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.     

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.     

Preparation,radiolabeling and biodistribution of a new class of bisaminothiol (BAT) ligands as possible imaging agents

In developing new ligands as potential brain and heart perfusion imaging agents two ligands based upon N₂S₂ donor atoms with the biphenyl backbone were synthesized. Biphenyl-2,2′-bis(N-1-amino-2-methyl-propane-2-thiol) (BP-BAT-TM) and biphenyl-2,2′-bis(N-1-amino-2-ethyl-butane-2-thiol) (BP-BAT-TE) form stable, neutral and lipid soluble complexes with [^99mTc]pertechnetate in the presence of tin(II) tartarate as a reducing agent. The [^99mTc]BP-BAT-TM complex penetrates the blood-brain barrier following i.v. injection into rats. Washout from the brain is fast, indicating no retention. The biodistribution of [^99mTc]BP-BAT-TE in rats showed an intitial heart uptake (0.8% /organ, at 2 min) and a slow washout (0.74% at 15 min). No brain uptake was found (0.05%). Significant uptake and retention in liver was observed. An imaging study of [^99mTc]BP-BAT-TE in a monkey showed no brain uptake and a clear indication of liver uptake and gall bladder clearance. These results indicate that this ligand system may be suitable as the basic core structure for the development of new imaging agents. Further studies with structural variations in the biphenyl backbone are warranted to develop new ^99mTc imaging agents for clinical applications.     

Preparation and evaluation of a 99mTcN–PNP complex of sanazole analogue for detecting tumor hypoxia

A sanazole derivative, having a favorable single electron reduction potential (SERP) value compared to that of misonidazole, was synthesized and radiolabeled with [^99mTcN(PNP)] precursor to evaluate its potential as a hypoxia imaging agent. The complex, which was lipophilic, could be prepared in good yields and challenging studies with cysteine showed stability of the complex against trans-chelation. However, despite being lipophilic as well as possessing favorable SERP value, biodistribution studies of this complex in fibrosarcoma tumor bearing Swiss mice showed low uptake in tumor. This observation is possibly attributed to fast clearance of the complex from blood, whereby the complex spends insufficient time in tumor to get reduced and trapped. Though uptake in tumor was low, slow clearance of activity from tumor suggests reduction and trapping of the complex in hypoxic cells. The present ^99mTc-complex demonstrated acceptable values of tumor to blood (TBR) and tumor to muscle (TMR) ratios. However, low uptake in tumor which may not be indicative of the actual hypoxic status of the tumor, limit the utility of the complex to detect tumor hypoxia.     

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 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.     

Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of <Superscript>99m</Superscript>Tc-labeled recombinant Affibody molecules

Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide ^99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of ^99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied ^99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of ^99mTc. The biodistribution of all ^99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of ^99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.     

Synthesis and in vivo testing of a bromobutyl substituted 1,2-dithia-5,9-diazacycloundecane: a versatile precursor for new 99mTc-bis(aminoethanethiol) complexes

7-(4′-Bromobutyl)-3,3,11,11-tetramethyl-1,2-dithia-5,9-diazacycloundecane (6) an intermediate for the preparation of new ^99mTc-bis(aminoethanethiol) complexes (^99mTc-BAT) was synthesized from the corresponding 7-(4′-phenoxybutyl) derivative (5) by ether cleavage with HBr/AcOH. To demonstrate its versatility as an alkylating agent, 6 was reacted with the amines piperidine, morpholine, NH₃ and l-phenyl-1,3,8-triazaspiro(4,5)decan-4-one, yielding the corresponding N-alkylated amines. Mild ring opening affording the BAT-ligands was achieved by reductive cleavage of the disulphide bonds with threo-2,3-dihydroxy-1,4-dimercaptobutane. The ^99mTc-BAT complexes prepared by the tin-reduction method proved to be stable under in vitro conditions. With the exception of the 7-(4′-aminobutyl) substituted one, the ^99mTc-BAT complexes revealed octanol-buffer partition coefficients (P) of log P > 1 at physiological pH. The complexes proved to be neutral and the amount of ultrafiltrable ^99mTc-BAT complex varied between 8–18%. In contrast to the good in vitro characteristics, the brain uptake values in CD-1 mice were comparably low.     

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.     

Biolocalization and cell labeling properties of neutral-lipophilic 99mTc-amine-phenol chelates

^99mTc-diamine-diphenol chelates are neutral lipophilic chelates exhibiting good stability in aqueous solutions. The cell labeling and biolocalization properties of four different ^99mTc-amine-phenol complexes were determined. All four chelates readily labeled leukocytes and RCBs in high yields. Even though ^99mTc was retained by the cells, the elution rate of ^99mTc from the labeled cells in plasma at 37 °C was unacceptably high for potential utility in scintigraphic imaging. The uptake of ^99mTc in brain or heart following i.v. injection of the chelates in rats was low and clearance of activity from the blood was slow.     

Influence of ligand denticity on the properties of novel ⁹⁹mTc(I)-carbonyl complexes. Application to the development of radiopharmaceuticals for imaging hypoxic tissue

An important issue in the development of metal-based radiopharmaceuticals is the selection of the labelling strategy in order to couple the metal to the pharmacophore without losing the biological activity. With the aim to evaluate the correlation between ligand denticity and biological behaviour of the corresponding (99m)Tc complexes, we designed a tridentate and a bidentate 5-nitroimidazole derivatives suitable for (99m)Tc(I) tricarbonyl complexation and with potential use as radiopharmaceuticals towards hypoxic tissue diagnosis. Ligands were synthesized using metronidazol, a pharmaceutical containing the bioreductive pharmacophore as starting material. The chelating units were connected to the pharmacophore using the click reaction of Huisgen. Both (99m)Tc complexes were obtained in high yield and were hydrophilic and stable in labelling milieu. The complex obtained from the tridentate ligand exhibited high stability in human plasma, low protein binding and a favourable biodistribution characterized by low blood and liver uptake, fast elimination and negligible uptake in other organs or tissues. Selective uptake and retention in tumour together with favourable tumour/muscle ratio makes this (99m)Tc-complex a promising candidate for further evaluation as potential hypoxia imaging agent in tumours. The bidentate ligand, on the other hand, yielded a less stable (99m)Tc-complex that experimented hydrolysis in vitro and decomposition in human plasma and showed high protein binding, high blood and liver uptake and moderate excretion. Although selective uptake and retention in tumour was also observed physicochemical and biological behaviour are inadequate for in vivo use, demonstrating that denticity of the ligand is particularly important and that tridentate ligands are preferable in order to prepare (99m)Tc-tricarbonyl complexes for Nuclear Medicine imaging.     

First example of well-characterized Re and Tc tricarbonyl complexes of ciprofloxacin and norfloxacin in the development of infection-specific imaging agents

Aiming at the development of ^99mTc-based infection-specific imaging agents, the synthesis and characterization of rhenium and technetium-99m tricarbonyl complexes with derivatized ciprofloxacin and norfloxacin is hereby reported. The ligands were prepared by coupling the tridentate chelator picolylamino-N,N-diacetic acid (PADA) with the piperazinyl (NH) nitrogen of ciprofloxacin or norfloxacin, through the employment of the PADA anhydride. The corresponding rhenium complexes were synthesized using the fac-[NEt₄]₂[ReBr₃(CO)₃] precursor and were fully characterized by elemental analysis and NMR spectroscopy. X-ray crystallography of the ciprofloxacin complex showed that the geometry about rhenium is distorted octahedral defined by the NNO donor atom set of the tridentate chelator and the three carbonyl groups. The analogous technetium-99m complexes were prepared quantitatively through the use of the fac-[^99mTc(H₂O)₃(CO)₃]⁺ precursor and their structure was established by comparative HPLC studies using the well-characterized rhenium complexes as reference. Preliminary studies with the technetium-99m complexes showed high bacterial uptake in vitro.     

99mTc/Re complexes based on flavone and aurone as SPECT probes for imaging cerebral β-amyloid plaques

Two ^99mTc/Re complexes based on flavone and aurone were tested as potential probes for imaging β-amyloid plaques using single photon emission computed tomography. Both ^99mTc-labeled derivatives showed higher affinity for Aβ(1–42) aggregates than did ^99mTc-BAT. In sections of brain tissue from an animal model of AD, the Re-flavone derivative 9 and Re-aurone derivative 19 intensely stained β-amyloid plaques. In biodistribution experiments using normal mice, ^99mTc-labeled flavone and aurone displayed similar radioactivity pharmacokinetics. With additional modifications to improve their brain uptake, ^99mTc complexes based on the flavone or aurone scaffold may serve as probes for imaging cerebral β-amyloid plaques.     

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

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

The effect of ligand structure on glutathione-mediated decomposition of propylene amine oxime derivatives

Tetramethylpropyleneamine oxime (TMPAO) was synthesized and complexed to ^99mTc. ^99mTc-TMPAO samples, when challenged with reduced glutathione (GSH), were shown to have two GSH sensitive components, similar to a mixture of d,l and meso ^99mTc-HMPAO. One component had a GSH-induced second-order dissociation rate constant (K₂) similar to ^99mTc-meso-HMPAO. Despite the presence of a large fraction of this component in these samples, brain uptake and autoradiographic studies with ^99mTc-TMPAO were equivalent to ^99mTc-d,l-HMPAO suggesting that both the d,l and meso ^99mTc-TMPAO isomers are efficiently trapped in brain.     

Lipophilic 99mTc-nitride radiopharmaceuticals as potential myocardial imaging agents

Monocationic ^99mTc-nitrido complexes of a variety of diphosphine ligands have been prepared and the in vivo distribution of such cations has been examined in Sprague-Dawley rats. These complexes show initially high myocardial uptake with subsequent wash-out in this animal model. The lack of myocardial retention can be attributed to the facile in vivo reduction of these cations.     

Effect of alterations in erythropoiesis on distribution of 99mTc sulfur colloid in the rat

The distribution of ^99mTc sulfur colloid and ⁵⁹Fe was assessed in rats following long or short term stimulation or suppression of erythropoiesis. Acute stimulation of erythropoiesis did not alter ^99mTc sulfur colloid distribution, whereas, long term stimulation resulted in increased marrow colloid uptake. Suppression of erythropoiesis by hypoxia-induced plethora or hypertransfusion did not alter the marrow uptake of ^99mTc sulfur colloid. ^99mTc sulfur colloid blood clearance was not altered by any of the experimental conditions utilized. These observations suggest that marrow RE activity as assessed by ^99mTc sulfur colloid uptake increases with erythropoietic stimulation and varies with the duration and intensity of the stimulus.     

Replacement of the Lys linker with an Arg linker resulting in improved melanoma uptake and reduced renal uptake of Tc-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptide

The purpose of this study was to reduce the non-specific renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide through structural modification or l-lysine co-injection. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys^3,4,10, d-Phe⁷, Arg¹¹] α-MSH_3-13 {(Arg¹¹)CCMSH} through the Arg linker (substituting the Lys linker) to generate a novel RGD-Arg-(Arg¹¹)CCMSH hybrid peptide. The melanoma targeting and pharmacokinetic properties of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The effect of l-lysine co-injection on the renal uptake was determined through the co-injection of l-lysine with ^99mTc-RGD-Arg-(Arg¹¹)CCMSH or ^99mTc-RGD-Lys-(Arg¹¹)CCMSH. Replacement of the Lys linker with an Arg linker exhibited a profound effect in reducing the non-specific renal uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH, as well as increasing the tumor uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH compared to ^99mTc-RGD-Lys-(Arg¹¹)CCMSH. ^99mTc-RGD-Arg-(Arg¹¹)CCMSH exhibited high tumor uptake (21.41 ± 3.74% ID/g at 2 h post-injection) and prolonged tumor retention (6.81 ± 3.71% ID/g at 24 h post-injection) in B16/F1 melanoma-bearing mice. The renal uptake values of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH were 40.14–64.08% of those of ^99mTc-RGD-Lys-(Arg¹¹)CCMSH (p <0.05) at 0.5, 2, 4 and 24 h post-injection. Co-injection of l-lysine was effective in decreasing the renal uptakes of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH by 27.7% and ^99mTc-RGD-Lys-(Arg¹¹)CCMSH by 52.1% at 2 h post-injection. Substitution of the Lys linker with an Arg linker dramatically improved the melanoma uptake and reduced the renal uptake of ^99mTc-RGD-Arg-(Arg¹¹)CCMSH, warranting the further evaluation of ¹⁸⁸Re-labeled RGD-Arg-(Arg¹¹)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.     

A developmental analysis of differences in the uptake of [123I]isopropyliodoamphetamine versus99mTc-pertechnetate by the choroid plexus and brain

The uptake of intravascular [¹²³I]isopropyliodoamphetamine (IMP) and^99mTc-pertechnetate into choroid plexus (CP) and brain (frontal cortex) was studied by an indicator fractionation method applied to immature, ketamine-anesthetized Sprague-Dawley rats (1.5, 2, and 3 wk). Assessment of the rate and extent of uptake of these indicators provides functional information (eg blood flow; transport) about various regions of the developing CNS. IMP uptake by lateral ventricle CP was 1.15 ml/g/min in 1.5-wk-old infant rats and gradually increased to 3.9 ml/g/min by adulthod (7–8 wk) (P<0.05); over the same postnatal period,^99mTc uptake went from 2.82 to 3.18 ml/g/min. IMP uptake by cortex was 0.39 and 0.99 ml/g/min in infants and adults, respectively (P<0.05); however,^99mTc uptake by cortex was only 0.07±0.01 ml/g/min at all ages, reflecting early development of blood-brain barrier (BBB) to pertechnetate. Overall, our findings indicated a progressive increase with age in the rate of uptake of IMP by CP and brain; and that^99mTc penetration into CP was relatively constant and substantially greater than into cortex at all developmental stages. Thus the nature of uptake of IMP, relative to^99mTc, was markedy different at the blood-cerebrospinal fluid barrier (i.e., CP) vs. the blood-brain barrier.     

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

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