Radiolabeling and preliminary biological evaluation of a (99m)Tc(CO)(3) labeled 3,3'-(benzylidene)-bis-(1H-indole-2-carbohydrazide) derivative as a potential SPECT tracer for in vivo visualization of necrosis

N,N'-bis(diethylenetriamine pentaacetic acid)-3,3'-(benzylidene)-bis-(1H-indole-2-carbohydrazide) (bis-DTPA-BI) was radiolabeled with (99m)Tc(CO)(3). The resulting (99m)Tc(CO)(3)-bis-DTPA-BI was characterized (LC-MS) and evaluated as a potential SPECT tracer for imaging of necrosis in Wistar rats with a reperfused partial liver infarction and Wistar rats with ethanol induced muscular necrosis. To study the specificity, uptake of (99m)Tc(CO)(3)-bis-DTPA-BI was also studied in a mouse model of Fas-mediated hepatic apoptosis. The obtained results indicate that (99m)Tc(CO)(3)-bis-DTPA-BI displays selective uptake in necrotic tissue and can be used for in vivo visualization of necrosis by SPECT.     

Synthesis and preliminary evaluation of radiolabeled bis(zinc(II)-dipicolylamine) coordination complexes as cell death imaging agents

The aim of this study was the development of (??m)Tc labeled bis(zinc(II)-dipicolylamine) (Zn2?-DPA) coordination complexes, and the in vivo evaluation of their usefulness as radiotracers for the detection of cell death. DPA ligand 1 was labeled with (??m)Tc via the (??m)Tc-tricarbonyl core ([(??m)Tc(CO)?-1]3?) or via HYNIC ((??m)Tc-HYNIC-1) in good radiochemical yields. Highest in vitro stabilities were demonstrated for [(??m)Tc(CO)?-1]3?. A mouse model of hepatic apoptosis (anti-Fas mAb) was used to demonstrate binding to apoptotic cells. (??m)Tc-HYNIC-1 showed the best targeting of apoptotic hepatic tissue with a 2.2 times higher liver uptake in anti-Fas treated mice as compared to healthy animals. A rat model of ischemia-reperfusion injury was used to further explore the ability of the (??m)Tc-labeled Zn2?-DPA coordination complexes to target cell death. Selective accumulation could be detected for both tracers in the area at risk, correlating with histological proof of cell death. Area at risk to normal tissue uptake ratios were 3.82 for [(??m)Tc(CO)?-1]3? and 5.45 for (??m)Tc-HYNIC-1.     

Development and evaluation of a 68Ga labeled pamoic acid derivative for in vivo visualization of necrosis using positron emission tomography

In this study, we labeled N,N′-bis(diethylenetriamine pentaacetic acid)-pamoic acid bis-hydrazide (bis-DTPA-PA) with the generator produced PET radionuclide gallium-68 and evaluated ⁶⁸Ga-bis-DTPA-PA as a potential tracer for in vivo visualization of necrosis by positron emission tomography (PET). Radiolabeling was achieved with a decay-corrected radiochemical yield of 63%. Biodistribution and in vivo stability studies in normal mice showed that ⁶⁸Ga-bis-DTPA-PA is cleared faster from normal tissue than the previously reported ^99mTc(CO)₃ complex with bis-DTPA-PA which on the other hand is more stable in vivo. ⁶⁸Ga-bis-DTPA-PA showed a 3.5–5 times higher binding to necrotic tissue than to viable tissue as shown by in vitro autoradiography while no statistically significant increased hepatic uptake was found in a biodistribution study in a mouse model of hepatic apoptosis. Specificity and avidity for necrosis was further evaluated in rats with a reperfused partial liver infarction and ethanol induced muscular necrosis. Dynamic microPET images showed a fast and prolonged uptake of ⁶⁸Ga-bis-DTPA-PA in necrotic tissue with in vivo and ex vivo images correlating well with histochemical stainings. With necrotic to viable tissue activity ratios of 8–15 on ex vivo autoradiography, depending on the necrosis model, ⁶⁸Ga-bis-DTPA-PA showed a faster and higher uptake in necrotic tissue than the ^99mTc(CO)₃ analog. These results show that ⁶⁸Ga-bis-DTPA-PA specifically binds to necrotic tissue and is a promising tracer for in vivo visualization of necrosis using PET.     

(99m)Tc-labeling of ciprofloxacin and nitrofuryl thiosemicarbazone using fac-[(99m)Tc(CO)3(H2O)3] core: evaluation of their efficacy as infection imaging agents

The aim of this study was to radiolabel ciprofloxacin (Cip) and nitrofuryl thiosemicarbazone (NFT) with the fac-[(99m)Tc(CO)(3)(H(2)O)(3)](+) core and to evaluate the ability of the radiopharmaceuticals as tracers in detecting sites of infection. Cip and NFT were radiolabeled with the fac-[(99m)Tc(CO)(3)(H(2)O)(3)](+) core and characterized by RHPLC. The stabilities of the preparations were evaluated in saline and rat serum. In vitro binding studies of the radiopharmaceuticals with S. aureus were performed. Biodistribution studies were conducted at different time points after injecting (i.v.) the radiopharmaceuticals in rats (intramuscularly infected with S. aureus) as well as in rats with sterile inflammation. To assess the infection targeting capacity of (99m)Tc-tricarbonyl ciprofloxacin and nitrofuryl thiosemicarbazone, (99m)Tc(v)O-Cip and (99m)Tc(v)O-NFT were used as control. Scintigraphic imaging studies of tricarbonyl compounds and (99m)Tc(v)O-Cip were performed at 4 h after injection. The radiochemical purities of (99m)Tc(CO)(3)-Cip and (99m)Tc(CO)(3)-NFT were between 97-98% as determined by thin layer chromatography (TLRC) and RHPLC; no further purification is necessary before injection. The radiopharmaceuticals exhibited substantial stability when incubated in isotonic saline and serum up to 24 h. Biodistribution studies showed maximum uptake in the infected rat thigh muscle at 4 h post injection and washing out at slower rate from the infected site than the oxo technetium chelate. The mean ratios of uptake in infected/non-infected thighs were 3.87:1, 3.41:1 and 3.17:1 for (99m)Tc(CO)(3)-Cip, (99m)Tc(CO)(3)-NFT and (99m)Tc(v)O-Cip respectively. During scintigraphic studies, infection sites appeared quite distinctly with (99m)Tc(CO)(3)-Cip and (99m)Tc(CO)(3)-NFT, comparable to the behaviour with (99m)Tc(v)O-Cip. These results encouraged us for further development of infection imaging radiopharmaceuticals based on the (99m)Tc-tricarbonyl core.     

Characterisation and biodistribution of two neutral 99mTc(CO)3 complexes with a tridentate ligand

N-(2-Mercapto-propyl)-1,2-phenylenediamine (MPPDA) and N-beta-aminoethylglycine (AEG) were labelled with 99mTc(CO)3(+) to form the neutral complexes [99mTc(CO)3(MPPDA)] and [99mTc(CO)3(AEG)]. Both complexes were formed in excellent yields and their identity was confirmed by LC-MS. In mice, none of the new tracer agents showed brain uptake. [(99m)Tc(CO)3(MPPDA)] was trapped mainly in the liver and excreted via the hepatobiliary system, whereas [99mTc(CO)3(AEG)] was excreted rapidly via the kidneys to the urine.     

Preparation and properties of 99mTc(CO)3+-labeled N,N-bis(2-pyridylmethyl)-4-aminobutyric acid

Labeling biomolecules with (99m)Tc(CO)(3)(+) ((99m)Tc tricarbonyl) is attracting increasing attention. Although histidine is often considered an ideal bifunctional chelator for (99m)Tc (or (188)Re) tricarbonyl, the family of dipicolylamine carboxylate chelators may be a useful alternative because of the expected ease of synthesis and because the structure provides a pendent carboxylate for potential conjugation to biomolecules. The dipicolylamine chelator N,N-bis(2-pyridylmethyl)-4-aminobutyric acid (BPABA) was synthesized using 4-aminobutyric acid in place of glycine or aminopropionic acid in the literature, to avoid possible involvement of the carboxylate in the complex formation process by forming five- or six-membered chelation rings. Using a commercial tricarbonyl kit (Mallinckrodt), the complex formation properties of both BPABA and commercial histidine with (99m)Tc tricarbonyl were investigated, and the in vitro complex stabilities in saline and in serum were compared. Stability in vivo was also examined following i.v. administration to normal mice. BPABA was synthesized simply and quantitatively by reacting picolyl chloride with aminobutyric acid in one step. On RP HPLC, the product eluted essentially in one peak and the structure was confirmed by ESI-MS. After labeling, both BPABA and histidine were shown by RP HPLC to form tricarbonyl complexes. In both cases, after incubation at 100 degrees C for 20 min, only one predominant peak of (99m)Tc(CO)(3)(+)-histidine or (99m)Tc(CO)(3)(+)-BPABA was apparent, and both complexes were stable at room temperature in saline for at least 24 h. After incubation for 24 h in 37 degrees C serum, by SE HPLC, 20% of the (99m)Tc(CO)(3)(+)-histidine was bound to serum protein compared to less than 10% for (99m)Tc(CO)(3)(+)-BPABA. A 5000 molar excess of histidine at 100 degrees C for 6 h was unable to dissociate (99m)Tc(CO)(3)(+)-BPABA. By contrast, BPABA easily dissociated (99m)Tc(CO)(3)(+)-histidine under the same conditions. Both complexes were stable in vivo in mice, and (99m)Tc(CO)(3)(+)-BPABA showed rapid and specific hepatobiliary clearance while (99m)Tc(CO)(3)(+)-histidine was cleared through the kidneys. In conclusion, BPABA was easily synthesized and was shown to possess properties comparable to histidine for labeling of biomolecules with (99m)Tc tricarbonyl. However, it was found that the chelator concentration required for quantitative (99m)Tc tricarbonyl labeling with both BPABA and histidine were 2 orders higher than that required with more conventional labeling using MAG(3). Finally, the complex (99m)Tc(CO)(3)(+)-BPABA itself was found to clear exclusively via the hepatobiliary pathway and may have value as a potential hepatobiliary imaging agent.     

Carbohydrate-appended 3-hydroxy-4-pyridinone complexes of the [M(CO)3]+ core (M = Re, 99mTc, 186Re)

This work describes the use of 3-hydroxy-4-pyridinone ligands for binding the [M(CO)(3)](+) core (M = Re, Tc) in the context of preparing novel Tc(I) and Re(I) glucose conjugates. Five pyridinone ligands bearing pendent carbohydrate moieties, HL(1-5), were coordinated to the [M(CO)(3)](+) core on the macroscopic scale (M = Re) and on the tracer scale (M = (99m)Tc, (186)Re). On the macroscopic scale the complexes, ReL(1-5)(CO)(3)(H(2)O), were thoroughly characterized by mass spectrometry, IR spectroscopy, UV-visible spectroscopy, elemental analysis, and 1D/2D NMR spectroscopy. Characterization confirmed the bidentate coordination of the pyridinone and the pendent nature of the carbohydrate and suggests the presence of a water molecule in the sixth coordination site. In preliminary biological evaluation, both the ligands and complexes were assessed as potential substrates or inhibitors of hexokinase, but showed no activity. Labeling via the [(99m)Tc(CO)(3)(H(2)O)(3)](+) precursor gave the tracer species (99m)TcL(1-5)(CO)(3)(H(2)O) in high radiochemical yields. Similar high radiochemical yields when labeling with (186)Re were facilitated by in situ preparation of the [(186)Re(CO)(3)(H(2)O)(3)](+) species in the presence of HL(1-5) to give (186)ReL(1-5)(CO)(3)(H(2)O). Stability challenges, incubating (99m)TcL(1-5)(CO)(3)(H(2)O) in the presence of excess cysteine and histidine, confirmed complex stability up to 24 h.     

Characterization of novel histidine-tagged Tat-peptide complexes dual-labeled with (99m)Tc-tricarbonyl and fluorescein for scintigraphy and fluorescence microscopy

To enable concurrent whole body scintigraphy and direct imaging of subcellular localization of permeation peptides, dual-labeled Tat-peptides useful for both radiometric analysis and fluorescence microscopy are desired for molecular imaging applications. Thus, novel dual-labeled D-Tat-peptides comprising Tat-basic domain (hgrkkrrqrrrgc), C-terminus conjugated with fluorescein-5-maleimide (FM) and N-terminus chelated with [(99m)Tc(CO)(3)] via histidine coordination, were synthesized and characterized. In human Jurkat cells, radiotracer uptake and washout studies revealed concentration-dependent accumulation of the dual-labeled Tat-peptide within cells. Subcellular localization of Tat-peptide was confirmed by fluorescence microscopy using an analogous [Re(CO)(3)] dual-labeled Tat-peptide. As seen with C-terminus single-labeled Tat-peptides, localization to the nucleoli was observed with the dual-labeled Tat-peptide, suggesting that the mechanism of Tat-peptide uptake and localization was not dependent on free peptide termini at either end. In Balb/c mice, biodistribution studies performed with the dual-labeled Tat-peptide showed fluorescence intensity by microscopic analysis that visually confirmed and correlated directly with scintigraphic and radiometric data. Of note, following intravenous administration, little brain penetration of these permeation sequences was observed in vivo. His[(99m)Tc(CO)(3)]-, DTPA[(99m)Tc(CO)(3)]-, and epsilon-lys-gly-cys[(99m)Tc(O)]-labeled Tat-peptides showed significant pharmacokinetic differences in liver and kidney depending on labeling strategy, indicating that Tat-peptide biodistribution can be impacted by the chelation moiety coordinated with (99m)Tc. Thus, we have shown that dual-labeled (99m)Tc-tricarbonyl Tat-peptide-FM conjugates can be conveniently synthesized and enable direct comparison of quantitative radiometric and qualitative fluorescence data both in vitro as well as in vivo.     

Influence of the denticity of ligand systems on the in vitro and in vivo behavior of (99m)Tc(I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules

Functionalization of biologically relevant molecules for the labeling with the novel fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) precursor has gained considerable attention recently. Therefore, we tested seven different tridentate (histidine L(1)(), iminodiacetic acid L(2)(), N-2-picolylamineacetic acid L(3)(), N, N-2-picolylaminediacetic acid L(4)()) and bidentate (histamine L(5)(), 2-picolinic acid L(6)(), 2,4-dipicolinic acid L(7)()) ligand systems, with the potential to be bifunctionalized and attached to a biomolecule. The ligands allowed mild radiolabeling conditions with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) (30 min, 75 degrees C). The ligand concentrations necessary to obtain yields of >95% of the corresponding organometallic complexes 1-7 ranged from 10(-)(6) to 10(-)(4) M. Complexes of the general formula "fac-[(99m)TcL(CO)(3)]" (L = tridentate ligand) and "fac-[(99m)Tc(OH(2))L'(CO)(3)]" (L' = bidentate ligand), respectively, were produced. Challenge studies with cysteine and histidine revealed significant displacement of the ligands in complexes 5-7 but only little exchange with complexes 1-4 after 24 h at 37 degrees C in PBS buffer. However, no decomposition to (99m)TcO(4)(-) was observed under these conditions. All complexes showed a hydrophilic character (log P(o/w) values ranging from -2.12 to 0.32). Time-dependent FPLC analyses of compounds 1-7 incubated in human plasma at 37 degrees C showed again no decomposition to (99m)TcO(4)(-) after 24 h at 37 degrees C. However, the complexes with bidentate ligands (5-7) became almost completely protein bound after 60 min, whereas the complexes with tridentate coordinated ligands (1-4) showed no reaction with serum proteins. The compounds were tested for their in vivo stability and the biodistribution characteristics in BALB/c mice. The complexes with tridentate coordinated ligand systems (1-4) revealed generally a good and fast clearance from all organs and tissues. On the other hand, the complexes with only bidentate coordinated ligands (5-7) showed a significantly higher retention of activity in the liver, the kidneys, and the blood pool. Detailed radiometric analyses of murine plasma samples, 30 min p.i. of complex fac-[(99m)TcL(1)(CO)(3)], 1, revealed almost no reaction of the radioactive complex with the plasma proteins. By contrast, in plasma samples of mice, which were injected with complex fac-[(99m)Tc(OH(2))L(5)(CO)(3)](+), 5, the entire radioactivity coeluded with the proteins. On the basis of these in vitro and in vivo experiments, it appears that functionalization of biomolecules with tridentate-chelating ligand systems is preferable for the labeling with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+), since this will presumably result in radioactive bioconjugates with better pharmacokinetic profiles.     

99mTc-labeled bombesin(7-14)NH2 with favorable properties for SPECT imaging of colon cancer

In this report, we present the synthesis and evaluation of the (99m)Tc-labeled beta-Ala-BN(7-14)NH2 (ABN = beta-Ala-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) as a new radiotracer for tumor imaging in the BALB/c nude mice bearing HT-29 human colon cancer xenografts. The gastrin releasing peptide receptor binding affinity of ABN and HYNIC-ABN (6-hydrazinonicotinamide) was assessed via a competitive displacement of (125)I-[Tyr4]BBN bound to the PC-3 human prostate carcinoma cells. The IC 50 values were calculated to be 24 +/- 2 nM and 38 +/- 1 nM for ABN and HYNIC-ABN, respectively. HYNIC is the bifunctional coupling agent for (99m)Tc-labeling, while tricine and TPPTS (trisodium triphenylphosphine-3,3',3'-trisulfonate) are used as coligands to prepare the ternary ligand complex [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] in very high yield and high specific activity. Because of its high hydrophilicity (log P = -2.39 +/- 0.06), [(99m)Tc(HYNIC-ABN)(tricine)(TPPS)] was excreted mainly through the renal route with little radioactivity accumulation in the liver, lungs, stomach, and gastrointestinal tract. The tumor uptake at 30 min postinjection (p.i.) was 1.59 +/- 0.23%ID/g with a steady tumor washout over the 4 h study period. As a result, it had the best T/ B ratios in the blood (2.37 +/- 0.68), liver (1.69 +/- 0.41), and muscle (11.17 +/- 3.32) at 1 h p.i. Most of the injected radioactivity was found in the urine sample at 1 h p.i., and there was no intact [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] detectable in the urine, kidney, and liver samples. Its metabolic instability may contribute to its rapid clearance from the liver, lungs, and stomach. Despite the steady radioactivity washout, the tumors could be clearly visualized in planar images of the BALB/c nude mice bearing the HT-29 human colon xenografts at 1 and 4 h p.i. The favorable excretion kinetics from the liver, lungs, stomach, and gastrointestinal tract makes [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] a promising SPECT radiotracer for imaging colon cancer.     

Cell uptake and radiotoxicity studies of an nuclear localization signal peptide-intercalator conjugate labeled with [99mTc(CO)3]+

A trifunctional bioconjugate consisting of the SV40 nuclear localization signal (NLS) peptide, an aliphatic triamine ligand, and the DNA intercalating pyrene has been synthesized and quantitatively labeled with [(99m)Tc(OH(2))(3)(CO)(3)](+). The radiotoxicity of the resulting nucleus-targeting radiopharmaceutical on B16F1 mouse melanoma cells has been investigated to evaluate the activity of Auger and Coster-Kronig electrons on the viability of cells. We found a dose-dependent significant radiotoxicity of the nucleus-targeting radiopharmaceutical clearly related to the low energy decay of (99m)Tc. These principal results imply a possible therapeutic strategy based on the use of the low-energy Auger electron-emitting (99m)Tc radionuclide attached to nucleus-targeting molecules and comprising an intercalator. Highly efficient DNA targeting vectors could complement the usual role of (99m)Tc in diagnostic applications. The Auger electrons emitted by the (99m)Tc nuclide induce DNA damage leading ultimately, through a mitotic catastrophe pathway, to necrotic cell death. Non-DNA-targeting (99m)Tc complexes display much lower radiotoxicity.     

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.     

Experimental pretargeting studies of cancer with a humanized anti-CEA x murine anti-[In-DTPA] bispecific antibody construct and a (99m)Tc-/(188)Re-labeled peptide

The aim of this study was to localize (99m)Tc and (188)Re radionuclides to tumors, using a bispecific antibody (bsMAb) in a two-step approach where the radionuclides are attached to novel peptides incorporating moieties recognized by one arm of the bsMAb. A chemically cross-linked human/murine bsMAb, hMN-14 x 734 (Fab' x Fab'), anti-carcinoembryonic antigen [CEA] x anti-indium-DTPA was prepared as a prelude to constructing a fully humanized bsMAb for future clinical application. N,N'-o-Phenylenedimaleimide was used to cross-link the Fab' fragments of the two antibodies at their hinge regions. This construct was shown to be >92% pure and fully reactive with CEA and a divalent (indium)DTPA-peptide. For pretargeting purposes, a peptide, IMP-192 [Ac-Lys(In-DTPA)-Tyr-Lys(In-DTPA)-Lys(TscG-Cys-)-NH(2) ?TscG = 3-thiosemicarbazonylglyoxyl?], with two indium-DTPAs and a chelate for selectively binding (99m)Tc or (188)Re, was synthesized. IMP-192 was formulated in a "single dose" kit and later radiolabeled with (99m)Tc (94-99%) at up to 1836 Ci/mmol and with (188)Re (97%) at 459-945 Ci/mmol of peptide. [(99m)Tc]IMP-192 was shown to be stable by extensive in vitro and in vivo testing and had no specific uptake in the tumor with minimal renal uptake. The biodistribution of the hMN-14 x murine 734 bsMAb was compared alone and in a pretargeting setting to a fully murine anti-CEA (F6) x 734 bsMAb that was reported previously [Gautherot, E., Bouhou, J., LeDoussal, J.-M., Manetti, C., Martin, M., Rouvier, E., and Barbet, J. (1997) Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten. Cancer 80 (Suppl.), 2618-2623]. Both bsMAbs maintained their integrity and dual binding specificity in vivo, but the hMN-14 x m734 was cleared more rapidly from the blood. This coincided with an increased uptake of the hMN-14 x m734 bsMAb in the liver and spleen, suggesting an active reticuloendothelial cell recognition mechanism of this mixed species construct in naive mice. Animals bearing GW-39 human colonic cancer xenografts were injected with bsMAb (15 microg) and after allowing 24 or 72 h for the bsMAb constructs to clear from the blood (hMN-14 and murine F6 x 734, respectively), [(188)Re]IMP-192 (7 microCi) or [(99m)Tc]IMP-192 (10 microCi) was injected at a bsMAb:peptide ratio of 10:1. Tumor uptake of [(99m)Tc] or [(188)Re]IMP-192 was 12.6 +/- 5.2 and 16.9 +/- 5.5% ID/g at 3 h postinjection, respectively. Tumor/nontumor ratios were between 5.6 and 23 to 1 for every major organ, indicating that early imaging with (99m)Tc will be possible. Radiation absorbed doses showed a 4.8-, 7.2-, and a 12.6 to 1.0 tumor to blood, kidney, and liver ratios when (188)Re was used. Although this new bsMAb pretargeting approach requires further optimization, it already shows very promising targeting results for both radioimmunodetection and radioimmunotherapy of colorectal cancer.     

A Tc-99m-labeled long chain fatty acid derivative for myocardial imaging

C-11- and I-123-labeled long chain fatty acid derivatives have been reported as useful radiopharmaceuticals for the estimation of myocardial fatty acid metabolism. We have reported that Tc-99m-labeled N-[[[(2-mercaptoethyl)amino]carbonyl]methyl]-N-(2-mercaptoethyl)-6-aminohexanoic acid ([(99m)Tc]MAMA-HA), a medium chain fatty acid derivative, is metabolized by beta-oxidation in the liver and that the MAMA ligand is useful for attaching to the omega-position of fatty acid derivatives as a chelating group for Tc-99m. On the basis of these findings, we focused on developing a Tc-99m-labeled long chain fatty acid derivative that reflected fatty acid metabolism in the myocardium. In this study, we synthesized a dodecanoic acid derivative, MAMA-DA, and a hexadecanoic acid derivative, MAMA-HDA, and performed radiolabeling and biodistribution studies. [(99m)Tc]MAMA-DA and [(99m)Tc]MAMA-HDA were prepared using a ligand-exchange reaction. Biodistribution studies were carried out in normal mice and rats. Then, a high initial uptake of Tc-99m was observed, followed by a rapid clearance from the heart. The maximum heart/blood ratio was 3.6 at 2 min postinjection of [(99m)Tc]MAMA-HDA. These kinetics were similar to those with postinjection of p-[(125)I]iodophenylpentadecanoic acid. Metabolite analysis showed [(99m)Tc]MAMA-HDA was metabolized by beta-oxidation in the body. In conclusion, [(99m)Tc]MAMA-HDA is a promising compound as a long chain fatty acid analogue for estimating beta-oxidation of fatty acid in the heart.     

Biological evaluation of glucose and deoxyglucose derivatives radiolabeled with [99mTc(CO)3(H2O)3]+ core as potential melanoma imaging agents

Glucose 9 and 2-deoxyglucose 10 were successfully synthesized and radiolabeled with [(99m)Tc(CO)(3)(H(2)0)(3)](+) intermediate in high yield. The complexes were characterized by HPLC and its stability with histidine over time was challenged. Cell uptake and biodistribution studies in melanoma-bearing C57BL/6 mice were performed. Both compounds showed accumulation in tumor tissue with high tumor-to-muscle ratios. Thus, D-glucose- and D-2-deoxyglucose-(99m)Tc complex could be considered as agents for melanoma diagnosis.     

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.     

A novel ternary ligand system useful for preparation of cationic (99m)Tc-diazenido complexes and (99m)Tc-labeling of small biomolecules

This report describes a novel ternary ligand system composed of a phenylhydrazine, a crown ether-containing dithiocarbamate (DTC), and a PNP-type bisphosphine (PNP). The combination of three different ligands with (99m)Tc results in cationic (99m)Tc-diazenido complexes, [(99m)Tc(NNAr)(DTC)(PNP)]+, with potential radiopharmaceuticals for heart imaging. Synthesis of cationic (99m)Tc-diazenido complexes can be accomplished in two steps. For example, the reaction of phenylhydrazine with (99m)TcO4- at 100 degrees C in the presence of excess stannous chloride and 1,2-diaminopropane-N,N,N',N'-tetraacetic acid (PDTA) results in the [(99m)Tc(NNPh)(PDTA)n] intermediate, which then reacts with sodium N-(dithiocarbamato)-2-aminomethyl-15-Crown-5 (L4) and N,N-bis[2-(bis(3-ethoxypropyl)phosphino)ethyl]ethoxyethylamine (PNP6) at 100 degrees C for 15 min to give the complex, [(99m)Tc(NNPh)(L4)(PNP6)]+ in high yield (>90%). Cationic complexes [(99m)Tc(NNPh)(DTC)(PNP)]+ are stable for > or = 6 h. Their composition was determined to be 1:1:1:1 for Tc:NNPh:DTC:PNP using the mixed-ligand experiments on the tracer ((99m)Tc) level and was further confirmed by the ESI-MS spectral data of a model compound [Re(NNPh)(L4)(L6)]+. It was found that both DTCs and bisphosphines have a significant impact on the lipophilicity of their cationic (99m)Tc-diazenido complexes. Results from a (99m)Tc-labeling efficiency experiment showed that 4-hydrazinobenzoic acid (HYBA) might be useful as a bifunctional coupling agent for (99m)Tc-labeling of small biomolecules. However, the (99m)Tc-labeling efficiency of HYBA is much lower than that of 6-hydrazinonicotinic acid (HYNIC) with tricine and trisodium triphenylphosphine-3,3',3'-trisulfonate (TPPTS) as coligands.     

Pyrazolyl derivatives as bifunctional chelators for labeling tumor-seeking peptides with the fac-[M(CO)3]+ moiety (M = 99mTc, Re): synthesis, characterization, and biological behavior

Radiolabeling of biologically active molecules with the [(99m)Tc(CO)(3)](+) unit has been of primary interest in recent years. With this in mind, we herein report symmetric (L(1)) and asymmetric (L(2)-L(5)) pyrazolyl-containing chelators that have been evaluated in radiochemical reactions with the synthon [(99m)Tc(H(2)O)(3)(CO)(3)](+) (1a). These reactions yielded the radioactive building blocks [(99m)Tc(CO)(3)(k(3)-L)](+) (L = L(1)-L(5), 2a-6a), which were identified by RP-HPLC. The corresponding Re surrogates (2-6) allowed for macroscopic identification of the radiochemical conjugates. Complexes 2a-6a, with log P(o/w) values ranging from -2.35 to 0.87, were obtained in yields of > or =90% using ligand concentrations in the 10(-5-)10(-4) M range. Challenge studies with cysteine and histidine revealed high stability for all of these radioactive complexes, and biodistribution studies in mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion, occurring primarily through the renal-urinary pathway. Based on the framework of the asymmetric chelators, the novel bifunctional ligands 3,5-Me(2)-pz(CH(2))(2)N((CH(2))(3)COOH)(CH(2))(2)NH(2) (L(6)) and pz(CH(2))(2)N((CH(2))(3)COOH)(CH(2))(2)NH(2) (L(7)) have been synthesized and their coordination chemistry toward (NEt(4))(2)[ReBr(3)(CO)(3)] (1) has been explored. The resulting complexes, fac-[Re(CO)(3)(k(3)-L)]Br (L(6)(7), L(7)(8)), contain tridentate ancillary ligands that are coordinated to the metal center through the pyrazolyl and amine nitrogen atoms, as observed for the other related building blocks. L(6) and L(7) were coupled to a glycylglycine ethyl ester dipeptide, and the resulting functionalized ligands were used to prepare the model complexes fac-[Re(CO)(3)(kappa(3)-3,5-Me(2)-pz(CH(2))(2)N(glygly)(CH(2))(2)NH(2))](+) (9/9a) and fac-[Re(CO)(3)(kappa(3)-pz(CH(2))(2)N(CH(2))(3)(glygly)(CH(2))(2)NH(2))](+) (10/10a) (M = Re, (99m)Tc). These small conjugates have been fully characterized and are reported herein. On the basis of the in vitro/in vivo behavior of the model complexes (2a-6a, 9a, 10a), we chose to evaluate the in vitro/in vivo biological behavior of a new tumor-seeking Bombesin pyrazolyl conjugate, [(L(6))-G-G-G-Q-W-A-V-G-H-L-M-NH(2)], that has been labeled with the [(99m)Tc(CO)(3)](+) metal fragment. Stability, in vitro cell binding assays, and pharmacokinetics studies in normal mice are reported herein.     

Extension of the single amino acid chelate concept (SAAC) to bifunctional biotin analogues for complexation of the M(CO)3(+1) Core (M = Tc and Re): syntheses, characterization, biotinidase stability, and avidin binding

Biotin and avidin form one of the most stable complexes known (K(D) = 10(-15) M(-1)) making this pairing attractive for a variety of biomedical applications including targeted radiotherapy. In this application, one of the pair is attached to a targeting molecule, while the other is subsequently used to deliver a radionuclide for imaging and/or therapeutic applications. Recently, we reported a new single amino acid chelate (SAAC) capable of forming stable complexes with Tc(CO)3 or Re(CO)3 cores. We describe here the application of SAAC analogues for the development of a series of novel radiolabeled biotin derivatives capable of forming robust complexes with both Tc and Re. Compounds were prepared through varying modification of the free carboxylic acid group of biotin. Each 99mTc complex of SAAC-biotin was studied for their ability to bind avidin, susceptibility to biotinidase, and specificity for avidin in an in vivo avidin-containing tumor model. The radiochemical stability of the 99mTc(CO)3 complexes was also investigated by challenging each 99mTc-complex with large molar excesses of cysteine and histidine at elevated temperature. All compounds were radiochemically stable for greater than 24 h at elevated temperature in the presence of histidine and cysteine. Both [99mTc(CO)3(L6)]+1 [TcL6; L6 = biotinylamidopropyl-N,N-(dipicolyl)amine] and [99mTc(CO)3(L12a)]+1 (TcL12; L12 = N,N-(dipicolyl)biotinamido-Boc-lysine; TcL12a; L12a = N,N-(dipicolyl)biotinamide-lysine) readily bound to avidin whereas [99mTc(CO)3(L9)]+1 [TcL9; L9 = N,N-(dipicolyl)biotinamine] demonstrated minimal specific binding. TcL6 and TcL9 were resistant to biotinidase cleavage, while TcL12a, which contains a lysine linkage, was rapidly cleaved. The highest uptake in an in vivo avidin tumor model was exhibited by TcL6, followed by TcL9 and TcL12a, respectively. This is likely the result of both intact binding to avidin and resistance to circulating biotinidase. Ligand L6 is the first SAAC analogue of biotin to demonstrate potential as a radiolabeled targeting vector of biotin capable of forming robust radiochemical complexes with both 99mTc and rhenium radionuclides. Computational simulations were performed to assess biotin-derivative accommodation within the binding site of the avidin. These calculations predict that deformation of the surface domain of the binding pocket can occur to accommodate the transition metal-biotin derivatives with negligible changes to the inner-beta-barrel, the region most responsible for binding and retaining biotin and its derivatives. The biological activity and biodistribution of the technetium complexes TcL6, TcL9, and TcL12a were examined in an avidin tumor model. In the avidin bead tumor localization model, TcL6 demonstrated the most favorable localization with a 7:1 ratio of avidin bead implanted muscle versus normal muscle, while TcL9 exhibited a 2:1 ratio. However, TcL9 displayed no specificity for avidin.     

HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [⁹⁹(m)Tc(CO)₃](+), and shows improved biodistribution with reduced hepatic radioactivity accumulation

Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [??(m)Tc(CO)?](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER)?(:)??? were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [??(m)Tc(CO)?](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER)?(:)???-H? and (HE)?-Z(HER)?(:)???, respectively, could be efficiently purified using IMAC and stably labeled with [??(m)Tc(CO)?](+) and were subsequently compared with the parental H?-Z(HER)?(:)??? having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [??(m)Tc(CO)?](+)-Z(HER2:342)-H? compared to [??(m)Tc(CO)?](+)-H?-Z(HER)?(:)???, and more than 10-fold lower with [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)???. These differences translated into appreciably superior tumor-to-liver ratio for [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)??? compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.