Synthesis and biological properties of the lipophilic technetium-99m complex 99mTc(acac)3

In the development of technetium-99m radiopharmaceuticals for the evaluation of regional cerebral perfusion, one series of complexes that has remained unexplored is the neutral lipophilic tris complexes formed with β-diketonato ligands. The prototype complex of this series, tris(2,4-pentanedionato) technetium(III), has been prepared via a new synthetic route and chemically characterized using ⁹⁹Tc and the biodistribution of the no-carrier-added ^99mTc complex has been determined. The ^99mTc complex was found to be distributed throughout the body with persistant high blood levels indicative of a high degree of protein binding. The primary route of excretion was the hepatobiliary system as indicated by the appearance of ^99mTc in the gut and feces at longer sample times post-injection. Although this complex was not retained by the brain, it does provide a starting point from which a more effective agent might be developed.     

Synthesis,radiochemistry and biological evaluation of technetium-99m complexes with 1,8-diamine-3,6-dithiaoctane (DDO) ligands

The synthesis, radiochemical analysis and biological characteristics of some 1,8-diamine-3,6-dithiaoctane derivatives labelled with Tc-99m are reported. Analysis by HPLC shows that most of the ^99mTc-chelates are multicomponent. Furthermore, almost all ^99mTc-complexes isolated by HPLC are lipophilic and stable in vitro. The biodistributions of the most lipophilic of these complexes were evaluated in mice. The N-morpholinylethyl and N,N'′-bisalicylyl derivatives of 1,8-diamine-3,6-dithiaoctane yielded ^99mTc-complexes which exhibit considerable uptake and retention in organs of interest, such as the heart and the brain.     

Synthesis and biological evaluation of EC20: a new folate-derived, (99m)Tc-based radiopharmaceutical

A new peptide derivative of folic acid was designed to efficiently coordinate (99m)Tc. This new chelate, referred to as EC20, was found to bind cultured folate receptor (FR)-positive tumor cells in both a time- and concentration-dependent manner with very high affinity (K(D) approximately 3 nM). Using an in vitro relative affinity assay, EC20 was also found to effectively compete with (3)H-folic acid for cell binding when presented either alone or as a formulated metal chelate. Following intravenous injection into Balb/c mice, (99m)Tc-EC20 was rapidly removed from circulation (plasma t(1/2) approximately 4 min) and excreted into the urine in a nonmetabolized form. Data from gamma scintigraphic and quantitative biodistribution studies performed in M109 tumor-bearing Balb/c mice confirmed that (99m)Tc-EC20 predominantly accumulates in FR-positive tumor and kidney tissues. These results suggest that (99m)Tc-EC20 may be clinically useful as a noninvasive radiodiagnostic imaging agent for the detection of FR-positive human cancers.     

Synthesis and biological evaluation of technetium-99m labeled galactose derivatives as potential asialoglycoprotein receptor probes in a hepatic fibrosis mouse model

Two galactose derivatives, a monovalent ^99mTc-MAMA-MGal galactoside and a divalent ^99mTc-MAMA-DGal galactoside, were synthesized and radiolabeled in high radiochemical purity (>98%). Dynamic microSPECT imaging and biodistribution study of two traces in normal and liver fibrosis mice showed that the ^99mTc-MAMA-DGal revealed higher specific binding to asialoglycoprotein receptors in liver and then rapidly excreted via both hepatobiliary system and renal clearance. The results suggest that ^99mTc-MAMA-DGal may be used as SPECT probes for noninvasive evaluation of asialoglycoprotein receptor-related liver dysfunction.     

Bombesin derivative radiolabeled with technetium-99m as agent for tumor identification

A bombesin derivative was successfully radiolabeled in high labeling yield. Biodistribution studies and scintigraphic images in Ehrlich tumor-bearing mice were performed. This compound showed high accumulation in tumor tissue with high tumor-to-muscle and tumor-to-blood ratios. Thus, ^99mTc-HYNIC-Bombesin_(7–14) could be used as an agent for tumor diagnosis.     

Synthesis and biological evaluation of diethylenetriamine pentaacetic acid-polyethylene glycol-folate: a new folate-derived, (99m)Tc-based radiopharmaceutical

(99m)Technetium-labeled diethylenetriamine pentaacetic acid-polyethylene glycol-folate (DTPA-PEG-folate) was synthesized and tested as a radiopharmaceutical agent, which targeted the lymphatic system with metastatic tumor. Folic acid was reacted with H2N-PEG-NH2 to yield H2N-PEG-folate. After purification by anion-exchange chromatography, the product was reacted with cyclic DTPA. By removal of unreacted DTPA by size-exclusion chromatography, DTPA-PEG-Folate was obtained. Fluorescein-5-isothiocyanate (FITC)-labeled DTPA-PEG-folate and DTPA-PEG-OCH3 were prepared via a dicyclohexylcarbodiimide-mediated coupling. In vitro competitive binding test showed that the uptake of [125I] folic acid was inhibited by DTPA-PEG-folate and the 50% inhibitory concentration was 4.37 pmol/L (R2 = 0.9922). The relative affinity of DTPA-PEG-FITC was 0.18 for human folate receptor comparing with folic acid. In cultured tumor cells, uptake of fluorescence-labeled DTPA-PEG-folate was found to increase significantly in folate-deficient medium compared with that of untargeted DTPA-PEG-OCH3 and FITC-ethylenediamine. The competition with free folic acid blocked the cell uptake of DTPA-PEG-folate. These results confirmed the DTPA-PEG-folate entered into KB cells through the folate receptor endocytosis pathway in vitro. The radiolabeled yield of [(99m)Tc] DTPA-PEG-folate was in excess of 98%, and specific activities of 7.4 kBq (0.2 microCi/microg) were achieved. After subcutaneous injection, [(99m)Tc] DTPA-PEG-folate exhibited an initial increase and successive decline of accumulation in popliteal nodes in normal Wistar rats. Expect for the kidney, uptake by other tissues was rather low. In a normal rabbit imagine study, the lymphatic vessels were readily visualized by single-photon-emission computed tomography following subcutaneous injection of [(99m)Tc] DTPA-PEG-folate. In conclusion, the [(99m)Tc] DTPA-PEG-folate conjugate may have a potential as a lymphatic tumor-targeted radiopharmaceutical.     

Synthesis and biological evaluation of technetium-99m-labeled deoxyglucose derivatives as imaging agents for tumor

Three deoxyglucose (DG) derivatives, S-DG, MAG(3)-DG and MAMA-BA-DG, were synthesized and labeled successfully with high labeling yields and high radio-chemical purities. Biodistribution in tumor-bearing mice demonstrated that these three new (99m)Tc-deoxyglucose derivatives showed accumulation in tumor and high tumor-to-muscle ratios. Among them, the (99m)Tc-MAG(3)-DG showed the best characteristics as a potential tumor marker for single photon emission computed tomography (SPECT).     

Synthesis and biodistribution studies of carbohydrate derivatives radiolabeled with technetium-99m

Three carbohydrate derivatives, MAG₃-Gl, MAG₃-Ga, MAG₃-NG, were synthesized and radiolabeled in high yields. These substances were injected in health Swiss mice and their biodistribution were evaluated. Among them, ^99mTc-MAG₃-Ga displayed higher accumulation in hepatic tissue, due to the presence of specific receptors in the liver for this carbohydrate. Thus, the use of ^99mTc-MAG₃-Ga to assess hepatic function can be considered.     

Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.     

A pre-targeting strategy for imaging glucose metabolism using technetium-99m labelled dibenzocyclooctyne derivative

During the last four decades, nuclear medicine has undergone enormous growth, and positron emission tomography (PET) has been in the driving seat for most of the time. ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) is the most widely used agent for the detection of hibernating myocardium and metabolically active cancer tissue. But its cost and limited availability are the main limitations. For a long time different researchers and groups of pharmacists have tried to label glucose with a cheaper and long-acting radionuclide like ^99mTc. However, they failed to achieve this goal owing to the chemical complexity of ^99mTc and the lack of maintaining the physiological activity of diagnostic compounds. A pre-targeting strategy based on strain-promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) reaction was applied to solve this problem. Functional click synthons were synthesized: 2-azido-2-deoxy-d-glucose (GlucN₃) as a glucose analogue, and N- (2- (2- (2- (bis (pyridin-2-ylmethyl) amino) ethoxy) ethoxy) ethyl-2- (6H-11,12-didehydrodibenzo [a,e] cycloocten-5-ylideneaminooxy) acetamide (C7) as a ^99mTc(CO)₃ labeling and azido-binding group. The results of biodistribution experiments in mice bearing S180 tumor show the relatively high tumor/blood ratio (up to 2.95) and tumor/muscle ratio (up to 6.37), and both of them decreases significantly in the glucose blocking experiment. It indicates that GlucN₃ behaves similarly to glucose and that in vivo SPAAC reactions can occur effectively. It is supposed that this pre-targeting strategy can indeed enhance target specificity and may be used for glucose metabolism imaging in tumor diagnosis.     

Synthesis and biological characterization of novel 2-quinolinecarboxamide ligands of the peripheral benzodiazepine receptors bearing technetium-99m or rhenium

Potential receptor imaging agents based on Tc-99m for the in vivo visualization of the peripheral benzodiazepine receptor (PBR) have been designed on the basis of the information provided by the previously published structure-affinity relationship studies, which suggested the existence of tolerance to voluminous substituents in the receptor area interacting with 3-position of the quinoline nucleus of 2-quinolinecarboxamides 5. In the first step of the investigation, the stereoelectronic features of the above-indicated receptor area were also probed by means of 4-phenyl-3-[(1-piperazinyl)methyl]-2-quinolinecarboxamide derivatives bearing different substituents on the terminal piperazine nitrogen atom (compounds 6a-f). The structure-affinity relationship data confirmed the existence of a tolerance to bulky lipophilic substituents and stimulated the design of bifunctional ligands based on the 4-phenyl-3-[(1-piperazinyl)methyl]-2-quinolinecarboxamide moiety (compounds 6h,j,k,m). The submicromolar PBR affinity of rhenium complexes 6j,m suggests that the presence of their metal-ligand moieties with encaged rhenium is fairly compatible with the interaction with the PBR binding site. Thus, in order to obtain information on the in vivo behavior of these bifunctional ligands, (99m)Tc-labeled compounds 6h,k were synthesized and evaluated in preliminary biodistribution and single photon emission tomography (SPET) studies. The results suggest that both tracers do not present a clear preferential distribution in tissues rich in PBR, probably because of their molecular dimensions, which may hamper both the intracellular diffusion toward PBR and the interaction with the binding site.     

Single isomer technetium-99m tamoxifen conjugates

To produce an imaging agent for breast cancer using a technetium-99m-labeled agent specific for estrogen receptors, an N(2)S(2) bifunctional chelator was conjugated to Z- and E-aminotamoxifens through an amide linker. These bioconjugates have been chelated with both technetium-99m and rhenium. For the Z-isomer, chelation with rhenium in the presence of sodium acetate yields a mixture of two isomers, anti and syn, in a 1:1 ratio and in the presence of hydroxide results in only the anti isomer. Both the Z- and E-tamoxifen conjugates have been chelated with technetium-99m at the tracer level yielding a single isomer product, which is assigned as anti based on chromatographic comparison to the rhenium complexes. Radiochemical yields were consistently greater than 80%, with Sep-Pak column purification yielding a final product with >99% radiochemical purity and no residual starting material. Both in vitro and in vivo biological evaluation of the tamoxifen chelates indicated very limited estrogen receptor binding.     

Synthesis and evaluation of a novel (99m)Tc-labeled bioreductive probe for tumor hypoxia imaging

Tumor hypoxia is closely associated with the malignant progression and/or the high metastatic ability of tumors and often induces resistance to chemo- and/or radiotherapy. Thus, the detection and evaluation of hypoxia is important for the optimization of cancer therapy. We designed a novel (99m)Tc-labeled probe for tumor hypoxia imaging that utilizes bioreductive reactions in hypoxic cells. This probe, which contains a 4-nitrobenzyl ester group, is reduced in hypoxic cells to produce a corresponding carboxylate anion that cannot penetrate cell membranes because of its hydrophilicity and negative charge; therefore, it is expected to be trapped inside hypoxic cells. Based on this unique strategy, we synthesized the Technetium-99m ((99m)Tc)-labeled probe (99m)Tc-SD32. The uptake of (99m)Tc-SD32 in tumor cells was investigated under normoxic and hypoxic conditions. (99m)Tc-SD32 showed sufficient accumulation and good retention in hypoxic cells. In addition, we demonstrated that (99m)Tc-SD32 was subjected to bioreduction in hypoxic cells and was trapped as the corresponding carboxylate anion. These results indicated that (99m)Tc-SD32 would be a promising agent for in vivo hypoxia imaging.     

Synthesis of a diaminedithiol bifunctional chelating agent for incorporation of technetium-99m into biomolecules

The synthesis of a bifunctional chelating agent (BCA), 1, based on the diaminedithiol (DADT) ligand system, is described. The six-step synthetic sequence has been accomplished in 16% overall yield, affording 1, which contains a thiolactone as a reactive moiety, which permits direct coupling to nucleophiles without the formation of byproducts. The reactivity of 1 toward benzylamine and subsequent labeling of the ligand with technetium-99m has been evaluated as a model for preparation of various bioconjugates. Both coupling and exchange labeling occur in high yield under mild conditions, and competition reactions with diethylenetriaminepentaacetic acid (DTPA) indicate the superior stability of the technetium-99m-DADT complex. Preparation of BCA 1 thus provides a new avenue into technetium-labeled radiopharmaceuticals.     

Synthesis of a technetium-99m labeled tricyclic ganciclovir analog for non-invasive reporter gene expression imaging

A potential radiopharmaceutical and HSV1-TK substrate, 3-((1,3-dihydroxypropan-2-yloxy)-methyl)-6-(4-(3-((2-mercaptoethyl)(2-(2-mercaptoethyl-amino)-ethyl)amino)propoxy)phenyl)-3H-imidazopurin-9(5H)-one-oxo-technetium(V), was synthesized via a converging approach and its chemical structure was comparatively characterized with a non-radioactive analog. The final radiochemical purity and yield were 97 and 73%, respectively.     

Synthesis and evaluation of technetium-99m monocationic mixed ligand complexes of phenyl substituted/condensed tetradentate schiff's bases and trimethylphosphine

Tc-99m monocationic mixed ligand complexes of phenyl substituted/condensed Schiff's bases, N,N′-ethylene-bis-(benzoylacetone imine) (L_b) or N,N′-ethylene-bis-(salicylaldehyde imine) (L_c) or N,N′-ethylene-bis-(2-hydroxyacetophenone imine) (L_d) and trimethylphosphine were synthesized to determine the influence of the presence of a phenyl group in these tracers on their heart uptake in rats. A new formulation procedure using aq. β-hydroxypropylcyclodextrin (HPB) solution was developed for intravenous administration of nonpolar ^99mTc complexes. Comparison of biodistribution data for the reference ^99mTc complex from N,N′-ethylene-bis-(acetylacetone imine) and trimethylphosphine using HPB formulation and alternate formulation (0.9% saline) showed the same results. Biodistribution of the title ^99mTc complexes, [^99mTc L_b (PMe₃)₂]⁺, [^99mTc L_c (PMe₃)₂]⁺ and [^99mTc L_d (PMe₃)₂]⁺ showed heart-to-blood activity ratios of 1.7, 2.1 and 1.7, respectively, at 15 min post-injection in rats.     

Nitroimidazoles and hypoxia imaging: synthesis of three technetium-99m complexes bearing a nitroimidazole group: biological results

Several Tc-99m complexes were synthesized, substituted with a nitroimidazole group, in order to visualize hypoxic tissues. The complexes were tested on rats (isolated hearts) and showed no significant uptake under hypoxic conditions.     

Synthesis and biological evaluation of a novel 99mTc labeled 2-nitroimidazole derivative as a potential agent for imaging tumor hypoxia

Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with ^99mTc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue. The biodistribution and scintigraphic image of the corresponding ^99mTc-complex showed accumulation in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.     

Synthesis of [(99m)Tc]DTPA-folate and its evaluation as a folate-receptor-targeted radiopharmaceutical

A DTPA-folate conjugate was radiolabeled with (99m)Tc by stannous chloride reduction of [(99m)Tc]sodium pertechnetate in an aqueous solution of DTPA-folate. The radiochemical purity of the product consistently exceeded 97%, as assessed by thin-layer chromatography employing conditions analogous to those for radiochemical quality control of the radiopharmaceutical [(99m)Tc]DTPA. HPLC demonstrated that the radiolabeled product resulted from the intact DTPA-folate conjugate and not unconjugated DTPA. The ability of [(99m)Tc]DTPA-folate to target folate receptors in vivo was assessed in biodistribution studies with athymic mice bearing subcutaneous folate-receptor-positive human KB cell tumors. As an internal control, previously studied [(111)In]DTPA-folate was coinjected with the [(99m)Tc]DTPA-folate, along with varying amounts of DTPA-folate (0.38 mg/kg, 1.6 mg/kg, or 14 mg/kg). At each DTPA-folate dose, [(99m)Tc]DTPA-folate exhibited tumor uptake comparable to that of the coadministered [(111)In]DTPA-folate, with radiotracer levels declining at the higher DTPA-folate doses due to competitive receptor binding of the unlabeled conjugate. Tumor uptake of both tracers was also competitively blocked by preadministered folic acid dihydrate (2.9 mg/kg). Tumor-to-background tissue contrast obtained with [(99m)Tc]DTPA-folate was generally similar to that obtained with [(111)In]DTPA-folate. The (99m)Tc-labeled DTPA-folate conjugate may have utility as a targeted radiopharmaceutical for imaging neoplastic tissues known to overexpress the folate receptor.