Radiolabeling of Preformed Niosomes with [99mTc]: In Vitro Stability, Biodistribution, and In Vivo Performance.

Nanocarriers radiolabeled with [99mTc] can be used for diagnostic imaging and radionuclide therapy, as well as tracking their pharmacokinetic and biodistribution characteristics. Due to the advantages of niosomes as an ideal drug delivery system, in this study, the radiolabeling procedure of niosomes by [99mTc]-HMPAO complexes was investigated and optimized. Glutathione (GSH)-loaded niosomes were prepared using a thin-film hydration method. To label the niosomes with [99mTc], the preformed GSH-loaded niosomes were incubated with the [99mTc]-HMPAO complex and were characterized for particle size, size distribution, zeta potential, morphology, and radiolabeling efficiency (RE). The effects of GSH concentration, incubation time, incubation temperature, and niosomal composition on RE were investigated. The biodistribution profile and in vivo SPECT/CT imaging of the niosomes and free [99mTc]-HMPAO were also studied. Based on the results, all vesicles had nano-sized structure (160-235 nm) and negative surface charge. Among the different experimental conditions that were tested, including various incubation times, incubation temperatures, and GSH concentrations, the optimum condition that resulted in a RE of 92% was 200-mM GSH and 15-min incubation at 40°C. The in vitro release study in plasma showed that about 20% of radioactivity was released after 24 h, indicating an acceptable radiolabeling stability in plasma. The biodistribution of niosomes was clearly different from the free radiolabel. Niosomes carrying radionuclide were successfully used for tracking the in vivo disposition of these carriers and SPECT/CT imaging in rats. Furthermore, biodistribution studies in tumor-bearing mice revealed higher tumor accumulation of the niosomal formulation as compared with [99mTc]-HMPAO.

Influence of radiolytic-produced hydrogen peroxide on the stability of commercial kit (99m) technetium-exametazime preparation.

99mTc-d,l-hexamethylpropylene amine oxime ((99m) Tc-d,l-HMPAO) is a widely used radiopharmaceutical that suffers from an inherent instability with a shelf life of 30 min that constrains its availability for clinical use. A protocol for improving the stability of the kit with minimal modification of manufacturer's instructions and no chemicals addition to the commercial formulation is proposed. The protocol is based on the displacement of the oxygen present in the preparation, preventing free radicals build up and free pertechnetate formation. Although the degradation of (99m) Tc-d,l-HMPAO cannot be explained solely by the radiolytic production of free radicals, it appears to be an important factor in the shelf stability of the complex.

Synthesis, radiolabeling and biological evaluation of propylene amine oxime complexes containing nitrotriazoles as hypoxia markers.

Two propylene amine oxime (PnAO) complexes, 1, containing a 3-nitro-1,2,4-triazole and 2, containing two 3-nitro-1,2,4-triazoles, were synthesized and radiolabeled with (99m)Tc in high labeling yields. Cellular uptakes of (99m)Tc-1 and (99m)Tc-2 were tested using a S180 cells line. Under anoxic conditions, the cellular uptakes of (99m)Tc-1 and (99m)Tc-2 were 33.7 ± 0.2% and 35.0 ± 0.7% at 4 h, whereas the normoxic uptakes of the two complexes were 6.0 ± 1.6% and 4.6 ± 0.9%, respectively. Both (99m)Tc-1 and (99m)Tc-2 displayed significant anoxic/normoxic differentials. The cellular uptakes were highly dependent on oxygen and temperature. Biodistribution studies revealed that both (99m)Tc-1 and (99m)Tc-2 showed a selective localization in tumor and slow clearance from it. At 4 h, the tumor-to-muscle ratios (T/M) were 3.79 for (99m)Tc-1 and 4.58 for (99m)Tc-2. These results suggested that (99m)Tc-labeled PnAO complexes (99m)Tc-1 and (99m)Tc-2 might serve as novel hypoxia markers. By introducing a second nitrotriazole redox center, the hypoxic accumulation of the marker was slightly enhanced.

Labelling and Clinical Performance of Human Leukocytes Labelled with 99mTc-HMPAO Using Leukokit® with Gelofusine versus Leukokit® with HES as Sedimentation Agent.

The scintigraphy with radiolabelled autologous leukocytes (WBCs) is considered the gold-standard technique for imaging infections. Leukokit® is a commercially available, disposable, sterile kit for labelling WBCs ex vivo. In this kit, WBCs isolation from red blood cells (RBCs) was performed using poly(O-2-hydroxyethyl)starch (HES) as the RBCs sedimentation agent. Due to its poor availability, HES has been recently replaced by Gelofusine as the RBC sedimentation agent. The aim of this study was to compare the labelling efficiency and the diagnostic accuracy of WBCs labelled with Leukokit® with HES vs Leukokit® with Gelofusine. WBCs were isolated using HES or Gelofusine for 45 minutes and then purified from platelets (PLTs) and labelled with 1.1 ± 0.3 GBq of freshly prepared 99mTc-HMPAO. The following parameters were evaluated: the number and type of recovered WBCs, RBCs contamination, PLTs contamination, vitality of neutrophils, and chemotactic properties of neutrophils. Clinical comparison was performed between 80 patients (33 males; age 67.5 ± 14.2) injected with 99mTc-HMPAO-WBCs, using HES as the sedimentation agent, and 92 patients (38 males; age 68.2 ± 12.8) injected with 99mTc-HMPAO-WBCs using Gelofusine as the sedimentation agent. Patients were affected by prosthetic joint infections, peripheral bone osteomyelitis, or vascular graft infection. We compared radiolabelling efficiency (LE), final recovery yield (RY), and diagnostic outcome based on microbiology or 2-year follow-up. Results showed that HES provides the lowest RBCs and PLTs contamination, but Gelofusine provides the highest WBC recovery. Both agents did not influence the chemotactic properties of WBCs, and no differences were found in terms of LE and RY. Sensitivity, specificity, and accuracy were also not significantly different for WBCs labelled with both agents (diagnostic accuracy 90.9%, CI = 74.9-96.1 vs 98.3%, CI = 90.8-100, for HES and Gelofusine, respectively). In conclusion, Gelofusine can be considered a suitable alternative of HES for WBCs separation and labelling.

99mTc-HMPAO-labeled liposomes: an investigation into the effects of some formulation factors on labeling efficiency and in vitro stability.

Technetium-99m complex of hexamethyl-propylene-amineoxime (HMPAO) is used as an efficient agent to label liposomes. For this, 99mTc-HMPAO is incubated with preformed liposomes that contain glutathione (GSH). Effect of GSH and lipid concentration on labeling efficiency, as well as the effect of lipid composition on in vitro stability of labeled liposomes, was investigated in the present study. d,l-HMPAO was synthesized and kits including d,l-HMPAO and SnCl2.2H2O were optimized at 0.5 mg HMPAO, 5.0 microg SnCl2.2H2O and pH 7, and lyophilized. DSPC/CHOL (molar ratio 2:1) liposomes encapsulating GSH were labeled with 99mTc-HMPAO prepared kits. Increase of GSH concentration in hydration buffer from 5 to 200 mM during liposome preparation resulted in a broad labeling efficiency of liposomes ranging from 4.16% to 69.81%. An initial approximate concentration of 100 mM GSH in the hydration buffer seems to be appropriate for a good labeling efficiency. At the optimum concentration of GSH, change of the total initial lipid concentration from 10 to 70 mM did not produce a remarkable difference in labeling efficiency. Study of the effect of lipid composition on the stability of liposomes showed that all three kinds of labeled liposomes composed of DSPC/CHOL, DPPC/CHOL and DMPC/CHOL (molar ratio 2:1) had good in vitro stability in human plasma at 37 degrees C for 48 h; however, employing DSPC resulted in the most stable ones.

PLA-PEG nanocapsules radiolabeled with 99mTechnetium-HMPAO: release properties and physicochemical characterization by atomic force microscopy and photon correlation spectroscopy.

The present work describes the preparation, characterization and labelling of conventional and surface-modified nanocapsules (NC) with 99m Tc-HMPAO. The size, size distribution and homogeneity were determined by photon correlation spectroscopy (PCS) and zeta potential by laser doppler anemometry. The morphology and the structural organization were evaluated by atomic force microscopy (AFM). The stability and release profile of the NC were determined in vitro in plasma. The results showed that the use of methylene blue induces significant increase in the encapsulation efficiency of 99m Tc-HMPAO, from 24.4 to 49.8% in PLA NC and 22.37 to 52.93% in the case of PLA-PEG NC (P<0.05) by improving the complex stabilization. The average diameter of NC calculated by PCS varied from 216 to 323 nm, while the average diameter determined by AFM varied from 238 to 426 nm. The AFM analysis of diameter/height ratios suggested a greater homogeneity of the surface-modified PLA-PEG nanocapsules compared to PLA NC concerning their flattening properties. The in vitro release of the 99m Tc-HMPAO in plasma medium was faster for the conventional PLA NC than for the surface-modified NC. For the latter, 60% of the radioactivity remained associated with NC, even after 12h of incubation. The results suggest that the surface-modified 99m Tc-HMPAO-PLA-PEG NC was more stable against label leakage in the presence of proteins and could present better performance as radiotracer in vivo.

Biodistribution study and identification of inflammation sites using 99mTc-labelled stealth pH-sensitive liposomes.

PURPOSE: To investigate the biodistribution and the ability of stealth pH-sensitive liposomes radiolabelled with 99mTc to identify inflammatory regions in a rat focal inflammation model. METHODS: Preformed glutathione-containing stealth pH-sensitive liposomes were labelled with 99mTc-hexamethylpropylene amine oxime (99mTc-HMPAO). The 99mTc-HMPAO radiolabelled stealth pH-sensitive liposomes (99mTc-SpHL) were administered intravenously in Wistar male rats with inflammation induced by injection subplantar of carrageenan in the right foot. At pre-established time intervals the animals were anaesthetized and tissues were removed and analysed for 99mTc content using an automatic scintillation apparatus. Scintigraphic imaging was also performed after 2, 4 and 8 h of intravenous injection of 99mTc-SpHL. RESULTS: The 99mTc-SpHL was significantly taken up by the spleen (19.21+/-2.98%ID/g at 30 min post-injection). Low radioactivity levels were found in the liver, lungs, and kidney. Moreover, the 99mTc-SpHL uptake was significantly higher in the inflamed foot when compared to the respective control (0.386+/-0.059 and 0.215+/-0.018%ID/g at 2 h post-injection, respectively). As early as 30 min after administration of 99mTc-SpHL, the focus of inflammation could be visualized scintigraphically. The value of the inflammatory and non-inflammatory site radioactivity counting ratio was greater than 5. CONCLUSION: This result indicates that the 99mTc-SpHL presents a high tropism for inflammatory regions and may be useful as a radiopharmaceutical to identify these foci.

Stability of stabilized 99mTc-D,L-HMPAO stored in vials and syringes.

UNLABELLED: Our objective was to determine the stability of stabilized (99m)Tc-hexamethylpropylene amine oxime ((99m)Tc-d,l-HMPAO) dispensed by vial and syringe, with the storage time and labeling activity varied. METHODS: (99m)Tc-d,l-HMPAO was labeled according to the manufacturer's instructions, but with modification of the (99m)TcO(4)Na activity. Two groups were prepared: 1,110 MBq (30 mCi) and 2,600-3,700 MBq (70.3-100 mCi). Five minutes after labeling, the radiochemical purity (RCP) of the vial content was determined. Afterward, the same activity was distributed into two 2-mL syringes and into the manufacturer's vial. In one of the syringes, the radiopharmaceutical stayed in contact with the needle for 4 h. At 2 and 4 h after labeling, the RCP of the vial and syringe content was checked and compared. RESULTS: The mean RCP of stabilized (99m)Tc-d,l-HMPAO labeled with 1,110 MBq (30 mCi) and stored in a vial decreased from 93.1% at 5 min to 92.1% at 2 h and to 91.1% at 4 h. With storage in a syringe, the RCP decreased from 89.8% at 2 h to 88.7% at 4 h. This diminution increased for labeling with higher activities (2,600-3,700 MBq [70.3-100 mCi]), ranging from 91.4% at 5 min, 89.0% at 2 h, and 85.3% at 4 h in a vial and from 85.9% at 2 h to 80.2% in a syringe. (99m)TcO(2) and secondary (99m)Tc-HMPAO were the main impurities at t = 0. (99m)TcO(4)(-) was an impurity that increased with time in both vials and syringes but significantly so in syringes. All these impurities were higher with labeling activities in the range of 2,600-3,700 MBq (70.3-100 mCi). Contact of the needle with (99m)Tc-d,l-HMPAO sharply decreased the RCP to 57.1% at 4 h. CONCLUSION: The RCP of stabilized (99m)Tc-d,l-HMPAO decreases significantly in both vials and syringes with high labeling activities. The product is less stable when stored in a syringe than in a vial. The fraction of dose in contact with the needle affects the RCP results.

Leukocyte Imaging of the Diabetic Foot.

BACKGROUND: Diagnosing diabetic foot infection is often difficult, despite several available diagnostic methods. Amongst these, several imaging modalities exist to evaluate the diabetic foot in case of a suspected osteomyelitis. Nuclear Medicine, in particular, offers a variety of radiopharmaceuticals and techniques. Nowadays the gold standard radionuclide procedure, when an osteomyelitis is suspected, is represented by the use of radiolabelled leukocytes with either 99mTc-HMPAO or 111In-oxine. METHODS: In this review, we describe the correct acquisition and interpretation of white blood cell scintigraphy and we provide an overview of the existing literature data of the use of this technique in the infected diabetic foot. If images are correctly acquired, displayed and interpreted, this modality reaches very high diagnostic accuracy (>95%) in detecting osteomyelitis and it allows the differential diagnosis with a soft tissue infection or inflammation. Single-photon emission computed tomography/computed tomography (SPECT/CT) in addition to planar images is mandatory to determine the extent and exact location of the infective process in both fore foot and midhint foot. With the addition of bone marrow scintigraphy using radiolabelled nanocolloids, radiolabelled white blood cell scintigraphy is also able to differentiate between Charcot neuroarthropathy and osteomyelitis, which is a challenge in the evaluation of diabetic foot. Radiolabelled anti-granulocyte monoclonal antibodies and their fragments can also be used instead of white blood cells although there is a limited experience on their usefulness in diabetic foot infection.

Quantitation of rat cerebral blood flow using 99mTc-HMPAO.

INTRODUCTION: Technetium-99m-hexamethylpropyleneamine oxime (99mTc-HMPAO) is potentially useful for the assessment of cerebral blood flow (CBF) in small animals. In this paper, a procedure for quantitation of rat CBF using 99mTc-HMPAO was determined. METHODS: Biodistribution of 99mTc-radioactivity in normal rats was determined after intravenous administration of 99mTc-HMPAO. Acetazolamide treated rats were intravenously administered with the mixture of 99mTc-HMPAO and N-isopropyl-[125I]iodoamphetamine ([125I]IMP), and arterial blood was then collected for 5min. After blood sampling, the brain radioactivity concentration was measured with the auto-well γ counter. RESULTS: The brain radioactivity concentration after intravenous administration of 99mTc-HMPAO was steady from 14s to 60min post-injection. A double tracer experiment using 99mTc-HMPAO and [125I]IMP showed that 19s was the average of the optimal integration interval of arterial blood 99mTc-radioactivity concentration to obtain CBF values measured by 99mTc-HMPAO identical to those determined by [125I]IMP. The CBF value determined by 99mTc-HMPAO, calculated by dividing the brain radioactivity concentration at 5min post-injection by the integrated arterial blood radioactivity concentration until 19s post-injection, was well correlated with CBF as determined by [125I]IMP. CONCLUSION: These results suggest that the CBF quantitation procedure described in this paper could be useful for rat CBF assessment.

New synthesis route of active substance d,l-HMPAO for preparation Technetium Tc99m Exametazime.

BACKGROUND: Technetium Tc99m Exametazime (99mTc-HMPAO) is currently used as a radiopharmaceutical for determining regional cerebral blood flow and for the labelling of autologous leucocytes for infection and inflammation imaging. The HMPAO ligand exists in two diastereomeric forms: d,l and meso. Usually, the substance is obtained in low chemical yield in a time consuming procedure. Furthermore, the final product still contains some amounts of the meso-form. The aim of this study was to develop the efficient, reliable and fast method for isolation of the d,l-HMPAO, which would provide the ligand with high purity and free from the meso-diastereomer. MATERIAL AND METHODS: The mixture of the meso- and d,l-HMPAO was synthesized in two-steps by condensation of propanediamine with keto-oxime and the reduction of the obtained bisimine. The d- and l-enantiomers were separated individually directly from this mixture by repeated crystallizations from ethanol as their tartrate salts and pooled together in equal proportions. That substance was characterized for its identity and isomeric purity using IR, HPLC and GC methods. The meso-free d,l-HMPAO was used for the preparation of the radiopharmaceutical freeze-dried kit for technetium-99m radiolabelling. Quality assessment of obtained 99mTc-d,l-HMPAO complex was performed according to the current Ph.Eur. monograph 1925 and USP monograph - Technetium Tc99m Exametazime Injection. To verify its biological activity, the kit-prepared 99mTc-d,l-HMPAO has been used for the white blood cell (WBC) labelling. RESULTS: According to the proposed synthesis route the d,l-HMPAO was obtained with around 18-20% yield in the total time of 10 days. The ligand identity was confirmed and the HPLC analysis revealed more than 99% chemical purity. The undesired meso-form was not detected. Freeze dried kit formulation for 99mTc-labelling of d,l-HMPAO has been established and four batches of kits were manufactured. The radiochemical purity of 99mTc-d,l-HMPAO complex was high (> 95% of lipophilic technetium-99m exametazime). Brain uptake in rats reached 2.1 ± 0.3%. The in vitro labelling of WBC resulted in 68.3 ± 6.6% yield. CONCLUSION: A new synthesis method of d,l-HMPAO, drug substance for technetium-99m exametazime preparation has been developed.

Miniaturized Radiochemical Purity Testing for 99mTc-HMPAO, 99mTc-HMDP, and 99mTc-Tetrofosmin.

Quick methods are functional in clinical practice to ensure the fastest availability of radiopharmaceuticals. For this purpose, we investigated the radiochemical purity of the widely used 99mTc-hydroxymethylene diphosphonate, 99mTc-hexamethylpropyleneamine oxime, and 99mTc-tetrofosmin by reducing time as compared with the manufacturer's method. Methods: We applied a miniaturized chromatographic method with a reduced strip development from 18 cm to 9 cm for all 3 radiopharmaceuticals. The specific support medium and solvent system of the manufacturer's methods was kept unchanged for 99mTc-hydroxymethylene diphosphonate and 99mTc-tetrofosmin, whereas for 99mTc-hexamethylpropyleneamine oxime the instant thin-layer chromatography (ITLC) polysilicic gel (silicic acid [SA]) was replaced with a monosilicic gel (silicic gel [SG]) in the chromatographic system that uses methyl ethyl ketone as solvent. The method was applied and compared with the routine ITLC insert method in a total of 30 batches for each radiopharmaceutical. The precision of repeated tests was determined by comparison with the results of 10 replications on the same batch. Small volumes of concentrated 99mTcO4-, and 99mTc-albumin nanocolloid were used to produce potential radiochemical impurities. Correlation between the quick methods and the insert methods was analyzed using a nonparametric 2-tailed test and a 2 × 2 contingency table with the associated Fisher exact test to evaluate sensitivity and specificity. A receiver-operating-characteristic analysis was performed to evaluate the best cutoff. Results: The percentage radiochemical purity of the quick methods agreed with the standard chromatography procedures. We found that 99mTcO4 and colloidal impurities are not the only common radiochemical impurities with 99mTc-tetrofosmin, and shortening of the ITLC strip with respect to the manufacturer's method will worsen system resolution and may produce inaccuracy. Conclusion: The miniaturized methods we described represent a fast and reliable alternative for 99mTc-exametazime and 99mTc-oxidronate quality control, with the upper cutoff for acceptable radiochemical purity values being 84% and 95%, respectively. For 99mTc-tetrofosmin radiochemical purity testing, a longer strip as described in the standard method is warranted.

Hyperthermia increases accumulation of technetium-99m-labeled liposomes in feline sarcomas.

The effect of hyperthermia on the accumulation of technetium-99m-labeled liposomes was studied in feline sarcomas. Each cat received two separate injections of liposomes. The first was used to quantify the amount of technetium-99m-labeled liposomes within the tumor under normothermic conditions. The second injection was made at the beginning of a 60-min hyperthermia procedure. Planar scintigraphy was used to measure the activity of technetium-99m-labeled liposomes within the tumor at predetermined times up to 18 h after injection. Regions of interest were drawn for the tumor, lungs, liver, kidney, and aorta. Counts in the regions of interest were decay corrected. Counts/pixel in the tumor under normothermic and hyperthermic conditions were normalized to aorta counts/pixel. A total of 16 cats were eligible for the study. In two of the 16 cats, incomplete count data precluded analysis. In the remaining 14 cats, hyperthermia resulted in a significant increase in liposome accumulation in the tumor (P = 0.001). Tumor volume ranged from 1.2 to 236.2 cm3, and thermal dose ranged from 2.0 to 243.3 CEM43CT90 (equivalent time that the 10th percentile temperature was equal to 43 degrees C). There was not a relationship between either tumor volume or hyperthermia dose on the magnitude of increased liposome accumulation, suggesting that this method has application across a range of tumor volumes and degrees of heatibility.

Succinylated gelatine: an alternative to hydroxyethyl starch for labelling leukocytes with 99mTc-HMPAO.

BACKGROUND: Hydroxyethyl starch (HES) is the most used plasma expander in the sedimentation of the erythrocytes during the radiolabelling procedure for leukocytes in vitro. AIM: To evaluate the usefulness of succinylated gelatine (GEL), another colloidal plasma expander, as an alternative to HES in this process. METHODS: Two identical blood samples were obtained from 30 patients referred to white blood cell scintigraphy. The first sample was used to label leukocytes with Tc-HMPAO using the routine procedure, with HES. The other sample was used to label leukocytes with Tc-HMPAO using the same procedure, with GEL. The cell concentration of the leukocyte-platelet-rich plasma (LPRP) achieved after blood sedimentation was analysed. Labelling efficiency was calculated and the eosin Y viability and red cell/leukocyte ratio were evaluated from the final labelled cell suspension. RESULTS: Leukocytes and platelets recovered in LPRP were not statistically different between both HES and GEL samples (leukocytes: 8.10x10/microl+/-3.82 and 7.80x10/microl+/-3.47; platelets: 411x10/microl+/-182 and 406x10/microl+/-172, respectively). There were no significant differences between both agents on the labelling efficiency (HES: 80.3%+/-6.6%; GEL: 80.1%+/-6.3%), the eosin Y viability (HES: 99.2%+/-1.3%; GEL: 99.3%+/-1.1%) and the red cell/leukocyte ratio (HES: 1.21+/-0.7; GEL: 0.9+/-0.5). CONCLUSION: These results show that succinylated gelatine can be used instead of hydroxyethyl starch in the labelling of leukocytes with Tc-HMPAO.

Noninvasive imaging of radiolabeled exosome-mimetic nanovesicle using (99m)Tc-HMPAO.

Exosomes known as nano-sized extracellular vesicles attracted recent interests due to their potential usefulness in drug delivery. Amid remarkable advances in biomedical applications of exosomes, it is crucial to understand in vivo distribution and behavior of exosomes. Here, we developed a simple method for radiolabeling of macrophage-derived exosome-mimetic nanovesicles (ENVs) with (99m)Tc-HMPAO under physiologic conditions and monitored in vivo distribution of (99m)Tc-HMPAO-ENVs using SPECT/CT in living mice. ENVs were produced from the mouse RAW264.7 macrophage cell line and labeled with (99m)Tc-HMPAO for 1 hr incubation, followed by removal of free (99m)Tc-HMPAO. SPECT/CT images were serially acquired after intravenous injection to BALB/c mouse. When ENVs were labeled with (99m)Tc-HMPAO, the radiochemical purity of (99m)Tc-HMPAO-ENVs was higher than 90% and the expression of exosome specific protein (CD63) did not change in (99m)Tc-HMPAO-ENVs. (99m)Tc-HMPAO-ENVs showed high serum stability (90%) which was similar to that in phosphate buffered saline until 5 hr. SPECT/CT images of the mice injected with (99m)Tc-HMPAO-ENVs exhibited higher uptake in liver and no uptake in brain, whereas mice injected with (99m)Tc-HMPAO showed high brain uptake until 5 hr. Our noninvasive imaging of radiolabeled-ENVs promises better understanding of the in vivo behavior of exosomes for upcoming biomedical application.

Improving Cerebral Blood Flow Through Liposomal Delivery of Angiogenic Peptides: Potential of ¹8F-FDG PET Imaging in Ischemic Stroke Treatment.

UNLABELLED: Strategies to promote angiogenesis can benefit cerebral ischemia. We determined whether liposomal delivery of angiogenic peptides with a known biologic activity of vascular endothelial growth factor benefitted cerebral ischemia. Also, the study examined the potential of (18)F-FDG PET imaging in ischemic stroke treatment. METHODS: Male Sprague-Dawley rats (n = 40) underwent 40 min of middle cerebral artery occlusion. After 15 min of reperfusion, the rats (n = 10) received angiogenic peptides incorporated into liposomes. Animals receiving phosphate-buffered solution or liposomes without peptides served as controls. One week later, (18)F-FDG PET imaging was performed to examine regional changes in glucose utilization in response to the angiogenic therapy. The following day, (99m)Tc-hexamethylpropyleneamine oxime autoradiography was performed to determine changes in cerebral perfusion after angiogenic therapy. Corresponding changes in angiogenic markers, including von Willebrand factor and angiopoietin-1 and -2, were determined by immunostaining and polymerase chain reaction analysis, respectively. RESULTS: A 40-min period of middle cerebral artery occlusion decreased blood perfusion in the ipsilateral ischemic cortex of the brain, compared with that in the contralateral cortex, as measured by (99m)Tc-hexamethylpropyleneamine oxime autoradiography. Liposomal delivery of angiogenic peptides to the ischemic hemisphere of the brain attenuated the cerebral perfusion defect compared with controls. Similarly, vascular density evidenced by von Willebrand factor-positive staining was increased in response to angiogenic therapy, compared with that of controls. This increase was accompanied by an early increase in angiopoietin-2 expression, a gene participating in angiogenesis. (18)F-FDG PET imaging measured at 7 d after treatment revealed that liposomal delivery of angiogenic peptides facilitated glucose utilization in the ipsilateral ischemic cortex of the brain, compared with that in the controls. Furthermore, the change in regional glucose utilization was correlated with the extent of improvement in cerebral perfusion (r = 0.742, P = 0.035). CONCLUSION: Liposomal delivery of angiogenic peptides benefits cerebral ischemia. (18)F-FDG PET imaging holds promise as an indicator of the effectiveness of angiogenic therapy in cerebral ischemia.

Glutathione-mediated metabolism of technetium-99m SNS/S mixed ligand complexes: a proposed mechanism of brain retention.

Two series of [99mTc](SNS/S) mixed ligand complexes each carrying the N-diethylaminoethyl or the N-ethyl-substituted bis(2-mercaptoethyl)amine ligand (SNS) are produced at tracer level using tin chloride as reductant and glucoheptonate as transfer ligand. The identity of [99mTc](SNS/S) complexes is established by high-performance liquid chromatographic (HPLC) comparison with authentic rhenium samples. The para substituent R on the phenylthiolate coligand (S) ranges from electron-donating (-NH2) to electron-withdrawing (-NO2) groups, to study complex stability against nucleophiles as a result of N- and R-substitution. The relative resistance of [99mTc](SNS/S) complexes against nucleophilic attack of glutathione (GSH), a native nucleophilic thiol of 2 mM intracerebral concentration, is investigated in vitro by HPLC. The reaction of [99mTc](SNS/S) complexes with GSH is reversible and advances via substitution of the monothiolate ligand by GS- and concomitant formation of the hydrophilic [99mTc](SNS/GS) daughter compound. The N-diethylaminoethyl complexes are found to be more reactive against GSH as compared to the N-ethyl ones. Complex reactivity as a result of R-substitution follows the sequence -NO2 >> -H > -NH2. These in vitro findings correlate well with in vivo distribution data in mice. Thus, brain retention parallels complex susceptibility to GSH attack. Furthermore, isolation of the hydrophilic [99mTc](SNS/GS) metabolite from biological fluids and brain homogenates provides additional evidence that the brain retention mechanism of [99mTc](SNS/S) complexes is GSH-mediated.

Use of stabilized technetium-99m-exametazime for radiolabeling leukocytes.

UNLABELLED: With a stabilizing agent (i.e., methylene blue and sodium phosphate buffer mixture), the in vitro stability of 99mTc-exametazime has been increased to 4-6 hr postreconstitution. However, it is not feasible to use the stabilized 99mTc-exametazime for leukocyte radiolabeling. This is due to the deep blue appearance of the mixture of stabilized 99mTc-exametazime and blood components, which makes it impossible to separate properly the supernatant from the leukocyte button. In our study, we have developed a practical methodology for overcoming this difficulty in order to use stabilized 99mTc-exametazime in leukocyte labeling. METHODS: The stabilized 99mTc-exametazime preparation used in our method consisted of 2 ml 7.4-8.0 GBq (200-215 mCi) 99mTc and 2 ml methylene blue/phosphate buffer solution. The separated leukocytes from 80-ml fresh venous blood were incubated with three different ages (i.e., 0-, 4-, or 6-hr postreconstitution) of stabilized 99mTc-exametazime (approximately 925 MBq, approximately 25 mCi; 0.5-1 ml) at room temperature for 15 min. After incubation, 3 ml of 12.6% ACD/NS solution (anticoagulant citrate dextrose, solution A, USP mixed with 0.9% NaCl, v/v) was added to the tube and centrifuged at 160 g for 5 min. Three milliliters of the dark blue supernatant were carefully removed, and the bottom 1 ml portion was resuspended with 9 ml of 12.6% ACD/NS solution. After centrifugation (160 g for 5 min), the supernatant was clear enough to be drawn off without disturbing the radiolabeled leukocyte button. The white cell button was then resuspended in 4 ml of platelet-poor plasma. RESULTS: The overall labeling efficiency (LE) of our new technique was 67.8%-91.9%, with the higher LE associated with fresher stabilized 99mTc-exametazime. During a 6-hr in vitro stability evaluation, radiolabeled leukocytes lost 1.2% +/- 0.3% (n = 24), 1.3% +/- 0.1% (n = 16) and 1.8% +/- 0.1% (n = 16) each hour of the cell-bound 0-, 4-, and 6-hr-old 99mTc-exametazime, respectively. The 99mTc-exametazime-labeled leukocytes examined by the trypan blue staining technique at 6-hr postradiolabeling yielded nonstained cells indicating viable leukocytes. CONCLUSION: We concluded that with a small volume of 99mTc-exametazime and double dilution steps with 12.6% ACD/NS solution, stabilized 99mTc-exametazime can be used effectively for leukocyte radiolabeling with a high LE and long in vitro stability.

Seven-hour stabilization of 99Tcm-exametazime (HMPAO) for cerebral perfusion.

The objective of this study was to increase the stability of 99Tcm-exametazime and to investigate the effects of relaxing the eluate restrictions imposed by the manufacturer. We added 1800 MBq freshly eluted pertechnetate to 0.5 ml aliquots of stannous-enhanced exametazime followed by the addition of 0.7 mg gentisic acid and 0.5 ml sterile absolute alcohol BP. The radiochemical purity as measured by thin-layer chromatography was maintained at over 80% (range 88-99%, n = 40) for up to 7 h after preparation. High-performance liquid chromatography confirmed that the primary complex was maintained at over 80% (ranges 89-92%) for up to 7 h. In a second series of studies using the first eluate from a newly delivered generator to prepare 99Tcm-exametazime, a radiochemical purity of more than 80% was achieved for up to 7 h (range 88-95%, n = 24). In a third series using a 3-hour-old generator eluate, a radiochemical purity of more than 80% (range 88-93%, n = 18) was achieved for up to 5 h (for logistic reasons, we were unable to continue readings beyond 5 h). These results suggest that the manufacturer's restrictions on the eluate may be relaxed. Clinical validation was performed in a blinded study of 21 patients using single photon emission tomography. Image quality was assessed on the basis of salivary activity, nasal activity and the overall (global) image quality. There was no significant difference between the images obtained using the stabilized exametazime and exametazime prepared without gentisic acid and ethanol (chi 2 = 2.85, P = 0.05). We conclude that stabilization of 99Tcm-exametazime can be achieved for up to 7 h by using gentisic acid and alcohol and that the eluate restrictions may be disregarded.

Stabilization of Tc-99m D,L-HMPAO preparations as a leucocyte labelling agent.

An attempt was made to use stabilized Tc-99m D,L-HMPAO (S-HMPAO) to label leucocytes. The radiochemical purity of Tc-99m D,L-HMPAO, labelling efficiency of leucocytes, cell viability of labelled leucocytes, and stability of S-HMPAO labelled leucocytes were calculated. In comparison with commercial Tc-99m D,L-HMPAO (C-HMPAO) without stabilization, immediately, at 0.5, 1, 2, 4, and 6 h after HMPAO preparation, the radiochemical purity of S-HMPAO and the labelling efficiencies of S-HMPAO labelled leucocytes were higher. S-HMPAO is more stable than C-HMPAO and can provide higher leucocyte labelling efficiency. S-HMPAO, therefore, has the potential to replace C-HMPAO as a leucocyte-labelling agent.