Comparative binding of 125I-and 99mTc-d-labeled native and glycated low-density lipoprotein to human microvascular endothelial cells-potential for atherosclerosis imaging?

Native (n), glycated (g), and glycoxidated (go) low-density lipoproteins (LDL) were labeled with 125I or 99mTc, and the labeling efficiency and binding were assessed for potential use of these LDL compounds in imaging analysis of atherosclerotic lesions (PPAR-gamma receptors) by determining the number of specific receptors for nLDL, gLDL or goLDL on human microvascular endothelial cells as well as the KDs using either 125I-or 99mTc-labeled LDLs. The specific activity of labeled gLDL and goLDL was much higher (for goLDL 20 times higher) than that of nLDL. Gel filtration of labeled LDLs revealed, however, that 99mTc-g/goLDL is significantly degraded by the labeling reaction. No fragmentation was observed for 99mTc-nLDL and all the 125I-labeled LDL forms. Binding studies using both 125I-and 99mTc-nLDL indicated a weak binding affinity (KD 10- 7mol/L) to human microvascular endothelial cells. The binding affinity of 125I-g/goLDL to these cells was significantly higher (KD 10- 9mol/L) and could be increased further by preactivation of the endothelial cells using TNFalpha. Incubation with 99mTc-goLDL, however, did not result in specific binding of the ligand, possibly as a consequence of the fragmentation of the lipoprotein during the labeling. Scatchard transformation of the binding data with 99mTc-gLDL revealed the presence of only a few binding sites. This was in contrast to the results obtained with 125I-labeled gLDL, which revealed a much higher membrane density of scavenger receptors for this ligand. We conclude that for in vitro binding studies as well as for potential in vivo imaging, only 125I-labeled goLDL should be used, whereas nLDL may be applied as 125I-or 99mTc-labeled ligand.     

Influence of antipyretic drugs on the labeling of blood elements with technetium-99m

Acetaminophen (AAP), acetylsalicylic acid (ASA) and dipyrone (DIP) are antipyretic and analgesics drugs that have wide use in health sciences. Some drugs can modify the labeling of blood elements with technetium-99m (99mTc). This work has evaluated the effect of AAP, ASA and DIP on the labeling of the blood elements with 99mTc. Blood was incubated with different concentrations of the drugs before the 99mTc-labeled process. Plasma (P), blood cells (BC), insoluble (IF-P, IF-BC) and soluble (SF-P, SF-BC) fractions were separated and percentage of radioactivity (%ATI) in each fraction was determined. Data have shown that the antipyretic drugs used in this study did not significantly modify the fixation of 99mTc on the blood elements when the experiments were carried out with the doses usually used in human beings. Although the experiments were carried out with rats, it is possible to suggest that AAP, ASA or DIP should not interfere with the procedures in nuclear medicine involving the labeling of blood elements with 99mTc.     

In vitro and in vivo evaluation of a Technetium-99m-labeled cyclic RGD peptide as a specific marker of alpha(V)beta(3) integrin for tumor imaging

Three amino acids residues, Arg-Gly-Asp (RGD), in vitronectin and fibronectin show affinity for alpha(V)beta(3) integrins expressed in vascular endothelial cells. That tumor growth can upregulate the expression of these integrins on tumor cells for invasion and metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD peptides as antagonists of alpha(V)beta(3) integrins for broad spectrum tumor specific imaging. The polypeptide RGD-4C, which contains four cysteine residues for cyclization, has shown preferential localization on integrins at sites of tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for 99mTc radiolabeling. After purification of the conjugated peptides by a C18 Sep-Pak cartridge with 20% methanol, both peptides were radiolabeled using tricine. For cell binding studies, both 99mTc peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/micromol was achieved. Both 99mTc complexes were stable in the labeling solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degrees C and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of 99mTc-labeled RGD to cells. The binding percentage of 99mTc-labeled RGD to purified alpha(V)beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the integrin while 99mTc-labeled RGE showed no binding. The association constant for 99mTc-RGD was modest at 7 x 10(6) M(-)(1). In both human renal adenocarcinoma (ACHN) and human colon cancer cell line (LS174T) nude mouse tumor models, the accumulation of 99mTc-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers of alpha(V)beta(3) integrin receptors per tumor cell and low binding affinity, radiolabeled RGD peptides may have limitations as tumor imaging agents.     

99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin

Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (Nalpha-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (Nalpha-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 microg of minigastrin I human (MGh). HYNIC-MG and (Nalpha-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/micromol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with <10% intact peptide after 60 min at 37 degrees C, with no considerable differences between the radiolabeled conjugates; a somewhat lower degradation rate was seen in liver homogenates. Protein binding varied considerably with lowest levels for 99mTc-EDDA/HYNIC-MG. Competition experiments of unlabeled conjugates on AR42J membranes versus [125I-Tyr12]-gastrin I showed high CCK-2 receptor affinity for all conjugates under study. Internalization behavior was very rapid for all radiolabeled conjugates in the order of 99mTc-(Nalpha-His)Ac-MG > 99mTc-EDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/g) and 99mTc-(Nalpha-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc-(Nalpha-His)Ac-MG showed low tumor uptake in vivo.     

99mTc-neolactosylated human serum albumin for imaging the hepatic asialoglycoprotein receptor

99mTc-labeled diethylenetriaminepentaacetic acid (DTPA)-coupled neogalactosyl human serum albumin (GSA) is used as an imaging agent for asialoglycoprotein receptor of the liver. However, its labeling is inconvenient because it should be incubated for 30 min at 50 degrees C. In addition, the conjugated DTPAs can cause decrease of pI and denaturation of protein. Therefore, we developed an improved agent 99mTc-neolactosyl human serum albumin (LSA) which contains a terminal galactose. LSA was synthesized by conjugating lactose to human serum albumin by the formation of a Schiff's base and successive reduction with sodium cyanoborohydride. The number of conjugated lactose molecules per LSA was 40.7 +/- 12.3. To simplify the labeling procedure, we used a direct labeling method that adopts a high affinity 99mTc binding site concept in antibody labeling. The produced LSA was reduced by beta-mercaptoethanol to generate sulfhydryl groups and purified by PD-10 size-exclusion column. The number of generated sulfhydryl groups per LSA was 21.9 +/- 3.0. Medronate and stannous chloride were added to the reduced LSA and freeze-dried. Finally, 99mTc-pertechnetate (37 MBq, 1 mL) was added to the vial and incubated for 10 min at room temperature. The labeling efficiency of 99mTc-LSA was higher than 98%, and the stability in human serum at 37 degrees C for 24 h was over 90%. Biodistribution study using balb/c mice and imaging study using SD rats showed high initial liver uptake and slow increase in the intestine due to hepatobiliary excretion after metabolism in the hepatocytes. Negligible spleen uptake was found while 99mTc-tin colloid showed significant amount of spleen uptake due to reticuloendothelial uptake. In conclusion, an improved agent, 99mTc-LSA, for imaging asialoglycoprotein receptor of the liver was successfully developed which showed a simple labeling procedure, high labeling efficiency, high stability, and high initial liver uptake.     

Technetium-99m-labeled medium-chain fatty acid analogues metabolized by beta-oxidation: radiopharmaceutical for assessing liver function.

External imaging of energy production activity of living cells with 99mTc-labeled compounds is a challenging task requiring good design of 99mTc-radiopharmaceuticals. On the basis of our recent findings that 11C- and 123I-labeled medium-chain fatty acids are useful for measuring beta-oxidation activity of hepatocytes, we focused on development of 99mTc-labeled medium-chain fatty acid analogues that reflect beta-oxidation activity of the liver. In the present study, monoamine-monoamide dithiol (MAMA) ligand and triamido thiol (MAG) ligand were chosen as chelating groups because of the stability and size of their complexes with 99mTc and their ease of synthesis. Each ligand was attached to the omega-position of hexanoic acid (MAMA-HA and MAG-HA, respectively). In biodistribution studies, [99mTc]MAMA-HA showed high initial accumulation in the liver followed by clearance of the radioactivity in the urine. Analysis of the urine revealed [99mTc]MAMA-BA as the sole radiometabolite. Furthermore, when [99mTc]MAMA-HA was incubated with living liver slices, generation of [99mTc]MAMA-BA was observed. However, [99mTc]MAMA-HA remained intact when the compound was incubated with liver slices in the presence of 2-bromooctanoate, an inhibitor of beta-oxidation. The findings in this study indicated that [99mTc]MAMA-HA was metabolized by beta-oxidation after incorporation into the liver. On the other hand, poor hepatic accumulation was observed after administration of [99mTc]MAG-HA.     

A simple two-step approach for introducing a protected diaminedithiol chelator during solid-phase assembly of peptides

A simple synthetic strategy is described to incorporate a protected diaminedithiol (N(2)S(2)) chelator during Fmoc solid-phase synthesis of short peptides. The resulting constructs could be efficiently labeled with technetium-99m (99mTc). The chelator was assembled at the N-terminus of peptides in a two-step procedure where the deprotected terminal amino group was first reacted with di-Fmoc-diaminopropionic acid (Fmoc-DAP-[Fmoc]-OH). The two protected amino groups were then simultaneously deprotected and subsequently reacted with S-benzoylthiolglycolic acid (TGA) to generate a protected N(2)S(2) chelator. This metal binding site was introduced into di- and tripeptides. Each peptide construct was composed of a C-terminal lysine residue and an N-terminal diaminopropionic moiety modified to create the chelator site. The epsilon-amino group at the C-terminal lysine was further derivatized with a nitroimidazole group to facilitate cellular retention. The resulting constructs were then cleaved from the resin support, purified, and labeled with [99mTc]pertechnetate. Six constructs were prepared differing by a single amino acid inserted between the diaminopropionic acid and lysine residues. Optimal labeling yields of >70% were achieved around neutral pH and heating at 75 degrees C for 10 min. Purified 99mTc-labeled constructs were found to accumulate in Chinese hamster ovary (CHO) cells in vitro as a function of charge and hydrophobicity.     

99mTc-labeled MIBG derivatives: novel 99mTc complexes as myocardial imaging agents for sympathetic neurons

Radioactive-iodine-labeled meta-iodobenzylguanidine (MIBG) is currently being used as an in vivo imaging agent to evaluate neuroendocrine tumors as well as the myocardial sympathetic nervous system in patients with myocardial infarct and cardiomyopathy. It is generally accepted that MIBG is an analogue of norepinephrine and its uptake in the heart corresponds to the distribution of norepinephrine and the density of sympathetic neurons. A series of MIBG derivatives containing suitable chelating functional groups N2S2 for the formation of [TcvO]3+N2S2 complex was successfully synthesized, and the 99mTc-labeled complexes were prepared and tested in rats. One of the compounds, [99mTc]2, tested showed significant, albeit lower, heart uptakes post iv injection in rats (0.21% dose/g at 4 h) as compared to [125I]MIBG (1.7% dose/g at 4 h). The heart uptake of the 99mTc-labeled complex appears to be specific and can be reduced by co-injection with nonradioactive MIBG or by pretreatment with desipramine, a selective norepinephrine transporter inhibitor. Further evaluation of the in vitro uptake of [99mTc]2 in cultured neuroblastoma cells displayed consistently lower, but measurable uptake (approximately 10% of that for [125I]MIBG). These preliminary results suggested that the mechanisms of heart uptake of [99mTc]2 may be related to those for [125I]MIBG uptake. If suitable 99mTc-labeled MIBG derivatives can be further developed, the prevalent availability of 99mTc in nuclear medicine clinics will allow them to be readily available for widespread application.     

99mTc-labeling of HYNIC-conjugated cyclic RGDfK dimer and tetramer using EDDA as coligand

In this study, EDDA (ethylenediamine- N, N'-diacetic acid) was used as the coligand for 99mTc-labeling of cyclic RGDfK conjugates: HYNIC-dimer (HYNIC = 6-hydrazinonicotinamide; dimer = E[c(RGDfK)]2) and HYNIC-tetramer (tetramer = E{E[c(RGDfK)]2}2). First, HYNIC-dimer was allowed to react with 99mTcO4 (-) in the presence of excess tricine and stannous chloride to form the intermediate complex [99mTc(HYNIC-dimer)(tricine)2], which was then allowed to react with EDDA to afford [99mTc(HYNIC-dimer)(EDDA)] with high yield (>90%) and high specific activity ( approximately 8.0 Ci/micromol). Under the same radiolabeling conditions, the yield for [99mTc(HYNIC-tetramer)(EDDA)] was always <65%. The results from a mixed-ligand experiment show that there is only one EDDA bonding to the 99mTc-HYNIC core in [99mTc(HYNIC-dimer)(EDDA)]. The athymic nude mice bearing subcutaneous U87MG human glioma xenografts were used to evaluate the impact of EDDA coligand on the biodistribution characteristics and excretion kinetics of the 99mTc-labeled HYNIC-dimer and HYNIC-tetramer. Surprisingly, [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] had almost identical tumor uptake over the 2 h period. The use of EDDA as coligand to replace tricine/TPPTS (TPPTS = trisodium triphenylphosphine-3,3',3'-trisulfonate) did not significantly change the uptake of the 99mTc-labeled HYNIC-dimer in noncancerous organs, such as the liver, kidneys, and lungs; but it did result in a significantly lower kidney uptake for the 99mTc-labeled HYNIC-tetramer due to faster renal excretion. It was also found that the radiotracer tumor uptake decreases in a linear fashion as the tumor size increases. The smaller the tumors are, the higher the tumor uptake is regardless of the identity of radiotracer.     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery. Failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, 99mTc, and an aqueous marker, 125I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped 125I-labelled poly(vinyl pyrrolidone) were labelled with 99mTc by the SnCl2 method. 99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than 125I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, 99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than 125I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60-70% of 125I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of 99mTc-radioactivity was co-eluted with the liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all 99mTc-radioactivity does not represent the 60-70% of intact liposomes present in lymph nodes. Using the aqueous marker 125I-labelled poly(vinyl pyrrolidone), the lymph node localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that 125I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier, where the authors used 99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became 'leaky' to low-molecular-weight drugs, for example, methotrexate (Mr 454) to a much greater extent than with a large-molecular-weight substance, 125I-labelled poly(vinyl pyrrolidone) (Mr 30 000-40 000), when incubated with rat lymph at 37 degrees C. Using the two markers 99mTc and 125I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining lambda-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with 99mTc rather than using the SnCl2 technique, or using other radionuclides as markers for gamma-scan imaging.     

Control of radioactivity pharmacokinetics of 99mTc-HYNIC-labeled polypeptides derivatized with ternary ligand complexes

An enhancement of the target/nontarget ratio of radioactivity levels enables reliable diagnosis and therapy using polypeptide radiopharmaceuticals in nuclear medicine. In the present study, we investigated the effects of the physicochemical properties of radiometabolites on the radioactivity pharmacokinetics after administration of 99mTc-labeled polypeptides using 6-hydrazinopyridine-3-carboxylic acid (HYNIC). Four ternary ligands (L) [3-benzoylpyridine (BP), 3-acetylpyridine (AP), 3-nicotinic acid (NIC), pyridine (PY)] with different lipophilicity were selected as coligands for the preparation of 99mTc-HYNIC-polypeptides. Each of the ternary ligands tested provided 99mTc-HYNIC-labeled galactosyl-neoalbumin (NGA) and Fab fragments of high stability with high radiochemical purity. Moreover, after administration of each 99mTc-HYNIC-labeled NGA into normal mice, the respective ternary ligand [99mTc](HYNIC-lysine)(tricine)(L) complexes were generated as final radiometabolites in the hepatic lysosome. The partition coefficients of [99mTc](HYNIC-lysine)(tricine)(BP), [99mTc](HYNIC-lysine)(tricine)(AP), [99mTc](HYNIC-lysine)(tricine)(NIC), and [99mTc](HYNIC-lysine)(tricine)(PY) were determined to be -2.21, -2.37, -2.93, and -2.73, respectively. Elimination rates of these radiometabolites from the lysosome were enhanced in the order of increasing lipophilicity of the radiometabolites. After injection of the four 99mTc-HYNIC-labeled Fab fragments into normal mice, blood clearances of radioactivity were similar while radioactivity elimination rates from the kidney were enhanced in the order of increasing lipophilicity of the radiometabolites. The present study indicated that the lipophilicity of the radiometabolites constitutes one important factor affecting their elimination rates from the tissues. Thus, as ternary ligands facilitate alteration of the physicochemical properties of radiometabolites, the use of ternary ligand complexes might be applicable for controlling the pharmacokinetics of 99mTc-labeled polypeptides.     

A new high affinity technetium-99m-bombesin analogue with low abdominal accumulation

99mTc-labeled bombesin analogues have shown promise for noninvasive detection of many tumors that express bombesin (BN)/gastrin-releasing peptide (GRP) receptors. 99mTc-labeled peptides, however, have a tendency to accumulate in the liver and intestines due to hepatobiliary clearance as a result of the lipophilicity of the 99mTc chelates. This makes the imaging of lesions in the abdominal area difficult. In this study, we have synthesized a new high affinity 99mTc-labeled BN analogue, [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN, having a built-in pharmacokinetic modifier, DTPA, and labeled with 99mTc using a hydrophilic diaminedithiol chelator (Pm-DADT) to effect low hepatobiliary clearance. In vitro binding studies using human prostate cancer PC-3 cell membranes showed that the inhibition constant (Ki) for [DTPA1, Lys3(99Tc-Pm-DADT), Tyr4]BN was 4.1 +/- 1.4 nM. Biodistribution studies of [DTPA1, Lys3(99mTc-Pm-DADT), Tyr4]BN in normal mice showed very low accumulation of radioactivity in the liver and intestines (1.32 +/- 0.13 and 4.58 +/- 0.50% ID, 4 h postinjection, respectively). There was significant uptake (7.71 +/- 1.37% ID/g, 1 h postinjection) in the pancreas which expresses BN/GRP receptors. The uptake in the pancreas could be blocked by BN, partially blocked by neuromedin B, but not affected by somatostatin, indicating that the in vivo binding was BN/GRP receptor specific. Scintigraphic images showed specific, high contrast delineation of prostate cancer PC-3 xenografts in SCID mice. Thus, the new peptide has a great potential for imaging BN/GRP receptor-positive cancers located even in the abdomen.     

Effect of Thuya occidentalis on the labeling of red blood cells and plasma proteins with technetium-99m.

Thuya occidentalis is used in popular medicine in the treatment of condyloma and has antibacterial action. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used for several evaluations in nuclear medicine. This labeling depends on a reducing agent, usually stannous ion. Any drug which alters the labeling of the tracer could be expected to modify the disposition of the radiopharmaceutical. We have evaluated the influence of T. occidentalis extract on the labeling of RBC and plasma proteins with 99mTc. Blood was withdrawn and incubated with T. occidentalis (0.25; 2.5; 20.5; and 34.1 percent v/v). Stannous chloride (1.2 micrograms/ml) was added and then 99mTc was added. Plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. The analysis of the results shows that there is a decrease in radioactivity (from 97.64 to 75.89 percent) in BC with 34.1 percent of the drug. In the labeling process of RBC with 99mTc, the stannous and pertechnetate ions pass through the membrane, so we suggest that the T. occidentalis effect can be explained (i) by an inhibition of the transport of these ions, (ii) by damage in membrane, (iii) by competition with the cited ions for the same binding sites, or (iv) by possible generation of reactive oxygen species that could oxidize the stannous ion.     

A propolis extract and the labeling of blood constituents with technetium-99m

Since ancient times propolis has been employed for many human purposes because to their favourable properties. Blood constituents labeled with technetium-99m (99mTc) have been used in nuclear medicine procedures. Some authors have reported that synthetic or natural drugs can interfere with the labeling of blood constituents with 99mTc. The aim of this work was to evaluate the action of a propolis extract on the labeling of blood elements with 99mTc. Samples of whole blood of male Wistar rats were incubated in sequence with an aqueous propolis extract at different concentrations, stannous chloride and 99mTc, as sodium pertechnetate. Blood samples were centrifuged to separate plasma and blood cells, soluble and insoluble fractions of plasma and blood cells were also separated after precipitation in trichloroacetic acid solution and centrifugation. The radioactivity was counted and the percentage of incorporated radioactivity (%ATI) for each fraction was calculated. The data obtained showed that the aqueous propolis extract used decreased significantly the %ATI in plasma proteins at higher concentration studied. Results suggest that at high concentration the constituents of this extract could alter the labeling of plasma proteins competing with same binding sites of the 99mTc on the plasma proteins or acting as antioxidant compounds.     

Assessment of the effect of antiseizure drugs on the labeling process of red blood cells and plasma proteins with technetium-99m.

It is estimated that about 2.5 million people only in the United States are affected by epilepsy. Labelled red blood cells (RBC) and plasma proteins (PP) are used for several evaluations in nuclear medicine and drugs affecting those labelings have previously been described. The aim of this study was to evaluate whether the most popular antiseizure drugs interfere with the 99mTc labeling process of RBC and PP. Heparinized blood withdrawn from Wistar rats was incubated with phenobarbital (0.2, 2, 20, 200, 2,000 microg/ml), phenytoin (0.15, 1.5, 15, 150, 1,500 microg/ml), carbamazepine (0.7, 7, 70 microg/ml), clonazepam (0.5, 5, 50, 500 microg/ml) or valproic acid (0.5, 5, 50, 500 microg/ml) for I hr. Stannous chloride (SnCl2), in two different concentrations (0.012 or 1.2 microg/ml) and 99mTc were added. Plasma and cellular fractions were isolated by centrifugation, soluble and insoluble fractions were separated by trichloroacetic acid precipitation. The percentage of radioactivity was calculated for each fraction. Statistical analysis was performed with ANOVA and Dunnet tests. The analysis of the results has shown that phenobarbital (2,000 microg/ml) and clonazepam (50 microg/ml) significantly have reduced the RBC labeling efficiency when it was used the optimal SnCl2 concentration (1.2 microg/ml) and clonazepam (5, 50 microg/ml) has significantly decreased the PP labeling efficiency with 99mTc. Phenytoin (1,500 microg/ml) has decreased the RBC labeling efficiency when the experiments were carried out with a small SnCl2 concentration (0.012 microg/ml). We can suggest that with this in vitro assay, at the therapeutic level of phenytoin, phenobarbital, carbamazepine and valproic acid will not interfere on the 99mTc labeling process of RBC. Interference is displayed at higher phenobarbital concentrations (2,000 microg/ml). However, humans do not tolerate this concentration. On the other hand, a decreased RBC and PP labeling efficiency with 99mTc may be expected for clonazepam at therapeutic levels.     

A novel approach to the high-specific-activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP = diphosphine ligand).

A new labeling approach for incorporating bioactive peptides into a technetium-99m coordination complex is described. This method exploits the chemical properties of the novel metal-nitrido fragment [99mTc(N)(PXP)]2+, composed of a terminal Tc[triple bond] N multiple bond bound to an ancillary diphosphine ligand (PXP). It will be shown that this basic, molecular building block easily forms in solution as the dichloride derivative [99mTc(N)(PXP)Cl2], and that this latter complex selectively reacts with monoanionic and dianionic, bidentate ligands (YZ) having soft, pi-donor coordinating atoms to afford asymmetrical nitrido heterocomplexes of the type [99mTc(N)(PXP)(YZ)]0/+ without removal of the basic motif [99mTc(N)(PXP)]2+. The reactions of the amino acid cysteine was studied in detail. It was found that cysteine readily coordinates to the metal fragment [99mTc(N)(PXP)]2+ either through the [NH2, S-] pair of donor atoms or, alternatively, through the [O-, S-] pair, to yield the corresponding asymmetrical complexes in very high specific activity. Thus, these results were conveniently employed to devise a new, efficient procedure for labeling short peptide sequences having a terminal cysteine group available for coordination to the [99mTc(N)(PXP)]2+ fragment. Examples of the application of this novel approach to the labeling of the short peptide ligand H-Arg-Gly-Asp-Cys-OH (H(2)1) and of the peptidomimetic derivative H-Cys-Val-2-Nal-Met-OH (H2) will be discussed.     

Methods for MAG3 conjugation and 99mTc radiolabeling of biomolecules

The chelator mercaptoacetyltriglycine (MAG3) forms a single stable chelate with technetium-99m (99mTc) oxotechnetate. The bifunctional N-hydroxysuccinimidyl ester of mercaptoacetyltriglycine with S-acetyl protection of the sulfhydryl group may be used to conjugate MAG3 to primary amine functionalized biomolecules for the purpose of radiolabeling with 99mTc for gamma detection or single photon emission computed tomography imaging (SPECT). We report here an improved MAG3 conjugation and 99mTc radiolabeling method capable of generating high radiochemical yield and high specific radioactivity. Post-labeling purification will not be needed if the protocol is followed as presented. Apart from the preparation of reagents, the conjugation and purification requires about 4 h, while the labeling with 99mTc requires about an additional 30 min.     

99mTc-labeled neuropeptide Y analogues as potential tumor imaging agents.

The possible use of neuropeptide Y (NPY) as a novel radiopeptide has been investigated. NPY is a 36-amino acid peptide of the pancreatic polypeptide family, which is expressed in the peripheral and central nervous system, and is one of the most abundant neuropeptides in the brain. Its receptors are produced in a number of neuroblastoma and the thereof derived cell lines. As structure-activity relationships of NPY are well-known, we could assume where a radionuclide might be introduced without affecting receptor affinity. We applied the novel [99mTc(OH2)3(CO)3]+ aqua complex and PADA (2-picolylamine-N,N-diacetic acid) as bifunctional chelating agent. The peptides were synthesized by solid-phase peptide synthesis, and PADA was coupled to the side chain of Lys4 of the resin-bound peptide. Upon postlabeling of [K4(PADA)]-NPY, 99mTc(CO)3 did not only bind to the desired PADA, but presumably as well to the His in position 26. Since the replacement of His26 by Ala only slightly decreased binding affinity, [K4(PADA),A26]-NPY was specifically postlabeled, and the 185Re surrogate maintained high binding affinity. Furthermore, the prelabeling approach has been applied for the centrally truncated analogue [Ahx5-24]-NPY, which is highly selective for the Y2 receptor. The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA)]-NPY was produced with a yield of only 16%. Therefore, postlabeling was applied for the short analogue as well, again substituting His26 by Ala. Competitive binding assays using (185)Re as a surrogate for 99mTc showed high binding affinity of Ac-[Ahx5-24,K4(185Re(CO)3-PADA),A26]-NPY. Internalization studies with the corresponding 99mTc-labeled analogue revealed receptor-mediated internalization. Furthermore, biodistribution studies were performed in mice, and stability was tested in human plasma. Our centrally truncated analogue revealed a 6-fold increased stability compared to the natural peptide NPY. We conclude that Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY has promising characteristics for future applications in nuclear medicine.     

Enhanced kidney clearance with an ester-linked 99mTc-radiolabeled antibody Fab'-chelator conjugate

Bifunctional chelators for labeling antibodies with 99mTc based on the N3S core of (mercaptoacetyl)-triglycine having ester or amide linking moieties were synthesized and site-specifically attached to the sulfhydryl groups of the Fab' fragment of antimyosin. Protein labeling was quantitative after 15 min; postlabeling purification was not necessary. The radiolabeled conjugates exhibited no loss of immunoreactivity. Under basic conditions, the ester-linked conjugate lost 95% of the radiolabel in the form of the 99mTc complex of (mercaptoacetyl)triglycine as determined by RP-HPLC, while the radioactivity in the amide-linked conjugate remained completely bound to the protein. In a mouse biodistribution study, the ester-linked conjugate showed a 2-fold enhancement in clearance from the kidney when compared to the amide-linked product.     

Evaluation of the in vitro effect of a Lantana camara extract on the labeling of blood constituents of rats with technetium-99m

Blood constituents labeled with technetium-99m (99mTc) have been used in nuclear medicine procedures and drugs are capable to interfere on this labeling. Lantana camara (lantana) has medicinal properties and it has been used in folk medicine. The aim is to verify the effect of a lantana extract on the labeling of blood constituents with 99mTc. Blood of rats was incubated with extract, stannous chloride and 99mTc, as sodium pertechnetate. Plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) were separated. The % of radioactivity (%ATI) in these samples was calculated. Samples of labeled BC were washed and the %ATI maintained (%ATI-M) in the BC was determined. The results showed that lantana extract decreased significantly (p < 0.05) in the IF-P from 70.24 +/- 2.59 to 11.95 +/- 3.07. This effect was not observed in the BC and IF-BC. The BC-%ATI-M was significantly (p < 0.05) decreased in all concentrations tested when the BC was washed. This fact was not observed in the control. Substances present on the extract should have redoxi action decreasing the concentration of the stannous ion and this condition could justify the effect on the IF-P. The results about the BC-%ATI-M should indicate a possible effect on the transport of ions through the erythrocyte membrane.