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.     

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.     

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.     

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.     

The use of diaminodithiol for labeling small molecules with technetium-99m

To investigate the labeling of small molecules with ^99mTc by the bifunctional chelate approach, we have synthesized both a fatty acid and an estrone derivative containing a chelator of the N₂S₂ type. In the case of the fatty acid, this was a diaminodithiol (DADT) while for the estrone, a diaminodisulfide (DADS) was attached. The estrone derivative (5-(2-methylene estrone 3-methyl ether)-3,3,10,10-tetramethyl-1, 2-dithia-5,8-diazacyclodecane hydrochloride, DADS-E) was prepared by alkylation of DADS while the fatty acid derivative (N-(11-undecanoic acid)-N,N′-bis(2-methyl-2-mercaptopropyl) ethylenediamine hydrochloride, DADT-FA) was synthesized by alkylation of DADS followed by reduction. DADS-E was labeled in ethanol at elevated temperatures while DADT-FA was labeled at room temperature, both by stannous reduction. Paper chromatography showed both to be labeled and reverse-phase HPLC showed multiple peaks for both. Serum stability studies were performed by incubation at 37 °C with aliquots removed at 1 min and 1 day for analysis by size-exclusion HPLC. Initially, little pertechnetate or binding to serum proteins was observed whereas after 1 day the majority of activity in both cases was protein bound with 20 and 38% pertechnetate appearing for DADT-FA and DADS-E respectively. In conclusion, small biologically active molecules may be labeled with ^99mTc through an attached diaminodithiol or diaminodisulfide group.     

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.     

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.     

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.     

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.     

Comparative evaluation of 99mTc-labeled aminothiols as possible brain perfusion imaging agents

Several new ^99mTc aminodithiols were prepared and evaluated comparatively in experimental animals. The ligands were diamine, triamine or tetramine dithiols. Substituents were either attached on one of the nitrogens or introduced in between the two nitrogens of diamino dithiol (DADT) backbone. ^99mTc-derivatives prepared by coupling DADT to secondary amines via ethylene group showed in mice high initial brain uptake and significant retention in brain tissue. These preparations were mixtures of more than one ^99mTc-complex differing in brain uptake and clearance from the brain. The highest brain retention (brain to blood ratio 2.53, 15 min p.i.) was achieved with the ^99mTc-complex prepared by coupling DADT with ethylene pyrrolidine. Lengthening the chain between the nitrogens of DADT moiety by introducing methyl or amino alkyl groups resulted in ^99mTc-complexes with poor brain accumulation.     

Similarity of copper and technetium binding sites in human IgG

Kits for direct labeling of IgG with ^99mTc were used without modification for the preparation of [⁶⁷Cu]IgG. The IgG was pre-treated to generate thiolate groups which would bind ⁶⁷Cu. The direct labeling of reduced IgG with ⁶⁷Cu was highly efficient, resulting in approx. 95% ⁶⁷Cu binding. Non-reduced IgG (negative control) had labeling efficiencies of less than 10%. IgG pre-exposed to Cu(II) had reduced amounts of ^99mTc bound to it. The results demonstrate a direct relationship between copper- and ^99mTc-binding sites in IgG.     

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.     

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 and SAR of 99mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates

Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel ^99mTc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells. IC₅₀ values ranged from 3.8 ± 2 to >2000 nM. A linker between the SAAC chelate and pharmacophore was required for high affinity binding. However, extending the length of the linker did not substantially improve binding. PSMA binding was also influenced by the nature of the SAAC chelate. One of the most potent compounds, 23b (IC₅₀ = 4.8 ± 2.7 nM), was radiolabeled with technetium tricarbonyl ({^99mTc(CO)₃}⁺) to afford the {^99mTc(CO)₃}⁺ complex in excellent yield and high purity. This effort has led to the identification of a diverse series of promising high affinity {^99mTc(CO)₃}⁺ radiolabeled PSMA inhibitors.     

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 new [Tc(N)(PS)]-based mixed-ligand compounds useful in the design of target-specific radiopharmaceuticals: the 2-methoxyphenylpiperazine dithiocarbamate derivatives as an example

This study presents the first application of a general procedure based on the use of the [Tc(N)Cl(PS)(PPh₃)] species (PS is an alkyl phosphinothiolate ligand) for the preparation of Tc(N) target-specific compounds. [Tc(N)Cl(PS)(PPh₃)] selectively reacts with an appropriate dithiocarbamate ligand (S^∧Y) to give [Tc(N)(PS)(S^∧Y)] compounds. 1-(2-Methoxyphenyl)piperazine, which displays a potent and specific affinity for 5HT_1A receptors, was selected as a functional group and conjugated to the dithiocarbamate unit through different spacers (L ⁿ ). [^99mTc(N)(PS)(L ⁿ )] complexes were prepared in high yield (more than 90%). The chemical identity of ^99mTc complexes was determined by high performance liquid chromatography comparison with the corresponding ^99gTc complexes. All complexes were found to be inert toward transchelation with an excess of glutathione and cysteine. No notable biotransformation of the native compound into different species by the in vitro action of the serum and liver enzymes was shown. Nanomolar affinity for the 5HT_1A receptor was obtained for [^99mTc(N)(PSiso)L³] (IC₅₀ = 1.5 nM); a reduction of the affinity was observed for the other complexes as a function of the shortening of the alkyl chain interposed between the dithiocarbamate and the pharmacophore. Negligible brain uptake was found from in vivo distribution data of [^99mTc(N)(PSiso)L³]. The key finding of this study is that the complexes maintained good affinity and selectivity for 5HT_1A receptors, and the IC₅₀ value for [^99gTc(N)(PSiso)L³] being comparable to the IC₅₀ value found for WAY 100635. This result confirmed the possibility of preparing [^99mTc(N)(PS)]-based target-specific compounds without affecting the affinity and selectivity of the bioactive molecules for the corresponding receptors.     

A simple method for producing a technetium-99m-labeled liposome which is stable In Vivo

A new method for labeling preformed liposomes with technetium-99m (^99mTc) has been developed which is simple to perform and stable in vivo. Previous ^99mTc-liposome labels have had variable labeling efficiencies and stability. This method consistently achieves high labeling efficiencies (> 90%) with excellent stability. A commercially available radiopharmaceutical kit—hexamethylpropyleneamine oxime (HM-PAO)—is reconstituted with ^99mTcO^−₄ and then incubated with preformed liposomes that encapsulate glutathione. The incubation takes only 30 min at room temperature. Liposomes that co-encapsulate other proteins such as hemoglobin or albumin, in addition to glutathione, also label with high efficiency. Both in vitro and in vivo studies indicate good stability of this label. Rabbit images show significant spleen and liver uptake at 2 and 20 h after liposome infusion without visualization of thyroid, stomach or bladder activity.This labeling method can be used to study the biodistribution of a wide variety of liposome preparations that are being tested as novel drug delivery systems. This method of labeling liposomes with ^99mTc may also have applications in diagnostic imaging.     

99mTc-nitrido radiopharmaceuticals based on nitrogen heterocyclic ligands containing a thiol group

The Chromatographic and in vivo behaviour in mice of the ^99mTcN- and ^99mTc(Sn)-complexes of 2-mercaptopyridine, 2-mercaptopyrimidine, thiouracil, 6-mercaptopurine and thioguanine is compared. The biological distribution of the ^99mTcN-complexes is generally different to that of the ^99mTc(Sn)-complexes of the same ligand with the difference being very dependent on the structure of the ligand. The ^99mTcN-mercaptopyrimidine complex has potential as a blood-cell labelling agent.     

99mTc(N)-DBODC(5), a potential radiolabeled probe for SPECT of multidrug resistance: in vitro study

[^99mTc(N)(DBODC)(PNP5)]⁺ [DBODC is bis(N-ethoxyethyl)dithiocarbamato; PNP5 is bis(dimethoxypropylphosphinoethyl)ethoxyethylamine], abbreviated as ^99mTc(N)-DBODC(5), is a lipophilic cationic mixed compound investigated as a myocardial imaging agent. The findings that this tracer accumulates in mitochondrial structures through a mechanism mediated by the negative mitochondrial membrane potential and that the rapid efflux of ^99mTc(N)-DBODC(5) from nontarget tissues seems to be associated with the multidrug resistance (MDR) P-glycoprotein (P-gp) transport function open up the possibility to extend its clinical applications to tumor imaging and noninvasive MDR studies. The rate of uptake at 4 and 37 °C of ^99mTc(N)-DBODC(5) was evaluated in vitro in selected human cancer cell lines and in the corresponding sublines before and after P-gp and/or MDR-associated protein (MRP) modulator/inhibitor treatment using ^99mTc-sestamibi as a reference. The results indicated that (1) the uptake of both ^99mTc(N)-DBODC(5) and ^99mTc-sestamibi is correlated to metabolic activity of the cells and (2) the cellular accumulation is connected to the level of P-gp/MRP expression; in fact, an enhancement of uptake in resistant cells was observed after treatment with opportune MDR inhibitor/modulator, indicating that the selective blockade of P-gp/MRP prevented efflux of the tracers. This study provides a preliminary indication of the applicability of ^99mTc(N)-DBODC(5) in tumor imaging and in detecting P-gp/MRP-mediated drug resistance in human cancer. In addition, the possibility to control the hydrophobicity and pharmacological activity of this heterocomplex through the variation of the substituents on the ligands backbone without affecting the P₂S₂ coordinating sphere makes ^99mTc(N)-DBODC(5) a suitable scaffold for the preparation of a molecular probe for single photon emission computed tomography of MDR.     

Preparation, Tc-labeling and biodistribution studies of a PNA oligomer containing a new ligand derivative of 2,2′-dipicolylamine

A new azido derivative of 2,2′-dipicolylamine (Dpa), 2-azido-N,N-bis((pyridin-2-yl)methyl)ethanamine, (Dpa-N₃) was readily prepared from the known 2-(bis(pyridin-2-ylmethyl)amino)ethanol (Dpa-OH). It was demonstrated that Dpa-N₃ could be efficiently labeled with both [Re(CO)₃(H₂O)₃]Br and [^99mTc(H₂O)₃(CO)₃]⁺ to give [Re(CO)₃(Dpa-N₃)]Br and [^99mTc(CO)₃(Dpa-N₃)]⁺, respectively. Furthermore, Dpa-N₃ was successfully coupled, on the solid phase, to a Peptide Nucleic Acid (PNA) oligomer (H-4-pentynoic acid-spacer-spacer-tgca-tgca-tgca-Lys-NH₂; spacer = -NH-(CH₂)₂-O-(CH₂)₂-O-CH₂-CO-) using the Cu(I)-catalyzed [2 + 3] azide/alkyne cycloaddition (Cu-AAC, often referred to as the prototypical “click” reaction) to give the Dpa-PNA oligomer. Subsequent labeling of Dpa-PNA with [^99mTc(H₂O)₃(CO)₃]⁺ afforded [^99mTc(CO)₃(Dpa-PNA)] in radiochemical yields > 90%. Partitioning experiments in a 1-octanol/water system were carried out to get more insight on the lipophilicity of [^99mTc(CO)₃(Dpa-N₃)]⁺ and [^99mTc(CO)₃(Dpa-PNA)]. Both compounds were found rather hydrophilic (log D_o/w values at pH = 7.4 are −0.50: [^99mTc(CO)₃(Dpa-N₃)]⁺ and −0.85: [^99mTc(CO)₃(Dpa-PNA)]. Biodistribution studies of [^99mTc(CO)₃(Dpa-PNA)] in Wistar rats showed a very fast blood clearance (0.26 ± 0.1 SUV, 1 h p.i.) and modest accumulation in the kidneys (5.45 ± 0.45 SUV, 1 h p.i.). There was no significant activity in the thyroid and the stomach, demonstrating a high in vivo stability of the ^99mTc-labeled Dpa-PNA conjugate.