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.     

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.     

Comparison of relative renal functions calculated with 99mTc-DTPA and 99mTc-DMSA for kidney patients of wide age ranges

Renal scintigraphy is an imaging method that uses small amount of radioactive materials called radiotracers, a Gamma camera and a computer to evaluate kidney functions and its anatomy. The present work reports the comparison of the relative renal functions (RRF) calculated with technetium-99m dimercaptosuccinic acid (^99mTc‑DMSA) and technetium-99m diethylenetriaminepentaacetic acid (^99mTc‑DTPA) for kidney patients of ages between 5 months and 71 years. A total of 50 patients including 29 male and 21 female has been selected and studied for renography. The mean RRFs have been found to be 52.68 ± 23.63% and 47.32 ± 23.63% respectively for the left and right kidneys with ^99mTc-DMSA measurement. With ^99mTc-DTPA the values are 52.74 ± 23.54% and 47.26 ± 23.54% for the same. In bivariate correlation analysis, a significant positive correlation (r = 0.996, P < .001) has been found between the RRFs calculated with the two methods. Following the patients’ diagnosis, in ANOVA test, no difference has been found between the RRFs calculated for the left and right kidneys. In Bland-Altman plots, the mean difference between the two methods has been found to be 0.1 and the correlation limit lies between −4.3 and 4.2. According to the result obtained in the present work, both the ^99mTc-DMSA and ^99mTc-DTPA scanning methods provide almost the same RRF values. It is, therefore, always not necessary to calculate the RRFs with both the methods. This study suggests that ^99mTc-DMSA may be the primary choice for the evaluation of RRF, but if the glomerular filtration rate (GFR) and renogram curve are required, ^99mTc-DTPA can be the obvious selection.     

Serum stability and non-specific binding of technetium-99m labeled diaminodithiol for protein labeling

Previously we investigated the use of DTPA-coupled proteins to simplify labeling with ^99mTc but especially to improve the stability of the label. These investigations have now been extended to include several N₂S₂ ligands such as N,N′-bis(2-methyl-2-mercaptopropyl)ethylenediamine (DADT) and a novel ligand of similar structure with a propylene bridge between two amines, 2-hydroxy-N,N′-bis(2-methyl-2-mercaptopropyl)propylenediamine (DADT-3C-2OH). The condition of labeling of free ligand (pH, buffer and tin concentration) was optimized to provide 100% chelation with ^99mTc at reasonable ligand concentrations (100 μg/mL or less). Labeling was determined by paper chromatography, reverse-phase and size-exclusion HPLC. After incubation in fresh serum, 37 °C for 24 h, repeat analysis showed less than 5% dissociation of the chelate. By contrast, the DTPA chelate shows instability towards oxidation during this period. DADT derivatized on an ethylene carbon showed almost identical serum stability as DADT itself whereas when derivatized on a nitrogen greater instabilities were apparent. Using identical labeling conditions, free DADT was chelated in the presence of IgG at different ligand: protein molar ratios. Non-specific binding of ^99mTc to IgG at a 10:1 DADT-HM:IgG molar ratio was as little as 5% and was essentially zero at a 2:1 DADT:IgG molar ratio when labeling was by transcomplexation from ^99mTc-EDTA. The DADT-3C-2OH ligand showed superior performance both in regard to serum stability and the absence of non-specific binding. In conclusion, the N₂S₂ ligands form more stable chelates with ^99mTc than does DTPA with reduced non-specific binding and may therefore represent an attractive alternative for labeling proteins with ^99mTc by the bifunctional chelate approach.     

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of ^99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed ^99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the ^99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The ^99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the ^99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between ^99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N₂S₂) compound. The Fab′ with IgG2a isotype displayed tighter binding to ^99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N₂S₂ challenge and incubation with human plasma. The in vivo biodistribution of five ^99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable ^99mTc labeling of antibody fragments from three of the major murine isotypes.     

Radiolabeling of lipid-based nanoparticles for diagnostics and therapeutic applications: a comparison using different radiometals

Radiolabeling of nanoparticles (NPs) has been performed for a variety of reasons, such as for studying pharmacokinetics, for imaging, or for therapy. Here, we describe the in vitro and in vivo evaluation of DTPA-derivatized lipid-based NP (DTPA-NP) radiolabeled with different radiometals, including ¹¹¹In and ^99mTc, for single-photon emission computed tomography (SPECT), ⁶⁸Ga for positron emission tomography (PET), and ¹⁷⁷Lu for therapeutic applications. PEGylated DTPA-NP with varying DTPA amounts, different composition, and size were radiolabeled with ¹¹¹In, ¹⁷⁷Lu, and ⁶⁸Ga, using various buffers. ^99mTc-labeling was performed directly and by using the carbonyl aquaion, [^99mTc(H₂O)₃(CO)₃]⁺. Stability was tested and biodistribution evaluated. High labeling yields (>90%) were achieved for all radionuclides and different liposomal formulations. Specific activities (SAs) were highest for ¹¹¹In (>4 MBq/μg liposome), followed by ⁶⁸Ga and ¹⁷⁷Lu; for ^99mTc, high labeling yields and SA were only achieved by using [^99mTc(H₂O)₃(CO)₃]⁺. Stability toward DTPA/histidine and in serum was high (>80 % RCP, 24 hours postpreparation).). Biodistribution in Lewis rats revealed no significant differences between NP in terms of DTPA loading and particle composition; however, different uptake patterns were found between the radionuclides used. We observed lower retention in blood (<3.3 %ID/g) and lower liver uptake (< 2.7 %ID/g) for ^99mTc- and ⁶⁸Ga, compared to ¹¹¹In-NP (blood, <4 %ID/g; liver, <3.6 %ID/g). Imaging potential was shown by both PET magnetic resonance imaging fusion imaging and SPECT imaging. Overall, our study shows that PEGylated DTPA-NP are suitable for radiolabeling studies with a variety of radiometals, thereby achieving high SA suitable for targeting applications.     

Synthesis of 99mTc-labeled 2-Mercaptobenzimidazole as a novel radiotracer to diagnose tumor hypoxia

Discovery of ^99mTc-labeled imidazole derivatives as a potential radiotracer for hypoxic tumor imaging is considered to be of great interest because of non-invasive detection capabilities. 2-Mercaptobenzimidazole (2-MBI) was successfully synthesized, characterized and radiolabeled with [^99mTc (CO)₃(H₂O)₃]⁺ intermediate to form ^99mTc-2-MBI complex with radiochemical purity of ≥95% yield as observed by instant-thin layer chromatography (ITLC) and radio-high performance liquid chromatography (radio-HPLC). The ^99mTc-2-MBI complex was observed to be stable in saline and serum with no noticeable decomposition at room temperature and 37 °C, respectively, over a time period of 24 h. Biodistribution results in Balb/c mice bearing S180 tumor show that ^99mTc-2-MBI highly internalized in tumor tissue, also possess preferably high tumor/muscle and tumor/blood ratios 4.14 ± 0.77 and 3.91 ± 0.63, respectively at 24 h incubation. Scintigraphic imaging study shows ^99mTc-2-MBI is visibly accumulated in hypoxic tumor tissue, suggesting it would be a promising radiotracer for early stage diagnosis of tumor hypoxia.     

Comparison of two methods of labeling proteins with 111In

DTPA N-hydroxysuccinimide pentaester (DHSE) can be used as an alternative to bicyclic anhydride for coupling proteins with DTPA.⁽¹⁾ However, in-vitro analysis of labeled proteins coupled with DHSE compared to bicyclic anhydride showed more high molecular weight product and less serum stability. We studied the magnitude of these differences in vivo by coupling HSA and IgG with DTPA using both methods, labeling with ¹¹¹In, and analyzing the biodistribution in mice. The results of this analysis showed comparable activity in the liver, kidneys and blood.     

Direct 99mTc labeling of monoclonal antibodies: Radiolabeling and in vitro stability

Direct labeling involves ^99mTc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind ^99mTc. The direct ^99mTc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability.Stable direct antibody labeling with ^99mTc requires the generation of sulfhydryl groups, which show high affinity binding sites for ^99mTc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the ^99mTc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution.Very good ^99mTc-antibody stability is obtained with activated IgG (IgGa) and Fab′ fragment, which makes these substances possible candidates for immunoscintigraphy use.     

Studies on the metabolic fate of 111In-labeled antibodies

Indium-111-labeled antibodies, though providing superior photon flux to iodine-labeled antibodies, can exhibit high levels of accumulation in some non-target organs. In an effort to understand the nature of this non-target uptake we have evaluated the molecular weight of ¹¹¹In species retained in several tissues by radio-FPLC (sizing chromatography) following injection of [¹¹¹In]DTPA 5G6.4, a murine monoclonal antibody, into normal mice. Blood, liver and kidneys were removed, and liver and kidneys were homogenized at several time points after antibody injection. The proportion of ¹¹¹In-containing species was found to vary with the tissue and with time. Analysis of blood showed only radiolabeled antibody. In the liver, several ¹¹¹In species were identified with molecular weights compatible with intact antibody, [¹¹¹In]transferrin, and low molecular weight complexes, with an increase in the proportion of [¹¹¹In]transferrin and low molecular weight species occurring over time. While the same molecular weight species were also identified in the kidneys, the kidneys contained the largest percentage of low molecular weight species which increased over time. When ¹²⁵I-labeled 5G6.4 was injected and the tissues similarly analyzed, only radioactive material with the molecular weight of intact antibody was detected. Comparison of two methods of purification of [¹¹¹In]labeled antibody after labeling revealed a significant difference in the organ uptake of radiolabeled products for ¹¹¹In. Although dialysis was sufficient for the removal of labile ¹¹¹In, as determined by TLC, subsequent sizing chromatography on Bio-Gel P-60 dramatically dropped the hepatic and renal uptake of ¹¹¹In relative to blood and diminished the proportion of the low molecular weight species present on sizing FPLC of extracts from tissues. These data indicate that low and intermediate molecular weight ¹¹¹In compounds are accreted in the liver and kidneys following the i.p. injection of ¹¹¹In-labeled monoclonal antibodies and that their uptake can be diminished by more stringent radioantibody purification. This knowledge may be valuable in developing methods for reducing non-target ¹¹¹In uptake.     

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.     

Preparation and Identification of HER2 Radioactive Ligands and Imaging Study of Breast Cancer-Bearing Nude Mice

OBJECTIVE: A micro-molecule peptide TP1623 of ^99mTc-human epithelial growth factor receptor 2 (HER2) was prepared and the feasibility of using it as a HER2-positive molecular imaging agent for breast cancer was evaluated. METHODS: TP1623 was chemically synthesized and labeled with ^99mTc. The labeling ratio and stability were detected. HER2 expression levels of breast cancer cells (SKBR3 and MDA-MB-231) and cell binding activity were measured. Biodistribution of ^99mTC-TP1623 in normal mice was detected. SKBR3/MDA-MB-231-bearing nude mice models with high/low expressions of HER2 were established. Tumor tissues were stained with hematoxylin–eosin (HE) and measured by immunohistochemistry to confirm the formation of tumors and HER2 expression. SPECT imaging was conducted for HER2-overexpressing SKBR3-bearing nude mice. The T/NT ratio was calculated and compared with that of MDA-MB-231-bearing nude mice with low HER2 expression. The competitive inhibition image was used to discuss the specific binding of ^99mTc- TP1623 and the tumor. RESULTS: The labeling ratio of ^99mTc-TP1623, specific activity, and radiochemical purity (RCP) after 6 h at room temperature were (97.39 ± 0.23)%, (24.61 ± 0.06) TBq/mmol, and (93.25 ± 0.06)%, respectively. HER2 of SKBR3 and MDA-MB-231 cells showed high and low expression levels by immunohistochemistry, respectively. The in vitro receptor assays indicated that specific binding of TP1623 and HER2 was retained. Radioactivity in the brain was always at the lowest level, while the clearance rate of blood and the excretion rate of the kidneys were fast. HE staining showed that tumor cells were observed in SKBR3- and MDA-MB-231-bearing nude mice, with significant heteromorphism and increased mitotic count. The imaging of mice showed that targeted images could be made of ^99mTc-TP1623 in high HER2-expressing tumors, while no obvious development was shown in tumors in low HER2-expressing nude mice. No development was visible in tumors in competitive inhibition of imaging, which indicates the combination of ^99mTc-TP1623 and tumor was mediated by HER2. CONCLUSION: High labeling ratio and specific activity of ^99mTc-TP1623 is successfully prepared; it is a molecular imaging agent for HER2-positive tumors that has potential applicative value.     

99mTc(Cu)—mannitol complex: a new agent for dynamic renal function studies

Mannitol has been labelled with ^99mTc by using cuprous chloride as a reducing agent. Blood and kidney clearance of ^99mTc(Cu)-mannitol was slightly faster than that of ^99mTc(Sn)-DTPA in rat and maximum radioactivity ratio of kidneys to blood was 84.6 at 5 min. A comparative study of ^99mTc(Cu)-mannitol, ^99mTc(Sn)-DTPA was made in rabbits by taking serial images of kidneys and bladder with a γ camera. Results show superiority of ^99mTc(Cu)-mannitol over other agents for dynamic renal function studies.     

Evaluation of kidney repair capacity using Tc-DMSA in ischemia/reperfusion injury models

Quantitative ^99mTc-DMSA renal uptake was studied in different renal ischemia/reperfusion (I/R) mice models for the assessment of renal repair capacity. Mice models of nephrectomy, uni- and bi-lateral I/R together with sham-operated mice were established. At 1 h, 1 d, 4 d, 1, 2 and 3 wk after I/R, ^99mTc-DMSA (27.7 ± 1.3 MBq) was injected via tail vein and after 3 h post-injection, the mice were scanned for 30 min with pinhole equipped gamma camera. Higher uptake of ^99mTc-DMSA was measured in normal kidneys of uni-lateral I/R model and nephrectomized kidney I/R model at 3 wk post-surgery. Comparing the restoration capacities of the affected kidneys of nephrectomy, uni- and bi-lateral I/R models, higher repair capacity was observed in the nephrectomized model followed by bi-lateral then uni-lateral models. The normal kidney may retard the restoration of damaged kidney in uni-lateral I/R model. Moreover, 3 wk after Uni-I/R, the size of injured kidney was significantly smaller than non-ischemic contralateral and sham operated kidneys, while nephrectomy I/R kidneys were significantly enlarged compared to all others at 3 wk post-surgery. Very strong correlation between ^99mTc-DMSA uptake and weight of dissected kidneys in I/R models was observed. Consistent with ^99mTc-DMSA uptake results, all histological results indicate that kidney recovery after injury is correlated with the amount of intact tubules and kidney sizes. In summary, our study showed good potentials of ^99mTc-DMSA scan as a promising non-invasive method for evaluation of kidney restoration after I/R injuries. Interestingly, mice with Bi-I/R injury showed faster repair capacity than those with uni-I/R.     

Analysis of elimination mechanisms of some 99mTc-complexes

The biological behaviour of complexes of ^99mTc with aminopolycarboxylic and aminocarbohydroxamic ligands EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), EDTAH (ethylenediaminetetraacetohydroxamic acid) and HIDAmH (N-2-hydroxyethyl-N-carboxymethyl-aminoacetohydroxamic acid) was studied in rabbits. The pharmacokinetic parameters determined in intact rabbits were compared with the results obtained in the study of renal and hepatic clearance of the complexes under study. Hepatobiliary excretion, which in [^99mTc]EDTA forms 20–30% of the total excreted amount, is of negligible magnitude in the other ^99mTc-complexes studied (<2%). Their renal clearance is not influenced by the inhibition of tubular secretion with probenecid. Binding to plasma proteins increases in the order [^99mTc]DTPA < [^99mTc]EDTA <[^99mTc]HIDAmH <[^99mTc]EDTAH and the elimination half-life increases in the same order. The value of renal clearance of the complexes studied related to inulin clearance correlates well with the fraction of the free drug in the plasma. In rabbits the complexes under study are excreted mainly by the mechanism of glomerular filtration in the kidney.     

Attachment of 99mTc to lipid vesicles containing the lipophilic chelate dipalmitoylphosphatidylethanolamine-DTTA

The binding of ^99mTc to negatively-charged and neutral unilamellar lipid vesicles was investigated in the absence and presence of the ligand diethylenetriaminepentaacetic acid (DTPA) covalently attached to the headgroup of phosphatidylethanolamine at the surface of the membrane. Even in the absence of DTPA on the membrane surface, ^99mTc reduced by Sn bound to the membrane surface but rapidly dissociated from the vesicles in the presence of plasma in vitro. When DTPA was present on the membrane surface, dissociation of ^99mTc from the vesicle surface in plasma was much reduced. The dissociation of ^99mTc from the surface of negatively-charged vesicles was less than for neutral vesicles in the absence of ligand but was markedly greater than for vesicles containing the ligand DTPA, suggesting that the binding of ^99mTc to vesicles with surface-attached DTPA could not be explained solely on the basis of the negative charge provided by the DTPA. In vitro experiments using ¹⁴C-labeled lipids as well as in vivo imaging studies indicated that dissociation of ^99mTc from the surface of the vesicle did not arise predominantly because of lipid exchange with plasma components or due to cleavage of Tc-DTPA from the vesicle surface. For vesicles with surface-attached DTPA, ^99mTc dissociation from the vesicle surface in plasma was further reduced by addition of the antioxidant ascorbate.     

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.     

A study of the concept of non-radioactive unit-dosed reagent kits [cold unit doses (CUDs)] as an efficient and cost-saving method for 99mTc radiopharmaceutical preparation

Traditionally, when preparing ^99mTc-labeled radiopharmaceuticals, [^99mTc]pertechnetate is added to the entire contents of a vial of reagent kit, and patient doses are subsequently withdrawn from the vial. This technique of compounding can be potentially wasteful for two reasons: (1) once reconstituted with ^99mTc, most reagent kits have a relatively short shelf-life, and thus the entire contents may not be used before expiration and (2) due to a need to conserve radioactivity in many hospitals, enough [^99mTc]pertechnetate is added to the reagent kit in order to retrieve only 1–2 patient doses, even though adequate chemicals (ligand, reducing agent, etc.) are present in the reagent kit to supply as many as 5–10 doses. Hence, a method for optimizing the efficient use of reagent kits would be desirable. The purpose of this study was to determine the feasibility of unit-dosing non-radioactive reagent kits and storing these cold unit doses (CUDs) for eventual labeling with ^99mTc. To evaluate this concept, unit doses were prepared from reagent kits of medronate (MDP) and pentetate (DTPA). The specific variables studied in this research were the effects of storage time, storage temperature and reconstitution volume (dilution) on the unit doses. These effects were monitored by measuring the radiochemical and biodistribution properties of the unit doses following their final reconstitution with [^99mTc]pertechnetate. The labeling efficiency was determined using instant thin layer chromatography (ITLC), and the biodistribution patterns of these radiolabeled CUDs were studied in mice. The results showed that MDP- and DTPA-CUDs stored at −18 °C retained the properties which resulted in acceptable radiochemical purity and biodistribution in mice for as long as 30 days. On the other hand, the radiochemical purity of MDP and DTPA unit doses stored at 25 °C deteriorated rapidly. Mean radiochemical purities as low as 0.58–19.4% were observed on day 30. Altered biodistributions were observed in a manner consistent with the decreased labeling efficiencies. The CUDs of lower dilution (3 mL) appeared to be more stable than the CUDs of higher dilution (10 mL). However, the effect of reconstitution volume was much less significant than the temperature effect on the CUDs. In conclusion, the concept of unit-dosing non-radioactive reagent kits appears to provide an efficient and cost-saving method for preparing infrequent and emergency radiopharmaceutical doses. The study also showed that the storage temperature of these unit doses is critical to the success of the procedure. The volume of reconstitution has a minimal impact on the stability of CUDs if stored at the appropriate temperature.     

Investigations of N-linked macrocycles for 111in and 90Y labeling of proteins

To simplify the synthesis of macrocyclic chelators, commercially available macrocyclic amines were condensed with halogenated acetic acid to prepare the five chelators 12N4 (DOTA), 14N4 (TETA), 15N4, 9N3 and 12N3. Only 12N4 and 9N3 showed efficient labeling of the free chelator with ¹¹¹In and ⁹⁰Y. Serum stability studies at 37 °C with In-labeled DTPA, 12N4 and 9N3 showed no loss of label over 2 days whereas, with ⁹⁰Y, only 12N4 showed stabilities comparable to DTPA. The 12N4 chelator was derivatized by attaching biotin on one N-acetate group to simulate the attachment to protein. The serum stability for both ¹¹¹In and ⁹⁰Y was identical to that of biotin derivatized DTPA and lower than that of the free chelators. Biodistribution studies in normal mice of a model protein (avidin) labeled with ⁹⁰Y via biotinylated 12N4 and biotinylated DTPA showed identical distribution at 1 day except in bone where the %ID/g for the macrocyclic-conjugated protein (3.4 ± 0.5, N = 8) was significantly (P < 0.001) lower than that of the DTPA-conjugated protein (9.4 ± 0.9, N = 7). In conclusion, macrocycles may be readily synthesized from the macrocyclic amines and several show useful stabilities with In and Y. When N-linked to a protein, the Y biodistribution was found to be superior to that of the corresponding DTPA-coupled protein.