The use of technetium-99m as a radioisotopic label to assess cell-mediated immunity in vitro

We have developed a radioisotopic microassay of cell-mediated immunity employing target cells prelabeled with technetium-99m (^99mTc), a high specific activity metastable gamma emitter. Labeling kinetics, release and reutilization, subcellular localization, and effects of ^99mTc on DNA and protein synthesis have been investigated. Target cells were optimally labeled with 10 mCi of ^99mTc at 37 °C for 10 min. Cyclic freezing and thawing released less than 10% of total bound radioisotope. Spontaneous leakage of ^99mTc by monolayer cells was negligible over 48 hr and that which was released appeared to be nonreutilizable. Cell fractionation revealed that nuclear, mitochondrial, and microsomal fractions all were labeled with ^99mTc. The incorporation of ³H-thymidine and ³H-amino acids was not impaired in ^99mTc-labeled cells.The alloimmune reactivity of C57BL/6 mice which had received A/J skin allografts was studied by means of the ^99mTc microcytotoxicity assay. Cell-mediated immunity was clearly evident at 7 days postgrafting, peaked at 14 days, and had declined to background levels by 21 days. These findings correlated well with initial acceptance and ultimate rejection of the allografts. The rapid labeling time without dependence upon cell division for incorporation, high specific activity, low spontaneous release, and nonreutilizability are important advantages of ^99mTc over other radionuclides which have been employed in in vitro assays of cell-mediated immunity.     

Synthesis,characterization and biodistribution studies of a neutral-lipophilic Tc-99m N3S2 chelate

Diaminedithiols (DADT) are known to form neutral-lipophilic complexes with ^99mTc in aqueous solutions, where they are readily formed in high yields and demonstrate excellent stability. A new triaminedithiol (TADT) ligand was synthesized, characterized and shown to form a neutral-lipophilic ^99mTc-chelate. The biodistribution of this ^99mTc chelate in rats showed that its uptake in brain or heart following i.v. injection of the ^99mTc chelate was low, but activity taken up was retained over a long period of time. The in vivo and in vitro properties of this chelate indicate the possibility that chemical modification of this TADT ligand may produce ligand systems that form ^99mTc chelates with suitable diagnostic properties.     

The synthesis of pteroyl-lys conjugates and its application as Technetium-99m labeled radiotracer for folate receptor-positive tumor targeting

Aiming to develop a new ^99mTc-labeled folate derivative for FR-positive tumor imaging, a simpler method has been established to synthesize the folate-drug conjugates with free α-carboxyl group. In this study, the conjugate pteroyl-lys-HYNIC was synthesized and labeled with ^99mTc using tricine and TPPTS as co-ligands. The radiochemical purity of the final complex ^99mTc(HYNIC-lys-pteroyl)(tricine/TPPTS), 5 was high (>98%), and it remained stable in saline and plasma over 6 h after preparation. The biologic evaluation results showed that the ^99mTc labeled pteroyl-lys conjugate was able to specifically target the FR-positive tumor cells and tissues both in vitro and in vivo, highlighting its potential as an effective folate receptor targeted agent for tumor imaging.     

Transchelation of 99mTc from low affinity sites to high affinity sites of antibody

An exclusive labeling of high affinity sites of IgG and its F(ab′)₂ fragments with ^99mTc was accomplished. Antibody was first labeled in 0.1 M acetate buffer at pH 4.5, using stannous chloride as a reducing agent. Thus, high capacity, low affinity sites and low capacity, high affinity sites were both labeled. These ^99mTc complexes were stable at pH 4.5 and 7.0; however, they became destabilized at pH 8.2 and 9.0. Transchelation of ^99mTc to DTPA took place at the higher pH values and leveled off at 54% ^99mTc-F(ab′)₂ and 73% ^99mTc-IgG. These results indicate that the majority of ^99mTc bound to the low affinity sites was transchelated to the high affinity sites rather than to DTPA since low affinity sites account for 84% of total F(ab′)₂ sites and 76% of IgG sites. Biodistribution data in mice at 2.5 h postinjection were consistent with this hypothesis in that tissue concentrations of ¹¹¹In-DTPA-F(ab′)₂ were similar to the reequilibrated ^99mTc-F(ab′)₂ but were much higher than that of the unequilibrated ^99mTc-F(ab′)₂.     

Synthesis,characterization and biodistribution of neutral and lipid-soluble 99mTc-PAT-HM and 99mTc-TMR for brain imaging

Two new ligand systems for complexation with ^99mTc were prepared. The two analogs of bisaminoethanethiol (BAT): N,N′-bis(2-methyl-2-mercaptopropyl)-2,2-dimethylpropylenediamine (PAT-HM) and N,N′-bis[2-(2-ethyl-1-mercaptopropyl)] ethylenediamine (TMR), form neutral and lipid soluble complexes with ^99mTc that readily penetrate the blood-brain barrier following i.v. injection into rats. Although the ^99mTc chelates do not display the prolonged brain retention required for use in single photon emission computed tomographic imaging studies, the fact that each ligand forms a neutral and lipid-soluble complex of high chemical stability when coordinated with ^99mTc warrants further investigation to increase the site- and organ-specificity of these agents.     

Aptamers directly radiolabeled with technetium-99m as a potential agent capable of identifying carcinoembryonic antigen (CEA) in tumor cells T84

Aptamers are small oligonucleotides that are selected to bind with high affinity and specificity to a target molecule. Aptamers are emerging as a new class of molecules for radiopharmaceutical development. In this study a new method to radiolabel aptamers with technetium-99m (^99mTc) was developed. Two aptamers (Apt3 and Apt3-amine) selected against the carcinoembryonic antigen (CEA) were used. Labeling was done by the direct method and the developed complex was subjected to quality control tests. Radiochemical purity and stability were monitored by Thin Layer Chromatography. Binding and specificity assays were carried out in the T84 cell line (CEA+) to evaluate tumor affinity and specificity after radiolabeling. Aptamers were successfully labeled with ^99mTc in high radiochemical yields, showing in vitro stability in presence of plasma and cystein. In binding assays the radiolabeled aptamer Apt3-amine showed the highest affinity to T84 cells. When evaluated with HeLa cells (CEA−), lower uptake was observed, suggesting high specificity for this aptamer. These results suggest that the Apt3-amine aptamer directly labeled with ^99mTc could be considered a promising agent capable of identifying the carcinoembryonic antigen (CEA) present in tumor cells.     

Technetium-99m-labeled platelets: Comparison of labeling with a new lipid-soluble Sn(II)-mercaptopyridine-N-oxide and 99mTc-HMPAO

Platelets pretinned with a neutral Sn(II)-2-mercaptopyridme-N-oxide (SN-MPO) were labeled with ^99mTc and compared to those labeled with ^99mTc-HMPAO. The conditions of labeling platelets, e.g. concentrations of platelets and Sn(II)-MPO, ^99mTc in ACD-saline or ACD-plasma media, pH and incubation time, were optimized using canine platelets. Moderate labeling efficiency was obtained with 20 μg of tin(II) chloride and 30 min incubation with Sn-MPO and pertechnetate. The viability of labeled platelets was determined by platelet recovery and platelet survival times in Beagle dogs. The labeling efficiency with platelets from 43 mL of blood was 62.8 ± 7.6%. The platelet recovery was 35.7 ± 5.0% and exponential survival time was 34.6 ± 3.1 h compared to 43.3 ± 12.0% and 29.5 ± 3.3 h for ^99mTc-HMPAO-labeled platelets. These values were significantly (P < 0.01) lower than ¹¹¹In-labeled platelets. Biodistribution in dogs indicates lower retention in blood, spleen and liver after some initial ^99mTc excretion in urine. The platelet deposition with ^99mTc platelets (Sn-MPO method) on polyurethane angio-catheters was similar to ^99mTc-HMPAO-labeled platelets. This study indicates that the platelets could be successfully labeled with pertechnetate in a cost-effective manner for the evaluation of thromboembolic complications.     

Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo

In this study ‘second generation’ AnxV was specifically labeled with ^99mTc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged ‘second generation’ AnxV labeled with ^99mTc(CO)₃ in comparison to ^99mTc-HYNIC-cys-AnxV and ^99mTc(CO)₃-DTPA-cys-AnxV.     

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the ‘click-to-chelate’ protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12, [^99mTc(CO)₃]13, and [^99mTc(CO)₃]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)₃]12, [Re(CO)₃]13, and [Re(CO)₃]14, were shown to have high binding affinities (0.13 μM, 0.06 μM, and 0.16 μM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12–14) to c-Met receptor positive U87MG cells.     

99mTc-labeled monomeric and dimeric NGR peptides for SPECT imaging of CD13 receptor in tumor-bearing mice

CD13 receptor plays a critical role in tumor angiogenesis and metastasis. We therefore aimed to develop ^99mTc-labeled monomeric and dimeric NGR-containing peptides, namely, NGR1 and NGR2, for SPECT imaging of CD13 expression in HepG2 hepatoma xenografts. Both NGR-containing monomer and dimer were synthesized and labeled with ^99mTc. In vivo receptor specificity was demonstrated by successful blocking of tumor uptake of ^99mTc-NGR dimer in the presence of 20 mg/kg NGR2 peptide. Western blot and immunofluorescence staining confirmed the CD13 expression in HepG2 cells. The NGR dimer showed higher binding affinity and cell uptake in vitro than the NGR-containing monomer, presumably due to a multivalency effect. ^99mTc-Labeled monomeric and dimeric NGR-containing peptides were subjected to SPECT imaging and biodistribution studies. SPECT scans were performed in HepG2 tumor-bearing mice at 1, 4, 12, and 24 h post-injection of ~7.4 MBq tracers. The metabolism of tracers was determined in major organs at different time points after injection which demonstrated rapid, significant tumor uptake and slow tumor washout for both traces. Predominant clearance from renal and hepatic system was also observed in ^99mTc-NGR1 and ^99mTc-NGR2. In conclusion, monomeric and dimeric NGR peptide were developed and labeled with ^99mTc successfully, while the high integrin avidity and long retention in tumor make ^99mTc-NGR dimer a promising agent for tumor angiogenesis imaging.     

Technetium-99m monoclonal antibody fragment (FAb) scintigraphy in the evaluation of small cell lung cancer: a preliminary report

Small cell lung cancer (SCC) has the most rapid growth rate of the four cell types and metastasizes early. Present imaging modalities for staging include chest x-ray, CT, MRI and bone scans. In this preliminary study, we assessed the clinical role of ^99mTc-monoclonal antibody (MOAB) scintigraphy in five patients with histologically proven SCC. Each patient was infused with 20–30 mCi of ^99mTc labeled Fab fragment of MOAB (NR-LU-10, NeoRx, Seattle, Wash.). Total body simultaneous anterior and posterior images were obtained 14–16 h post injection. SPECT images of the chest were obtained through a 360 ° rotation of the gamma camera and recorded on a 62 × 64 × 16 matrix. Images (1.2cm thick) were generated in transaxial, sagittal and coronal views.Fourteen of fifteen chest lesions detected by CT were confirmed by ^99mTc MOAB scintigraphy. Scintigraphy detected one additional chest lesion not seen by CT. Scintigraphy failed to detect a brain lesion (2 cm), a chest lesion, and two adrenal lesions, all of which were seen by CT. In one patient with multiple (more than 10) lesions in the liver, both scintigraphy and CT detected all lesions. Three spine lesions seen on ^99mTc MDP scan and positive for metastasis on MRI concentrated ^99mTc MOAB, but two rib lesions seen on ^99mTc MDP bone scan did not concentrate ^99mTc MOAB. It is concluded from these preliminary results that the potential usefulness of ^99mTc MOAB scintigraphy as a complementary imaging modality in the staging of small cell lung cancer should be investigated further.     

Preparation and biodistribution of a 99mTc tricarbonyl complex with deoxyglucose dithiocarbamate as a tumor imaging agent for SPECT

The deoxyglucose dithiocarbamate (DGDTC) was successfully labeled with the ^99mTc(CO)₃ core to provide the corresponding ^99mTc(CO)₃–DGDTC complex in good yields. The radiochemical purity of the ^99mTc(CO)₃–DGDTC complex was over 90%, as measured by high performance liquid chromatography (HPLC). The complex possessed good stability in saline at room temperature and in mouse plasma at 37 °C. Its partition coefficient result indicated that it was a hydrophilic complex. The electrophoresis results showed the complex was neutral. The biodistribution of ^99mTc(CO)₃–DGDTC in mice bearing S 180 tumor showed that the complex clearly accumulated in tumor, exhibiting high tumor/blood and tumor/muscle ratios and good tumor retention. Single photon emission computed tomography (SPECT) image studies showed there was a visible uptake in tumor sites, suggesting ^99mTc(CO)₃–DGDTC could be considered as a potential tumor imaging agent.     

Synthesis of Tc-99m labeled glucosamino-Asp-cyclic(Arg-Gly-Asp-d-Phe-Lys) as a potential angiogenesis imaging agent

Angiogenesis imaging agents for single photon emission computed tomography (SPECT) play a role in diagnosing tumor-induced angiogenesis as well as tumor metastasis. We synthesized and evaluated radiolabeled RGD glycopeptides by incorporation of the [^99mTc(CO)₃(H₂O)₃]⁺. ^99mTc labeled glucosamino-D-c(RGDfK) ([^99mTc]2) was prepared in 90–93% radiochemical yields (decay corrected). In vitro cell binding assays demonstrated selective binding [^99mTc]2 to human umbilical vein endothelial (HUVE) cells, with inhibition of binding to 37.3% of control levels by 10 μM of cold authentic compounds. In addition, [^99mTc]2 was shown to have high binding affinity to purified α_vβ₃ integrin (IC₅₀ = 1.5 nM). These results suggest that these radiolabeled RGD glycopeptides may have value for non-invasive assessment of angiogenesis.     

Technetium-99m labeled monoclonal antibodies: Evaluation of reducing agents

We have evaluated five compounds, stannous chloride (SnCl₂), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythritol (DTE), and ascorbic acid (AA) to reduce monoclonal antibody MoAb (disulfide groups and compared their efficacy for labeling MoAbs with ^99mTc. The reduction of ^99mTc with dithionite at pH 11 was nearly quantitative. The use of AA, at a molar ratio of 3500:1, for three IgG and three IgM antibodies examined, gave a labeling efficiency greater than 95%. Hence no purification was needed. The immunospecificity of AA preparations determined by specific antigen assay was 84 ± 1% for an IgM and 82.6 ± 1.1% for an IgG, highest among all agents tested. The stability of the tracer was evaluated by challenging the product with such ^99mTc avid agents as cysteine, DTPA, and human serum albumin. By HPLC analysis, no ^99mTc was transchelated using chelating agent to protein molar ratios as high as 500:1. In two separate groups of five mice each, the liver uptake at 4 h post injection averaged 6.8 ± 2.9% per gram for ¹²⁵I-TNT-1 (IgG) and 6 ± 5.1% per gram for the same MoAb labeled with ^99mTc using AA. The AA technique promises to label antibodies with ^99mTc and perhaps with ¹⁸⁶Re, by a simple “kit” procedure.     

Use of liposomes as carriers for radiation synovectomy

Using [^99mTc]pertechnetate as an aqueous space marker, the permeability of liposomes composed of seven different mixtures of distearoylphosphatidylcholine (DSPC) and sphingomyelin (SM) was determined. Liposomes containing 20–33% SM were the least permeable in the presence of rheumatoid synovial fluid. Following injection of ^99mTc-containing liposomes into the knee joints of rabbits, retention of ^99mTc in the knee was more than 200 times greater than following injection of nonencapsulated [^99mTc]pertechnetate. The knee clearance biologic half time of ^99mTe with DSPC/SM (4:1) liposomes was 64 h. Most of the activity that had leaked from the knee was not found in extra-articular tissues, suggesting rapid excretion. When DSPC/SM (4:1) liposomes were labeled with ¹¹¹In(oxine), a knee clearance biologic half time of greater than 1200 h was observed.     

Use of Technetium (99mTc) as a Bacterial Label in Lung Clearance Studies

The suitability of technetium (^99mTc), a gamma emitter, for labeling of Diplococcus pneumoniae in studies of lung bacterial clearance was examined. A killed bacterial slurry with high specific activity was obtained with a ferric ascorbate reducing system. Approximately 5.5% of radioactive counts dissociated from labeled bacteria in 6 h. Rats were exposed to a uniformly mixed aerosol of untagged, viable pneumococci and killed, ^99mTc-tagged pneumococci. The aerodynamic behavior of labeled and unlabeled pneumococci was similar. Viable bacterial counts and radioactive counts were determined in lung homogenates at intervals following exposure, and rates of bacterial killing and disappearance of radioactive counts were plotted. Radioactive counts did not increase in the liver during the period of observation, suggesting that the decrease in lung radioactivity represents mucociliary clearance and not release of isotope to the systemic circulation. The use of ^99mTc for bacterial labeling provides advantages of technical simplicity and personnel safety compared to the use of beta-emitting isotopes.     

Tetraamine‐modified octreotide and octreotate: labeling with 99mTc and preclinical comparison in AR4‐2J cells and AR4‐2J tumor‐bearing mice

Two somatostatin analogues, [^99mTc]Demotide and [^99mTc]Demotate 4, were compared with [^99mTc]Demotate 1, a previously reported somatostatin receptor subtype 2 (sst₂) targeting tracer. Conjugates were prepared by coupling an open‐chain tetraamine chelator to D ‐Phe¹ of [Tyr³]‐octreotide or [Tyr³]‐octreotate, respectively, via a p‐benzylaminodiglycolic acid spacer adopting solid‐phase peptide synthesis techniques. Peptide conjugates were collected in a highly pure form after chromatographic purification. Eventually, [^99mTc]Demotide and [^99mTc]Demotate 4 were obtained in ～1 Ci/µmol specific activity and >96% purity after labeling under alkaline conditions. Demotide and Demotate 4 exhibited similar high binding affinities for the sst₂ expressed in AR4‐2J cells with IC₅₀ values 0.16 and 0.10 nM, respectively. The (radio)metallated analogues [^99mTc]Demotide and [^99mTc]Demotate 4 showed equally high affinities to the sst₂ during saturation binding assays in AR4‐2J cell membranes (K_ds 0.08 and 0.07 nM, respectively). During incubation at 37 °C with AR4‐2J cells, the radiopeptides internalized effectively via a receptor‐mediated process, with [^99mTc]Demotate 4 exhibiting a faster internalization rate than [^99mTc]Demotide. After injection in athymic mice bearing sst₂‐expressing AR4‐2J tumors, the radiotracers showed high and specific uptake in the tumor (>25%ID/g at 1 h) and in the sst₂–positive organs. However, both [^99mTc]Demotide and [^99mTc]Demotate 4 showed unfavorably higher background activity, especially in the abdomen, in comparison to [^99mTc]Demotate 1 and are, therefore, less suited than [^99mTc]Demotate 1 for sst₂‐targeted tumor imaging in man. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

99mTc-tricarbonyl labeled agents for cell labeling: Development,biodistribution in normal mice and preliminary in vitro evaluation

We have developed four ^99mTc(CO)₃-labeled lipophilic tracers as potential radiolabeling agents for cells based on a hexadecyl tail. ^99mTc(CO)₃-hexadecylamino-N,N′-diacetic acid (negatively charged), ^99mTc(CO)₃-hexadecylamino-N-α-picolyl-N′-acetic acid (uncharged), ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine (positively charged), ^99mTc(CO)₃-N-hexadecylaminoethyl-N′-aminoethylamine (positively charged) were prepared in a radiolabeling yield: >90%. Preliminary cell uptake studies were performed in mixed blood cells with or without plasma and were compared with ^99mTc-d,l-HMPAO and [¹⁸F]FDG. In plasma-free blood cells, maximum uptake (78%) was obtained for ^99mTc(CO)₃-N-hexadecylaminoethyl-N′-aminoethylamine after 60 min incubation (compared to 55% and 23% for ^99mTc-d,l-HMPAO and [¹⁸F]FDG, respectively) while in plasma-rich medium, ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine was best bound (54%, similar to the binding of ^99mTc-d,l-HMPAO). Biodistribution in normal mice showed mainly hepatobiliary clearance of the agents and initial high lung uptake. The radiolabeled compounds showed good blood clearance with maximally 7.9% injected dose per gram at 60 min post injection. While the least lipophilic agent (^99mTc(CO)₃-N,N′-dipicolylhexadecylamine, log P = 1.3) showed the best cell uptake, there appears to be no direct correlation between lipophilicity and tracer uptake in mixed blood cells. In view of its comparable cell uptake to well known cell labeling agent ^99mTc-d,l-HMPAO, ^99mTc(CO)₃-N,N′-dipicolylhexadecylamine merits further evaluation as a potential cell labeling agent.     

Synthesis and biological evaluation of a novel 99mTc(CO)3 complex of ciprofloxacin dithiocarbamate as a potential agent to target infection

The ciprofloxacin dithiocarbamate (CPFXDTC) was radiolabeled with [^99mTc(CO)₃(H₂O)₃]⁺ intermediate to form the ^99mTc(CO)₃–CPFXDTC complex in high yield. The ^99mTc(CO)₃–CPFXDTC complex was characterized by HPLC and its stability in serum was studied. Its partition coefficient indicated that it was a lipophilic complex. The bacterial binding efficiency of ^99mTc(CO)₃–CPFXDTC was almost the same as that of ^99mTcN–CPFXDTC, and was higher than that of ^99mTc–ciprofloxacin. Biodistribution results in induced infection mice showed ^99mTc(CO)₃–CPFXDTC had higher uptake at the sites of infection and better abscess/blood and abscess/muscle ratios than those of ^99mTc–ciprofloxacin and ^99mTcN–CPFXDTC. Single photon emission computed tomography (SPECT) static imaging study in infected rabbits demonstrated the uptake in the left thigh infection lesion was observable, while no accumulation in the right thigh muscle was found. These results suggested ^99mTc(CO)₃–CPFXDTC would be a promising candidate for further evaluation as infection imaging agent.     

Development,optimization, and biovalidation of <Superscript>99m</Superscript>Tc–insulin complex

Human biosynthetic insulin is a polypeptide hormone that plays an important and essential role in control of the level of carbohydrate, protein, and fat metabolism in the blood. Human pancreatic insulin was labeled with ^99mTc to form a new radiopharmaceutical with a labeling yield of 99 ± 1% under optimum conditions: 0.1 mL insulin, pH 7, 25 μg stannous chloride, 1 mL (19 mCi) of pertechnetate, room temperature, and 10 min reaction time. The ^99mTc–insulin complex was examined using paper chromatography, ITLC, electrophoresis, and HPLC. In addition, in vitro and in vivo study of ^99mTc–insulin complex was performed at different time intervals.