Radiosynthesis and micro-SPECT imaging of 99mTc-dendrimer poly(amido)-amine folic acid conjugate

Acetylated (Ac) dendrimer poly(amido)-amine (PAMAM) generation 5 (G5) reacted with folic acid (FA), followed by reacting with 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetria minepentaacetic acid (1B4M DTPA) to form the conjugate of Ac-G5-FA-1B4M DTPA which was further radiolabeled with ^99mTc. The radiochemical yield is up to 98.9% with excellent in vitro/in vivo stability, rapid blood clearance and certain tumor accumulation which was further confirmed by micro-SPECT imaging study.     

Novel Tat-peptide chelates for direct transduction of technetium-99m and rhenium into human cells for imaging and radiotherapy

Rapid and efficient delivery of radioactive metal complexes to the cell interior would enable novel applications in medical imaging and radiotherapy. Membrane permeant peptide conjugates incorporating HIV-1 Tat transactivation protein sequences (GRKKRRQRRR) and an appropriate peptide-based motif (epsilon-KGC) that provides an N(3)S donor core for chelating technetium and rhenium were synthesized. Oxotechnetium(V) and oxorhenium(V) Tat-peptide complexes were prepared by facile transchelation reactions with permetalates, tin(II) chloride and sodium glucoheptonate. RP-HPLC showed two major [(99m)Tc]Tat-peptide species (4) that differed in retention time by approximately 2 min corresponding to two [Re]Tat-peptide species (7) shown to have identical mass, consistent with formation of two isomers, likely the oxo-metal diastereomers. [(99m)Tc]Tat-peptides were stable to transchelation in vitro. In human Jurkat cells, [(99m)Tc]Tat-peptide 4 showed concentrative cell accumulation (30-fold greater than extracellular concentration) and rapid uptake kinetics (t(1/2) < 2 min) in a diastereomeric-comparable manner. Paradoxically, uptake was enhanced in 4 degrees C buffer compared to 37 degrees C, while depolarization of membrane potential as well as inhibition of microtubule function and vesicular trafficking showed no inhibitory effect. Cells preloaded with 4 showed rapid washout kinetics into peptide-free solution. Modification of [(99m)Tc]Tat-peptide by deletion of the N-terminus Gly with or without biotinylation minimally impacted net cell uptake. In addition, the C-terminus thiol of the prototypic Tat-peptide was labeled with fluorescein-5-maleimide to yield conjugate 8. Fluorescence microscopy directly localized conjugate 8 to the cytosol and nuclei (possibly nucleolus) of human Jurkat, KB 3-1 and KB 8-5 tumor cells. Preliminary imaging studies in mice following intravenous administration of prototypic [(99m)Tc]Tat-peptide 4 showed an initial whole body distribution and rapid clearance by both renal and hepatobiliary excretion. Analysis of murine blood in vivo and human serum ex vivo revealed >95% intact complex, while murine urine in vivo showed 65% parent complex. Thus, these novel Tat-peptide chelate conjugates, capable of forming stable [Tc/Re(V)]complexes, rapidly translocate across cell membranes into intracellular compartments and can be readily derivatized for further targeted applications in molecular imaging and radiotherapy.     

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.     

Avidin-dendrimer-(1B4M-Gd)(254): a tumor-targeting therapeutic agent for gadolinium neutron capture therapy of intraperitoneal disseminated tumor which can be monitored by MRI.

Peritoneal carcinomatosis is a late stage in cancer progress, for which no effective therapeutic modality exists. Targeting therapeutic agents to disseminated lesions may be a promising modality for treating peritoneal carcinomatosis. Gadolinium ((157,155)Gd) is known to generate Auger and internal conversion electrons efficiently by irradiation with a neutron beam. Auger electrons from neutron-activated Gd(III) are strongly cytotoxic, but only when Gd(III) atoms have been internalized into the cells. In the present investigation, we have developed a quickly internalizing tumor-targeting system to deliver large quantities of Gd(III) atoms into tumor cells to generate the Auger emission with an external neutron beam. Simultaneously, one would be able to image its biodistribution by MRI with a shortened T1 relaxation time. Avidin-G6-(1B4M-Gd)(254) (Av-G6Gd) was synthesized from generation-6 polyamidoamine dendrimer, biotin, avidin, and 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M). The Av-G6Gd was radiolabeled with Gd(III) doped with (153)Gd. All of the 1B4M's on the conjugate were fully saturated with Gd(III) atoms. An in vitro internalization study showed that Av-G6Gd accumulated and was internalized into SHIN3 cells (a human ovarian cancer) 50- and 3.5-fold greater than Gd-DTPA (Magnevist) and G6-(1B4M-Gd)(256) (G6Gd). In addition, accumulation of Gd(III) in the cells was detected by the increased signal on T1-weighted MRI. A biodistribution study was performed in nude mice bearing intraperitoneally disseminated SHIN3 tumors. Av-G6Gd showed specific accumulation in the SHIN3 tumor (103% ID/g) 366- and 3.4-fold greater than Gd-DTPA (0.28% ID/g, p < 0.001) and G6Gd (30% ID/g, p < 0.001) 1 day after i.p. injection. Seventy-eight percent of the tumor-related radioactivity of Av-G6Gd in the SHIN3 tumor was located inside the cells. The SHIN3 tumor-to-normal tissue ratio was greater than 17:1 in all organs and increased up to 638:1 at 1 day after i.p. injection. In conclusion, a sufficient amount (162 ppm) of Av-G6Gd was accumulated and internalized into the SHIN3 cells both in vitro and in vivo to kill the cell using (157/155)Gd with external irradiation with an appropriate neutron beam while monitoring with MRI. Thus, Av-G6Gd may be a promising agent for Gd neutron capture therapy of peritoneal carcinomatosis. This reagent also has the potential to permit monitoring of its pharmacokinetic progress with MRI.     

Synthesis and biodistribution of [99mTc]-N-[4-nitro-3-trifluoromethyl-phenyl] cyclopentadienyltricarbonyltechnetium carboxamide, a nonsteroidal antiandrogen flutamide derivative

In our efforts to develop a novel class of SPECT imaging agents based on nonsteroidal androgen receptor (AR) antagonists, we have synthesized N-cyclopentadienyltricarbonyltechnetium-N-[4-nitro-3-trifluoromethyl-phenyl] carboxamide (NF(99m)Tc), an analog of the AR antagonist ligand flutamide. NF(99m)Tc was obtained in 82% yield from the reaction of N-[4-nitro-3-trifluoromethyl-phenyl]-ferrocenecarboxamide (NFFe) with fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+) in DMF-water at pH 1 and at 150 °C for 1 h. The corresponding Re analog was also prepared. In vitro assays demonstrated high stability of NF(99m)Tc under physiological conditions, buffer and blood. The tissue biodistribution in mature male Wistar rats showed a significant selective uptake by prostate but this uptake was not blocked by an excess of testosterone acetate. A higher uptake by lung tissues was observed.     

Dramatic effect of the tridentate ligand on the stability of 99mTC "3 + 1" oxo complexes bearing arylpiperazine derivatives

Mixed-ligand model complexes of general formula [(99m)Tc(O)(kappa(3)-PNX)(kappa(1)-SPh))] [X = O (1a), S (2a)] were prepared in a one-step procedure from [(99m)TcO(4)(-)] using stannous chloride as reducing agent. Stability studies and challenge experiments with glutathione showed that complex 2a presented promising features for pursuing animal studies. The activity in the brain (% dose injected/organ) at 5 min (0.14% +/- 0.03) and 120 min (0.11% +/- 0.02) pi encouraged the synthesis of several mixed-ligand "3 + 1" oxo complexes of general formula [M(O)(kappa(3)-PNS)(kappa(1)-SL))] (M = (99m)Tc, 3a-6a, Re, 3-6), in which the tridentate ligand is the heterofunctionalized phosphine 2-(diphenylphosphanyl)-N-(2-thioethyl)benzamide (PNS) and the co-ligands are different arylpiperazine derivatives (HSL1-HSL4). The (99m)Tc complexes have been characterized by comparison of their retention times in the HPLC chromatogram (gamma-detection) with the retention times of the analogous Re complexes (UV detection at 254 nm). The (99m)Tc complexes, obtained with radiochemical purity higher than 95%, after HPLC purification, are stable in saline, 0.01 M PBS (pH 7.4), rat plasma (4 h, 37 degrees C), and glutathione (10 mM solutions, 2h, 37 degrees C). Binding affinity and selectivity for 5-HT(1A) receptors (relative to the 5-HT(2A) receptor) were determined, complex 5 demonstrating the best values (IC(50) for the 5-HT(1A) 2.35 +/- 0.02 nM; competitor 5-HT(2A) 372 +/- 11 nM). Biodistribution and stability studies in mice indicated a preferred hepatobiliary excretion, a high in vivo stability, but a poor brain uptake.     

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.     

Necrosis avidity of (99m)Tc(CO)3-labeled pamoic acid derivatives: synthesis and preliminary biological evaluation in animal models of necrosis

In a search for an infarct avid tracer agent with improved properties, we have observed that bis-DTPA derivatives of pamoic acid have a high avidity for necrotic tissue. Here, we report the synthesis, radiolabeling, and preliminary evaluation in normal mice and rats with hepatic infarction of the (99m)Tc-tricarbonyl complexes of N, N'-bis(diethylenetriaminopentaacetato)-4,4'-methylene bis(2-hydroxy-3-naphthoic hydrazide) ( (99m)Tc(CO) 3-bis-DTPA-pamoate) and [ N-(5-aminopentyl)pyridin-2-yl-methylamino]methylacetato-4,4'-methylene-2-hydroxy-3-napthalenecarboxamide-(2'-hydroxy-3'-naphthoic acid methyl ester) ( (99m)Tc(CO) 3 -12). Radiolabeling with (99m)Tc(CO) 3 (+) was achieved with a radiochemical yield of over 95% for both tracer agents. In normal mice, the polar (99m)Tc(CO) 3-bis-DTPA-pamoate was cleared from plasma via both the liver and the kidneys, while the more lipophilic (99m)Tc(CO) 3 -12 was rapidly cleared via the liver. Blood clearance in mice was faster for (99m)Tc(CO) 3 -12 (0.1% injected dose per gram at 4 h postinjection) than for (99m)Tc(CO) 3-bis-DTPA-pamoate (9.3% injected dose per gram at 4 h postinjection). Affinity and specificity of the tracers for necrotic tissue was studied in rats with hepatic infarction and ethanol-induced necrosis of the liver or muscles. Activity ratios of infarct to viable liver tissue of (99m)Tc(CO) 3-bis-DTPA-pamoate quantified by autoradiography of tissue slices ranged from 4 to 18, depending on the necrosis model and time postinjection of the tracer. Infarcts were also visualized in vivo by (99m)Tc(CO) 3-bis-DTPA-pamoate planar gamma imaging. After injection of (99m)Tc(CO) 3-bis-DTPA-pamoate, in vivo and ex vivo images correlated well with histochemical staining with triphenyltetrazolium chloride and hematoxylin and eosin. (99m)Tc(CO) 3 -12 on the other hand showed no uptake in necrotic tissue. Stability of the tracers was determined in vitro after storage at room temperature and by histidine challenge experiments, and in vivo in mouse plasma and in urine (for (99m)Tc(CO) 3-bis-DTPA-pamoate). (99m)Tc(CO) 3-bis-DTPA-pamoate was unstable in vitro to histidine challenge, while (99m)Tc(CO) 3 -12 was 98% stable in vitro in the same conditions. Both tracers showed good in vivo stability. (99m)Tc(CO) 3-bis-DTPA-pamoate shows high specificity for necrotic tissue and merits further evaluation as a necrosis avid imaging agent. (99m)Tc(CO) 3 -12 is not useful for visualization of necrotic tissue.     

The [(99m)Tc(N)(PNP)](2+) metal fragment: a technetium-nitrido synthon for use with biologically active molecules. The N-(2-methoxyphenyl)piperazyl-cysteine analogues as examples

The incorporation of a bioactive molecule into a nitrido-containing (99m)Tc-complex has been successfully achieved by using the [TcN(PNP)](2+) metal fragment. In this strategy, the strong electrophilic [TcN(PNP)](2+) metal fragment efficiently reacts with bifunctional chelating ligands having a pi-donor atom set, such as N-functionalized O,S-cysteine. The 2-methoxyphenylpiperazine (2-MPP) pharmacophore, which displays preferential affinity for 5HT(1A) receptors, was conjugated to the amino group of cysteine to obtain 2-MPPP-cys-OS, where 2-MPPP is 3-[4-(2-methoxyphenyl)piperazin-1-yl]propionate. The asymmetric Tc(V)-nitrido complexes, [(99g/99m)Tc(N)(PNP)(2-MPPP-cys-OS)] (PNP = PNP3, PNP4), were obtained in high yield (95%), by simultaneous addition of PNP and 2-MPPP-cys-OS ligand to a solution containing a starting (99g)/(99m)Tc-nitrido precursor. A mixture of syn and anti isomers was observed, the latter being the thermodynamically favored species. In vitro challenge experiments using the anti isomers with glutathione and cysteine indicated that no transchelation reaction occurs. Assessment of the in vitro 5HT(1A) receptor-affinity of the technetium complexes revealed that only the anti-PNP4 complex possesses some affinity for the receptor, but displayed negligible brain uptake in biodistribution studies in rats in vivo.     

Evaluation of 99mTc-labeled cyclic RGD dimers: impact of cyclic RGD peptides and 99mTc chelates on biological properties

The main objective of this study is to explore the impact of cyclic RGD peptides and (99m)Tc chelates on biological properties of (99m)Tc radiotracers. Cyclic RGD peptide conjugates, HYNIC-K(NIC)-RGD(2) (HYNIC = 6-hydrazinonicotinyl; RGD(2) = E[c(RGDfK)](2) and NIC = nicotinyl), HYNIC-K(NIC)-3G-RGD(2) (3G-RGD(2) = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)](2)), and HYNIC-K(NIC)-3P-RGD(2) (3P-RGD(2) = PEG(4)-E[PEG(4)-c(RGDfK)](2)), were prepared. Macrocyclic (99m)Tc complexes [(99m)Tc(HYNIC-K(NIC)-RGD(2))(tricine)] (1), [(99m)Tc(HYNIC-K(NIC)-3G-RGD(2))(tricine)] (2), and [(99m)Tc(HYNIC-K(NIC)-3P-RGD(2))(tricine)] (3) were evaluated for their biodistribution and tumor-targeting capability in athymic nude mice bearing MDA-MB-435 human breast tumor xenografts. It was found that 1, 2, and 3 could be prepared with high specific activity (～111 GBq/μmol). All three (99m)Tc radiotracers have two major isomers, which show almost identical uptake in tumors and normal organs. Replacing the bulky and highly charged [(99m)Tc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3″-trisulfonate) with a smaller [(99m)Tc(HYNIC-K(NIC))(tricine)] resulted in less uptake in the kidneys and lungs for 3. Surprisingly, all three (99m)Tc radiotracers shared a similar tumor uptake (1, 5.73 ± 0.40%ID/g; 2, 5.24 ± 1.09%ID/g; and 3, 4.94 ± 1.71%ID/g) at 60 min p.i. The metabolic stability of (99m)Tc radiotracers depends on cyclic RGD peptides (3P-RGD(2) > 3G-RGD(2) ～ RGD(2)) and (99m)Tc chelates ([(99m)Tc(HYNIC)(tricine)(TPPTS)] > [(99m)Tc(HYNIC-K(NIC))(tricine)]). Immunohistochemical studies revealed a linear relationship between the α(v)β(3) expression levels and tumor uptake or tumor/muscle ratios of 3, suggesting that 3 is useful for monitoring the tumor α(v)β(3) expression. Complex 3 is a very attractive radiotracer for detection of integrin α(v)β(3)-positive tumors.     

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.     

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the ‘click-to-chelate’ protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12, [^99mTc(CO)₃]13, and [^99mTc(CO)₃]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)₃]12, [Re(CO)₃]13, and [Re(CO)₃]14, were shown to have high binding affinities (0.13 μM, 0.06 μM, and 0.16 μM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12–14) to c-Met receptor positive U87MG cells.     

Uptake kinetics of 99Tc in common duckweed

The uptake of the nuclear waste product technetium-99 was studied in common duckweed (Lemna minor). In addition to measurements, a model involving two compartments in duckweed with different chemical forms of technetium was derived. The model was tested by chemical speciation, i.e. differentiating between reduced Tc-compounds and Tc(VII)O(4)(-). The TcO(4)(-) concentrations measured were in good agreement with those predicted by the model. Two processes determine technetium uptake: (1) transport of Tc(VII)O(4)(-) across the cell membrane, and (2) reduction of Tc(VII). The TcO(4)(-) concentration in duckweed reaches a steady state within 2 h while reduced Tc-compounds are stored, as a result of absence of release or re-oxidation processes. Bioaccumulation kinetic properties were derived by varying 99Tc concentration, temperature, nutrient concentrations, and light intensity. The reduction of technetium in duckweed was highly correlated with light intensity and temperature. At 25 degrees C the maximum reduction rate was observed at light intensities above 200 μmol m(-2) s(-1) while half of the maximum transformation rate was reached at 41 μmol m(-2) s(-1). Transport of TcO(4)(-) over the cell membrane requires about 9.4 kJ mol(-1), indicating an active transport mechanism. However, this mechanism behaved as first-order kinetics instead of Michaelis-Menten kinetics between 1x10(-14) and 2.5x10(-5) mol l(-1) TcO(4)(-). Tc uptake could not be inhibited by 10(-3) mol l(-1) nitrate, phosphate, sulphate or chloride.     

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.     

Synthesis and evaluation of a (99m)Tc-BAT-phenylbenzothiazole conjugate as a potential in vivo tracer for visualization of amyloid beta

We have conjugated S,S'-bis-trityl-N-BOC-N'-acetic acid-1,2-ethylenedicysteamine, a protected bis-amino-bis-thiol (BAT) tetraligand, with 2-(4'-aminophenyl)-1,3-benzothiazole, a derivative of thioflavin-T with known affinity for amyloid. The conjugate was efficiently labelled with (99m)Tc by heating of the protected precursor in diluted hydrochloric acid followed by neutralization and heating in the presence of (99m)Tc-tartrate. It was demonstrated that the (99m)Tc-BAT-phenylbenzothiazole conjugate binds in vitro to amyloid beta present in postmortem brain slices of Alzheimer's patients. Despite its high lipophilicity and neutral character, the radiolabelled conjugate did not cross the blood-brain barrier to a sufficient degree and therefore is not useful for detection of Alzheimer's disease. Further evaluation of this (99m)Tc-labelled tracer agent could elucidate its potential usefulness to visualize amyloid plaques in peripheral amyloidosis.     

Synthesis and biological evaluation of novel technetium-99m labeled phenylbenzoxazole derivatives as potential imaging probes for β-amyloid plaques in brain

Two uncharged (99m)Tc-labeled phenylbenzoxazole derivatives were biologically evaluated as potential imaging probes for β-amyloid plaques. The (99m)Tc and corresponding rhenium complexes were synthesized by coupling monoamine-monoamide dithiol (MAMA) and bis(aminoethanethiol) (BAT) chelating ligand via a pentyloxy spacer to phenylbenzoxazole. The fluorescent rhenium complexes 6 and 9 selectively stainined the β-amyloid plaques on the sections of transgenic mouse, and showed high affinity for Aβ((1-42)) aggregates (K(i)=11.1 nM and 14.3 nM, respectively). Autoradiography in vitro indicated that [(99m)Tc]6 clearly labeled β-amyloid plaques on the sections of transgenic mouse. Biodistribution experiments in normal mice revealed that [(99m)Tc]6 displayed moderate initial brain uptake (0.81% ID/g at 2 min), and quickly washed out from the brain (0.25% ID/g at 60 min). The preliminary results indicate that the properties of [(99m)Tc]6 are promising, although additional refinements are needed to improve the ability to cross the blood-brain barrier.     

(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.     

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.     

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.     

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.