A solid-phase technique for preparation of no-carrier-added technetium-99m radiopharmaceuticals: application to the streptavidin/biotin system

A high effective specific activity (HESA) formulation of a biotin-containing ^99mTc ligand [RP488: dimethyl-Gly-Ser-Cys(Acm)-Lys(Biotin)-Gly] conveniently prepared from solid phase was compared to a typical low effective specific activity (LESA) solution formulation to demonstrate improved targeting to streptavidin in an in vitro assay and in an in vivo rat model. RP488 was coupled to a maleimide-functionalized polyethylene glycol resin via a thiol ether linkage and labeled with ^99mTc-gluconate at room temperature, followed by elution of the HESA ^99mTc-RP488 in saline (minimum specific activity 1000 TBq/mmol by amino acid analysis). Both HESA and LESA ^99mTc-RP488 labeled at > 90% purity. In vitro, HESA ^99mTc-RP488 incubated with streptavidin-agarose was bound quantitatively, but there was competition from addition of increasing amounts of cold RP488. In rats, radiotracer uptake was evident at the site of implantation of streptavidin-agarose beads for the HESA dose, less uptake of low effective specific activity (LESA) material, and no appreciable uptake in the control rats of the LESA or HESA dose. The target-to-background ratio for HESA ^99mTc-RP488 was 5.4 times that of the control. The solid-phase technology offers a convenient way to prepare high specific activity receptor-targeting ^99mTc radiopharmaceuticals.     

Molecular recognition and stability of Tc-UBI 29–41 based on experimental and semiempirical results

^99mTc-UBI 29–41 is an antimicrobial peptide fragment that directly radiolabeled with ^99mTc shows high in vitro and in vivo stability, rapid background clearance, minimal accumulation in non-target tissues and rapid detection of infection sites. Molecular mechanics (MM) calculation has been an essential tool in explaining experimental results associated with molecular recognition and stability. This work is an attempt to explain the ^99mTc-UBI 29–41 specificity for bacteria and to understand from a structural point of view, the experimental results indicative of a molecular recognition and stability not well favored for two other cationic peptides (^99mTc-Tat-1-Scr and ^99mTc-Tat-2-Scr ) used as control. Structures of ^99mTc-UBI, ^99mTc-Tat-1-Scr, ^99mTc-Tat-2-Scr and of the corresponding free cationic peptides were built and the optimized structures, in the best stable configurations, were calculated by a MM procedure. In order to correlate the calculated and experimental results, in vitro stability tests with cysteine challenge and stability to dilution in human serum and in saline solution, were performed for the three labeled cationic peptides. The three complexes can be represented by the general formula [Tc(V)(O)(H₂O)₂(Lys_n=1,2-Arg_n=0,1-peptide)]^10+,11+. The potential energies were 104.5, 95.6 and 90.8 kcal/mol for ^99mTc-Tat-1-Scr, ^99mTc-Tat-2-Scr and ^99mTc-UBI 29–41, respectively. Experimental and calculated results were in good agreement. It is thus possible to predict and explain that in similar solution media ^99mTc-Tat-2-Scr would be more stable than ^99mTc-Tat-1-Scr and why ^99mTc-UBI shows the highest stability. In conclusion, the in vitro specific binding to bacteria and the accumulation at infection sites in humans of ^99mTc-labeled UBI could be the result of its high thermodynamic stability, selectivity and stereospecificity.     

Technetium-99m labeling of a molecule bearing easily reducible groups

Chemical stability of the naphthol-azo dye Evans Blue (EB) was examined in the presence of acidic stannous chloride (SnCl₂), with a view to preparing an instant cold kit. EB was found to be reactive toward this reducing agent, yielding the metal-chelating molecule 1,7-diamino-8-naphthol-2,4-disulfonic acid at high acidity and high stannous concentrations. This reduction reaction was undesirable in the cold kit preparation. The conditions were determined where reduction was inhibited, at pH = 5.0 and with a mole ratio of EB to SnCl₂ = [10:1], effecting the facile preparation of stable cold kits. Successful ^99mTc-labeling of an EB cold kit using these conditions resulted in the desired product with 98% radiochemical purity. Based on the radiolabeling efficiencies of chosen model compounds, it was rationalized that ^99mTc metal predominantly coordinated with the 1-amino-8-hydroxy groups in the EB molecule to form ^99mTc-EB.     

Uptake of Tc tetrofosmin in lymphoma cell lines: a comparative study with Tc sestamibi

Technetium-99m (^99mTc) tetrofosmin has been used as a tumor-seeking agent. However, its role in detecting lymphomas has not been widely investigated. The aim of the present study was to determine the uptake and clearance characteristics of ^99mTc tetrofosmin in lymphoma cell lines. ^99mTc sestamibi was also evaluated for comparison. Three lymphoma cell lines (U-937: monocyte-like, histiocytic lymphoma, human; RAMOS: B-lymphoma cell line, American Burkitt lymphoma, lymphoblastoid, human; Hs445: Hodgkin's disease, lymphoid, human) were studied. After incubation of radiotracers ^99mTc tetrofosmin and ^99mTc sestamibi in medium for 0, 10, 20, 30, 60, 120 and 180 min, the uptake and clearance of each radiotracer were measured in the three lymphoma cell lines. The uptake of ^99mTc tetrofosmin was lower than that of ^99mTc sestamibi in these lymphoma cell lines. Among the three cell lines, Hs445 showed the greatest ^99mTc tetrofosmin uptake capacity. RAMOS and U-937 showed similar ^99mTc tetrofosmin uptake capacities. ^99mTc tetrofosmin accumulated in the three tested lymphoma cell lines, especially in the Hodgkin's disease cell line. However, in comparison with ^99mTc sestamibi, ^99mTc tetrofosmin may not be the best radiotracer for detection of lymphoma.     

Evaluation of novel cationic 99mTc(I)-tricarbonyl complexes as potential radiotracers for myocardial perfusion imaging

This report describes the evaluation of three cationic ^99mTc(I)–tricarbonyl complexes — [^99mTc(CO)₃(L)]⁺ (L=N-methoxyethyl-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (ME-PNP), N-[15-crown-5)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (15C5-PNP) and N-[18-crown-6)-2-yl]-N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]amine (18C6-PNP)) — as potential radiotracers for myocardial perfusion imaging. Biodistribution, imaging and metabolism studies were performed using Sprague–Dawley rats. It was found that bisphosphine ligands have a significant impact on the biodistribution characteristics and clearance kinetics of their cationic ^99mTc(I)–tricarbonyl complexes. Among the three radiotracers evaluated in this study, [^99mTc(CO)₃(15C5-PNP)]⁺ has a very high initial heart uptake and is retained in the rat myocardium for >2 h. It also shows rapid clearance from the liver and lungs. The heart/liver ratio of [^99mTc(CO)₃(15C5-PNP)]⁺ is 2.5 times better than that of ^99mTc-sestamibi at 30 min postinjection. [^99mTc(CO)₃(15C5-PNP)]⁺ is almost identical to ^99mTcN-DBODC5 with respect to heart uptake, heart/lung ratio and heart/liver ratio. Results from metabolism studies show that there is no significant metabolism for [^99mTc(CO)₃(15C5-PNP)]⁺ in the urine, but it does show a small metabolite peak (<10%) in the radio high-performance liquid chromatography chromatogram of the feces sample at 120 min postinjection. Results planar imaging studies demonstrate that [^99mTc(CO)₃(15C5-PNP)]⁺ has a much better liver clearance profile than ^99mTc-sestamibi and might give clinically useful images of the heart as early as 30 min postinjection. [^99mTc(CO)₃(15C5-PNP)]⁺ is a very promising candidate for more preclinical evaluations in various animal models.     

Lowering of pI by acylation improves the renal uptake of Tc-labeled anti-Tac dsFv: effect of different acylating reagents

Anti-Tac disulfide-stabilized variable region fragment (dsFv) was labeled with ^99mTc by a preformed chelate approach using ^99mTc-MAG3-trifluorophenyl (TFP) ester. Simultaneously it was acylated with TFP-lactate or succinic anhydride to decrease the isoelectric point of dsFv (pI 10). Acylation of dsFv (0.04 mM) with the lactate at a 73 times molar excess reduced the pI to 5.0–6.7, whereas acylation with succinic anhydride at a 30 times molar excess reduced the pI to 4.9–8.7. Comparative biodistribution studies performed in mice (n = 5) showed the reduced renal accumulation of the ^99mTc proportional to the pI reduction. The effect of the pI on the reduced renal uptake was especially pronounced at 15 min postinjection. The reduced renal uptake was also reflected in the reduced whole-body retention, indicating that lowering the pI inhibited the tubular reabsorption of the labeled dsFv.     

New renal function imaging agents. I. Synthesis and biological characterization of a [Tc]diaminomercapto(thio)ether (DAMTE)

The present study describes the synthesis of a [^99mTc]diaminomercapto(thio)ether (DAMTE-derivative) as a first compound of a new class of ^99mTc-complexes which is tubular excreted.

10-Benzoyl-8-keto-7-aza-2-amino-4,10-dithia-decanoic acid (CO₂-DAMTE 3) was synthesized by the reaction of succinimidyl-S-benzoyl-thioglycolate and (S)-2-aminoethyl- -cysteine. The respective technetium complex, ^99mTc---CO₂---DAMTE was obtained in radiochemical yields of about 70% using stannous chloride as reducing agent. Hydrolysis of the protecting group was performed either prior to the complexation of pertechnetate (“cold kit”) or during the labelling reaction (“hot kit”). Organ distribution was determined in Wistar rats. Within 24 h 40% of the activity were excreted into the feces and 43% into the urine, whereas 10% were retained in the kidneys. In contrast, a first human study showed a very fast renal elimination of ^99mTc---CO₂---DAMTE, a low liver uptake (< 10%) and no retention in the kidneys. The renal clearance of approx. 240 mL/min/1.73 m² in addition to the protein binding of > 95% suggests an effective tubular excretion of the compound.     

Tc-2GAM: A tracer for renal imaging

We propose a renal imaging agent, the ^99mTc complex of the bidentate-N,S chelate N-(mercaptoacetyl) glycine (^99mTc-2GAM), with the imaging characteristics of ^99mTc-DMSA but a faster kidney uptake; chemical evidence supports the formulation of ^99mTc-2GAM as [Tc^V(O)(GAM)₂]⁻. After biodistribution and toxicity studies in animals, ^99mTc-2GAM was evaluated in five normal volunteers. ^99mTc-2GAM is rapidly cleared from the blood ( ) and 50% of the ID is excreted in the urine in the first 2 h. Dynamic data show a rapid renal uptake that increases up to 1 h with no significant wash-out between 1 and 8 h. The uptake in each kidney ranges from 11.3% to 20.7% ID. Low, stable liver uptake is observed. No significant activity is detected in other organs. We showed no differences between ^99mTc-2GAM and ^99mTc-DMSA compared in three patients with unilateral kidney disease. We conclude that ^99mTc-2GAM has good practical and dosimetric features for renal imaging.     

Oxotechnetium 99mTcO[SN(R)S][S] complexes as potential 5-HT1A receptor imaging agents

A series of ^99mTcO[SN(R)S][S] complexes carrying the 1-(2-methoxyphenyl)piperazine moiety on the tridentate ligand [SN(R)S] was synthesized. For structural characterization and for in vitro binding assays the analogous oxorhenium complexes were prepared. As demonstrated by appropriate competition binding tests in rat hippocampal preparations, all oxorhenium analogues showed affinity for the 5-HT_1A receptor binding sites with IC₅₀ values at the nanomolar range (IC₅₀= 5.8–103 nM). All ^99mTcO[SN(R)S]/[S] complexes showed significant brain uptake in rats at 2 min p.i. (0.24–1.31% ID). However, a clear correlation between distribution of radioactivity in the brain and distribution of 5-HT_1A receptors could not be established.     

Understanding the radiolabelling mechanism of Tc–antimony sulphide colloid

The chemistry of antimony trisulphide colloid (ATC) was examined to elucidate the radiolabelling mechanism with ^99mTcO₄⁻. Ion exchange chromatography and atomic absorption spectrophotometry techniques determined ATC to be resistant to hydrolysis in 0.1 M hydrochloric acid (HCl) at 25°C or 100°C (>97% recovery, Sb³⁺ absent). Hydrogen sulphide gas detected did not participate in the mechanism, where antimony trisulphide and ^99mTcO₄⁻ in HCl/100°C yielded 96% ^99mTc-product from a K₂S-free formulation (versus 98% when K₂S was present). ^99mTcO₄⁻ was reduced >90% by DMSA or dithiothreitol under the same conditions, identifying involvement of thiol groups. Infrared analysis of Re-ATC showed S=O bonds, indicating excess thiol groups at the colloid surface were oxidised at the expense of ^99mTcO₄⁻ reduction.     

Evaluation of Tc-labeled modified serum albumin for tumor detection

Serum albumin (SA) modified and labeled with ¹³¹l-tyramine N-1′-desoxysorbitol (¹³¹I-TDS) has been shown to localize in tumors [Sinn et al., (1990) Nucl. Med. Biol. Part B 17, 819–827]. We prepared similar TDS complexes labeled with ^99mTc and evaluated their potential for tumor imaging. Derivatization of SA with TDS was optimized using cyanuric chloride or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as coupling agents. A high TDS loading yield of 38 mol/mol SA was obtained with the latter reagent. Modified SA (8 and 38 mol TDS/mol SA) were labeled with ^99mTc via the stannous reduction method and injected i.v. into EMT-6 tumor bearing mice. ¹²⁵I-TDS-SA (8 mol ¹²⁵I-TDS/mol SA) revealed a high tumor uptake of 10% ID/g at 3 h post-injection. The ^99mTc-labeled SA and TDS-SA complexes lacked tumor specificity, instead TDS loading of SA resulted in increased liver/spleen uptake, suggesting colloid formation. This study confirms the potential of modified SA for tumor imaging but highlights the importance of choice of radioisotope, as well as site of attachment of the radiolabel to the modified SA for optimal tumor localization.     

Influence of technetium-99m-labeling conditions on physico-chemical and related biological properties of an acylated poly-galactosidic macrophage targeting agent for inflammation imaging

The potential of ^99m-Tc-J001 for the investigation of inflammatory lesions via the targeting of recruited macrophages (Mφ) has already been documented in several experimental models and in human diseases. To achieve a functional imaging of inflammation via Mφ targeting, minimal labeled colloid content and high in vivo stability of ^99mTc-J001 are essential. The actual specificity of such scintigraphy is closely dependent upon the radiolabeling of only the J001 molecules available for Mφ targeting. To develop an appropriate radiopharmaceutical kit, optimization of the labeling conditions was achieved from a series of pilot formulations that were evaluated for radiolabeling efficiency and both in vitro and in vivo ^99mTc-J001 stability. Colloids were characterized using autocorrelation spectroscopy and multiangle laser-light scattering, radioactive colloid content of the formulations being deduced from biodistribution studies. This work has made possible the definition of a formulation exhibiting a radiolabeling yield >97.0%, associated with in vivo stability and minimal colloid formation, thus greatly enhancing the specificity of such macrophage scintigraphy.     

A kit for labeling of [Re] human serum albumin microspheres for therapeutic use in nuclear medicine

Intra-arterial infusion of labeled particles is an effective method for local endoradiotherapy of tumors. In this study, we present an efficient method of radiolabeling biodegradable human serum albumin (HSA) microspheres with the short-lived beta-emitter ¹⁸⁸Re using a simple and reliable kit. Up to now, it was not possible to label particles with more than 85–90% efficiency. Under optimized reaction conditions, we achieved nearly 100% uptake of ¹⁸⁸Re. Using the proposed kit, we added tartrate solution and increased the pH from 2 to 4–5 after 1 h reaction time at 95 °C. The ¹⁸⁸Re binding to the particles was found to be stable in vitro.     

Influence of a TcN core on the biological and physicochemical behavior of Tc complexes of L,L-EC and L,L-ECD

^99mTc-nitrido complexes of L,L-ethylene dicysteine (^99mTcN-L,L-EC) and ^99mTcN-L,L-ethylene dicysteine diethylester (^99mTcN-L,L-ECD) were prepared and their characteristics compared to those of the respective ^99mTc-oxo complexes. ^99mTcN-L,L-EC and ^99mTcO-L,L-EC migrate to similar extents during electrophoresis at pH 12, but, at pH 6, ^99mTcN-L,L-EC migrates further than ^99mTcO-L,L-EC. Renal excretion of ^99mTcN-L,L-EC is inferior to that of ^99mTcO-L,L-EC, indicating that the TcN-glycine sequence has lower affinity for the renal tubular system. Both ^99mTcO-L,L-ECD and ^99mTcN-L,L-ECD are neutral, but ^99mTcN-L,L-ECD is hydrophilic and shows minimal brain uptake in both mice and the baboon.     

Facile synthesis of bis(hydroxamamide)-based tetradentate ligands for Tc-radiopharmaceutical

A facile, two-step synthesis of the bis(hydroxamamide)-based tetradentate ligands for ^99mTc-radiopharmaceuticals is described. Firstly, the hydroxamamide was converted to hydroximic acid chloride by reaction with sodium nitrite in hydrochloric acid at 0 °C. Secondly, treating the halide with the ethylenediamine or 1,3-propylenediamine in absolute ethanol formed the desired products, N,N′-ethylene bis(1-(4-nitroimidazole-1-yl)–propan-hydroxyiminoamide) (I) and N,N′-propylene bis(1-(4-nitroimidazole-1-yl)-propanhydroxyiminoamide) (II). The corresponding ^99mTc complexes showed high yields and were found by paper electrophoresis to be electrically neutral under physiological conditions. The partition coefficients indicated a distinct difference between the two complexes.     

Alternative quality control for technetium-99m IDA complexes

A quality control procedure for ^99mTc-IDA complexes based on the use of C₁₈ Sep-pak^TM cartridges is developed and the validation of the procedure presented. C₁₈ Sep-pak^TM cartridges are pretreated by washing with 95% ethanol followed by 10⁻³ N hydrochloric acid. A small amount of the ^99mTc-IDA complex is applied, washed with 10⁻³ N hydrochloric acid and eluted with 95% ethanol. The radiochemical purity values obtained for ^99mTc-mebrofenin and ^99mTc-disofenin using this Sep-pak^TM procedure are comparable to those obtained using the standard two strip (ITLC-SG/100% methanol, ITLC-SA/20% saline) procedure.     

In Vivo localization of diglycylcysteine-bearing synthetic peptides by nuclear imaging of oxotechnetate transchelation

A phenomenon of in vivo transchelation of oxotechnetate from a complex with glucoheptonic acid to synthetic peptides bearing oxotechnetate-binding motifs and a technique for in vivo visualization of these peptides are described. Using two model peptides bearing two tandem diglycylcysteine (GGC) motifs (P1) or three GGC motifs (P2), we demonstrated that: (i) these peptides efficiently transchelated oxo-[^99mTc]technetate from a complex with glucoheptonic acid in vitro (a complex with peptides was stable at least 24 h; radiochemical purity exceeded 95% by high performance liquid chromatography); (ii) injection of peptides into the rectus femoris muscle (at 0.5-1 μmol of SH groups) followed by an intravenous injection of ^99mTc-glucoheptonate (0.25-0.5 mCi per animal) yielded visualization of the injected muscle by nuclear imaging within 1 h after injection; (iii) the experimental/control (contralateral) thigh muscle ratio was 1.80 ± 0.05 for peptide P1 and 3.0 ± 0.1 for P2; (iv) the injection of a control peptide P2 with SH groups covalently modified with N-ethylmaleimide resulted in a ratio of 1.4 ± 0.2. These findings argue for specific association of oxo-[^99mTc]technetate with free thiols within the binding motif of injected peptides in vivo. In vivo transchelation of oxo-[^99mTc]technetate may be useful for the purpose of noninvasive imaging of gene expression, i.e., when the expression product bears GGC motifs.     

Scintigraphic imaging with Tc- exorphin C in rabbits

Exorphin C is a peptide with five amino acids [(Tyr-Pro-Ile-Ser-Leu) Trifluoroacetate salt] (Sigma) that has an affinity to opioid receptor-expressing tissues and tumors. Exorphin-C was labeled with ^99mTc using glucoheptonate (GH) as bifunctional chelating agent. Then, we investigated its radiopharmaceutical potential as opioid receptor-expressing tissue on rabbits.

Quality controls were performed by ITLC, paper electrophoresis and HPLC. Labeling efficiency was higher than 98%. The compound was stable for at least 5 h at room temperature. Scintigraphic imaging with ^99mTc–GH–exorphin C (^99mTc–GE) was performed on male Albino rabbits. Static images were obtained from anterior projection using a Camstar XR/T gamma camera at several time intervals. Although a significant amount of activity was seen in the brain, less activity was seen on receptor saturation studies at 30 min. Slight hepatobiliary excretion was seen, though the main excretion route was renal. After saturating, the receptor hepatobiliary excretion was not seen; the only excretion route was renal.     

A single-step kit formulation for the (99m)Tc-labeling of HYNIC-Duramycin

Introduction^99mTc-Duramycin is a unique radiopharmaceutical that binds specifically to phosphatidylethanolamine (PE). The current effort is to develop a single-step kit formulation for the ^99mTc labeling of HYNIC-Duramycin.MethodsA titration series of Tricine/TPPTS coligand systems were tested for an optimal formulation to produce ^99mTc-Duramycin with high radiochemical purity and specific activity. The radiopharmaceutical prepared using the kit formulation was tested for PE binding specificity using polystyrene microbeads coated with different phospholipid species. Radiochemical performance of the kits was assessed after storage at ? 20 °C, room temperature and 37 °C. Biodistribution profile of kit-prepared ^99mTc-Duramycin was characterized in healthy rats at 3, 10, 20, 60 and 180 min after intravenous injection. Binding studies were performed using the rat aortic arch and a rat model of myocardial ischemia/reperfusion, which represent scenarios of physiological and pathological PE externalization.ResultsA Tricine/TPPTS ratio of 10:1 led to a consistent production of ^99mTc-Duramycin with high radiochemical purity (> 90%), whereas a higher ratio at 40:1 produced radiopharmaceuticals with incomplete substitution of Tricine coligand. ^99mTc-Duramycin prepared using the single-step kit formulation retained PE-binding specificity. The kits are stable over long-term storage. The biodistribution profile of kit-prepared ^99mTc-Duramycin is consistent with HPLC purified radiopharmaceutical from prior studies. Binding studies on a tissue level indicate that the radiopharmaceutical is suitable for studying biological processes that involve PE distribution and redistribution in various physiological and pathological conditions.ConclusionA single-step kit formulation is developed for ^99mTc-labeling of HYNIC-Duramycin. The radiopharmaceutical has high radiochemical purity and specific activity, retained PE binding activities, amiable to long-term storage, and is injection-ready for in vivo applications.