stimulating hormone hybrid peptides with reduced renal uptake.

The purpose of this study was to examine whether the replacement of the positively-charged Lys or Arg linker with a neutral linker could reduce the renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), D-Phe(7), Arg(11)]α-MSH3-13 {(Arg(11))CCMSH} through the neutral ßAla or Ahx {aminohexanoic acid} linker (replacing the Lys or Arg linker) to generate novel RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH hybrid peptides. The receptor-binding affinity and cytotoxicity of RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH were determined in B16/F1 melanoma cells. The melanoma targeting and imaging properties of (99m)Tc-RGD-ßAla-(Arg(11))CCMSH and (99m)Tc-RGD-Ahx-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The replacement of the Lys or Arg linker with the ßAla or Ahx linker retained nanomolar receptor-binding affinities and remarkable cytotoxicity of RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH. The receptor-binding affinities of RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH were 0.8 ± 0.05 and 1.3 ± 0.1 nM. Three-hour incubation with 0.1 µM of RGD-ßAla-(Arg(11))CCMSH and RGD-Ahx-(Arg(11))CCMSH decreased the survival percentages of B16/F1 cells by 71 and 67 % as compared to the untreated control cells 5 days post the treatment. The replacement of the Arg linker with the ßAla or Ahx linker reduced the non-specific renal uptake of (99m)Tc-RGD-ßAla-(Arg(11))CCMSH and (99m)Tc-RGD-Ahx-(Arg(11))CCMSH by 62 and 61 % at 2 h post-injection. (99m)Tc-RGD-ßAla-(Arg(11))CCMSH displayed higher melanoma uptake than (99m)Tc-RGD-Ahx-(Arg(11))CCMSH at 0.5, 2, 4, and 24 h post-injection. Enhanced tumor to kidney uptake ratio of (99m)Tc-RGD-ßAla-(Arg(11))CCMSH warranted the further evaluation of (188)Re-labeled RGD-ßAla-(Arg(11))CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.

Effects of rehabilitation management on gastric emptying function in older adults with diabetes.

The relationship between gastric emptying dysfunction and blood glucose concentration in elderly with type 2 diabetes mellitus was investigated, and the effect of rehabilitation exercise prescription training on gastric emptying in the geriatric diabetic patients was evaluated. A total of 160 older type 2 diabetic adults and 30 cases of non-diabetic patients were studied with regard to the gastric half emptying time (GET1/2) of solid meals radiolabelled with 99mTc. Eighty delayed gastric emptying diabetic patients were randomly divided into 4 four groups: rehabilitation exercise + mosapride group (N = 20), rehabilitation exercise group (N = 20), mosapride group (N = 20), and control group (N = 20). The level of blood glucose was measured every six months in a two-year follow-up. The solid GET1/2 of regulated blood glycemic control patients showed no statistically significant differences from non-diabetic patients (P > 0.05). However, the value for poor blood glycemic control patients exhibited significant statistical differences compared with both non-diabetic (P < 0.01) and regulated blood glycemic control group patients (P < 0.01). It showed that the gastric emptying time improved in the rehabilitation exercise group, mosapride group and rehabilitation exercise group + mosapride group after two years of treatment (P < 0.05). Fasting blood glucose in both rehabilitation exercise group and rehabilitation exercise + mosapride group was significantly decreased. Postprandial blood glucose in the rehabilitation exercise group, mosapride group, rehabilitation exercise group + mosapride group was significantly decreased. High blood glucose level can delay gastric emptying in older type 2 diabetic patients. Gastric emptying and blood glucose control affect each other. It was shown that appropriate rehabilitation exercise combined with prokinetic agent may improve gastric emptying in some geriatric type 2 diabetic patients and help control their blood glucose.

[Study of heart region of interest setting method in the hepatic functional reserve index of (99m)tc-diethylenetriamine pentaacetic Acid-galactosyl human serum albumin].

In this study we analyzed the influence of region of interest (ROI) selection on the uptake ratio of the liver to the liver plus heart at 15 min (LHL15) during (99m)Tc-galactosyl human serum albumin (GSA) scintigraphy and determined the optimal ROI by evaluating the individual effects of different ROIs in the heart on LHL15. Twenty patients were randomly selected from those who had undergone (99m)Tc-GSA scintigraphy GSA between April 2008 and June 2009. The liver body (L/B) ratio, liver uptake 15 min (LU15), and LHL15 were analyzed and compared among the following ROIs: entire heart, both ventricles, right ventricle, and left ventricle. There were significant differences in the L/B ratio and LU15 values among the different ROIs. However, LHL15 showed a tendency to shift toward a normal value when the size of the ROI was small (only the right or left ventricle), resulting in a lack of distinction between normal and abnormal LHL15 values. Furthermore, setting the entire heart as the ROI was difficult and reproducibility was low. Our results suggest that the use of both ventricles as the ROI provides optimal LHL15 values during (99m)Tc-GSA scintigraphy.