Radio-LC-MS for the characterization of 99mTc-labeled bioconjugates

This report describes the first example of using radio-LC-MS for determining the composition of (99m)Tc radiopharmaceuticals at the tracer level. The in-line radiometric detector is a useful addition to a standard LC-MS and provides direct correlation between the MS data and the radioactive species in a radiopharmaceutical kit. Complexes [(99m)Tc(HYNICtide)(tricine)(L)] (RP444, L = TPPTS; RP445, L = TPPDS; and RP446, L = TPPMS) were prepared using a decayed generator eluant. All the ternary ligand (99m)Tc complexes show the expected monoprotonated molecular ions, (M + 1)(+), and diprotonated molecular ions, (M + 2)(2+). The LC-MS spectral data support the proposed structure and are consistent with those obtained for their corresponding (99)Tc analogues. Ternary ligand complexes [(99m)Tc(HYNICtide)(tricine)(L)] (L = ISONIC-HE and ISONIC-Sorb) are neutral, and the molecular weights are also lower than that of RP444. Using a fresh generator eluant (24 h prior elution), only 1-2 mCi of (99m)Tc [(7 x 10(-)(12))-(1.5 x 10(-)(11)) mol of technetium complex] are required to obtain a reasonably clean mass spectrum. Radio-LC-MS is a quick and accurate analytical tool for characterization of (99m)Tc radiopharmaceuticals at the tracer level.     

Synthesis, radiolabeling and in vitro and in vivo characterization of a technetium-99m-labeled alpha-M2 peptide as a tumor imaging agent.

In an effort to develop a peptide-based radiopharmaceutical for the detection of breast cancer, we have prepared an analog of alphaM2 peptide, modified to incorporate an N3S chelate system. Mercaptoacetyltriglycine (MAG)(3)-derivatized alphaM2 peptide was prepared by solid-phase synthesis and radiolabeled with (99m)Tc by an exchange method. In vitro cell-binding on human breast cancer cell lines, MDA-MB-231 and MCF-7, indicated the affinity and specificity of (99m)Tc-MAG(3)-alphaM2 toward breast cancer cells. Additionally, the radiolabeled peptide showed rapid internalization into human breast cancer cells. In vivo biodistribution in mice showed that the radiolabeled peptide cleared rapidly from the blood and most non-target tissues and was excreted significantly via the kidneys. Uptake of (99m)Tc-MAG(3)-alphaM2 in the tumor was moderate. The radiochemical and in vitro and in vivo characterization indicates that the radiolabeled peptide has certain favorable properties and it might be a useful radiopharmaceutical for the detection of breast cancer in vivo.     

99mTc-labeled cyclic RGDfK dimer: initial evaluation for SPECT imaging of glioma integrin alphavbeta3 expression

This report describes the evaluation of biodistribution properties of three radiotracers, [(99m)Tc(SQ168)(EDDA)], [(99m)Tc(SQ168)(tricine)(PDA)], and [(99m)Tc(SQ168)(tricine)(TPPTS)] (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-d-Phe})-cyclo{Lys-Arg-Gly-Asp-d-Phe}; EDDA = ethylenediamine-N,N'-diacetic acid; PDA = 2,5-pyridinedicarboxylic acid; TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate), and their potential to image the glioma integrin alpha(v)beta(3) expression in BALB/c nude mice bearing the U87MG human glioma xenografts. It was found that all three radiotracers were able to localize in glioma tumors with a relatively high tumor uptake and long tumor retention time by binding to the integrin alpha(v)beta(3) expressed on both tumor cells and endothelial cells of tumor neovasculature. It seems that the coligand has minimal effect on integrin alpha(v)beta(3) targeting capability of the (99m)Tc-labeled RGDfK dimer, but it has a significant impact on their biodistribution properties. For example, the complex [(99m)Tc(SQ168)(tricine)(TPPTS)] has the lowest liver uptake and the highest metabolic stability in normal BALB/c nude mice. Results from SPECT imaging studies show that the glioma tumors can be clearly visualized with all three radiotracers at 4 h postinjection. Among the three radiotracers evaluated in this study, [(99m)Tc(SQ168)(tricine)(TPPTS)] has the best imaging quality and is a promising candidate for more preclinical evaluations in the future.     

Phosphine-containing HYNIC derivatives as potential bifunctional chelators for (99m)Tc-labeling of small biomolecules

Two prototype phosphine-containing HYNIC chelators, HYNIC-Kp-DPPB and HYNIC-Ko-DPPB (HYNIC = 6-hydrazinonicotinamide; K = lysine; and DPPB = diphenylphosphine-benzoic acid), have been synthesized and characterized by NMR ((1)H, (13)C, and (31)P) and LC-MS. Macrocyclic (99m)Tc complexes, [(99m)Tc(HYNIC-Ko-TPPB)(tricine)] and [(99m)Tc(HYNIC-Kp-DPPB)(tricine)], were prepared by reacting the phosphine-containing HYNIC chelator with (99m)TcO(4)(-) in the presence of excess tricine and stannous chloride. Results from this study clearly demonstrated that both HYNIC-Kp-DPPB and HYNIC-Ko-DPPB are able to form highly stable macrocyclic (99m)Tc complexes, [(99m)Tc(HYNIC-Ko-TPPB)(tricine)] and [(99m)Tc(HYNIC-Kp-DPPB)(tricine)], when tricine is used as the coligand. Radio-HPLC data suggest that the complex [(99m)Tc(HYNIC-Kp-DPPB)(tricine)] exists as only one detectable isomer in solution while the complex [(99m)Tc(HYNIC-Ko-DPPB)(tricine)] has three isomers. It was also found that three isomers of [(99m)Tc(HYNIC-Ko-DPPB)(tricine)] interconvert at elevated temperatures, suggesting that the presence of these isomers might be due conformational changes in the macrocyclic Tc chelate. The LC-MS data for both macrocyclic (99m)Tc complexes are completely consistent with the proposed composition. The phosphine-containing HYNIC chelators described in this study may have the potential as bifunctional chelators for (99m)Tc labeling of small biomolecules.     

(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.     

Effect of coligands on biodistribution characteristics of ternary ligand 99mTc complexes of a HYNIC-conjugated cyclic RGDfK dimer

This report describes biodistribution characteristics of three ternary ligand complexes [(99m)Tc(SQ168)(tricine)(L)] (SQ168 = [2-[[[5-[carboonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonic acid]-Glu(cyclo{Lys-Arg-Gly-Asp-d-Phe})-cyclo{Lys-Arg-Gly-Asp-d-Phe}; L = TPPTS (trisodium triphenylphosphine-3,3',3' '-trisulfonate), ISONIC (isonicotinic acid) and PDA (2,5-pyridinedicarboxylic acid)) in athymic nude mice bearing MDA-MB-435 human breast cancer xenografts. Ternary ligand complexes [(99m)Tc(SQ168)(tricine)(L)] (L = TPPTS, ISONIC and PDA) were prepared and were analyzed by a reversed HPLC method. Surprisingly, coligands have little impact on log P values of their ternary ligand (99m)Tc complexes even though HPLC retention times suggest that [(99m)Tc(SQ168)(tricine)(PDA)] and [(99m)Tc(SQ168)(tricine)(ISONIC)] are more hydrophilic than [(99m)Tc(SQ168)(tricine)(TPPTS)]. The results from biodistribution studies indicated that excretion kinetics of the (99m)Tc-labeled cyclic RGDfK dimer can be modified by the choice of coligand. The fact that all three radiotracers show high tumor uptake during the 2 h study period suggests that the coligand has minimal effect on the tumor targeting capability of the (99m)Tc-labeled cyclic RGDfK dimer. Results from the blocking experiment suggest that the tumor localization of the (99m)Tc-labeled cyclic RGDfK dimer is integrin alpha(v)beta(3)-mediated. On the basis of their liver uptake and tumor/liver ratios, we believe that PDA has the advantage over TPPTS and ISONIC for the (99m)Tc-labeling of HYNIC-biomolecule conjugates.     

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.     

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.     

Synthesis, characterization, and biological evaluation of neutral nitrido technetium(V) mixed ligand complexes containing dithiolates and aminodiphosphines. A novel system for linking technetium to biomolecules

A new biomolecule labeling method that utilizes the [(99m)Tc(N)(PNP)](2+) metal fragment is presented. Thus, a series of nitrido mixed-ligand M(V) complexes (M = (99m)Tc, (99g)Tc, Re), [M(N)(Ln)(PNP)], where Ln is the dianionic form of a dithiolate or substituted-dithiolate ligand and PNP is an aminodiphosphine, is described. (99m)Tc complexes can be prepared using either a two-step or a three-step procedure starting from generator-eluted pertechnetate through a prereduced mixture of [(99m)Tc(N)]-containing species, followed by sequential or contemporary addition of the relevant dithiolate and aminodiphosphine. The reactions of 2,3-dimercaptopropionic acid (H(2)L1) with [Tc(N)(PNP)](2+) were investigated in detail. It was found that this bidentate ligand coordinated the metal fragment through the [S(-),S(-)] donor atom pair, to yield neutral mixed-ligand complexes [(99m)Tc(N)(L1)(PNP)] in high specific activity. The additional carboxylic functional group was not involved in metal coordination, thus remaining available for conjugation to target-specific molecules. Dithiolates incorporating pendant functional group(s) gave rise to a 1:1 diastereoisomeric mixture of syn-[M(N)(Ln)(PNP)] and anti-[M(N)(Ln)(PNP)] derivatives, depending on the relative orientation of the dithiolate substituent(s) with respect to the terminal nitrido group, and no isomeric conversion was detected. (99m)Tc species had been proven to be identical with the (99g)Tc complexes prepared at the macroscopic level by comparison of the corresponding radiometric and UV/vis HPLC profiles. Challenge experiments with cysteine or glutathione indicated that these physiological agents had no effect on the stability of this class of mixed-ligand (99m)Tc-complexes. Biodistribution studies in rats of selected (99m)Tc-complexes showed a rapid clearance from the blood and tissues after 60 min pi.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Development of 99mTc(CO)₃-dendrimer-FITC for cancer imaging

Study of fluorophore and technetium labeling of poly(amido)-amine (PAMAM) generation 4 (G4) dendrimer and its evaluation as potential molecular imaging agent in both normal and melanoma-bearing mice, are described. Dendrimers were first conjugated with FITC (fluorescein isothiocyanate). Dendrimer-FITC was then incubated with the intermediate [(99m)Tc(CO)(3)(H(2)O)(3)](+) and purified by gel filtration. Biodistribution and scintigraphy images were performed administrating (99m)Tc(CO)(3)-dendrimer-FITC to normal mice (NM) or melanoma-bearing mice (MBM). Cryostat tissue sections from MBM mice were analyzed by confocal microscopy. Radiolabeling yield of dendrimer was approx. 90%. The (99m)Tc(CO)(3)-dendrimer-FITC complex was stable for at least 24h. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with high tumor uptake that allowed tumor imaging. Confocal microscopy analysis showed cytoplasmic distribution of (99m)Tc(CO)(3)-dendrimer-FITC.     

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.     

RP463: a stabilized technetium-99m complex of a hydrazino nicotinamide derivatized chemotactic peptide for infection imaging.

A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with (99m)Tc using tricine and TPPTS as coligands. The combination of fMLFK-HYNIC, tricine, and TPPTS with (99m)Tc produced a ternary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). RP463 was synthesized either in two steps, in which the binary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(2)] (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of [(99m)Tc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS. The radiolabeling yield for RP463 was usually >/=90% using 10 microg of fMLFK-HYNIC and 100 mCi of [(99m)Tc]pertechnetate. Unlike RP469, which decomposed rapidly in the absence of excess tricine coligand, RP463 was stable in solution for at least 6 h. [(99)Tc]RP463 was prepared and characterized by HPLC and electrospray mass spectrometry. In an in vitro assay, [(99)Tc]RP463 showed an IC(50) of 2 nM against binding of [(3)H]fMLF to receptors on PMNs. [(99)Tc]RP463 also induces effectively the superoxide release of polymorphonuclear leukocytes (PMNs) with an EC(50) value of 0.2 +/- 0.2 nM. The localization of RP463 in the infection foci was assessed in a rabbit infection model. RP463 was cleared from the blood faster than RP469 and was excreted mainly through the renal system. As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 +/- 0.2 at 15 min postinjection to 7.5 +/- 0.4 at 4 h postinjection. Visualization of the infected area could be as early as 2 h. A transient decrease in white blood cell count of 35% was observed during the first 30 min after injection of the HPLC-purified RP463 in the infected rabbit. This suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes.     

Asymmetrical nitrido tc-99m heterocomplexes as potential imaging agents for benzodiazepine receptors

The design, synthesis, and biological evaluation of nitrido technetium-99m complexes for imaging benzodiazepine receptors are described. The design was performed by selecting the precursor biologically active substrate desmethyldiazepam, and the reactive metal-containing fragment [(99m)Tc(N)(PXP)](2+) (PXP = diphosphine ligand) as molecular building-blocks for assembling the structure of the final radiopharmaceuticals through the application of the so-called 'bifunctional' and 'integrated' approaches. This required the synthesis of the ligands H(2)BZ1, H(2)C1, and H(2)C2 (Figures 1 and 2) derived from desmethyldiazepam. In turn, these ligands were reacted with [(99m)Tc(N)(PXP)](2+) to afford the complexes [(99m)Tc(N)(PXP)(L)] (L = BZ1, C1, C2). The chemical nature of the resulting Tc-99m radiopharmaceuticals was investigated using chromatographic methods, and by comparison with the analogous complexes prepared with the long-lived isotope Tc-99g and characterized by spectroscopic and analytical methods. Results showed that the complexes [(99m)Tc(N)(PXP)(L)] are neutral and possess an asymmetrical five-coordinated structure in which two different bidentate ligands, PXP and L, are coordinated to the same Tc[triple bond]N core. With the ligand H(2)BZ1, two isomers were obtained depending on the syn or anti orientation of the pendant benzodiazepine group relative to the Tc[triple bond]N multiple bond. Biodistribution studies of Tc-99m complexes were carried out in rats, and affinity for benzodiazepine receptors was assessed through in vitro binding experiments on isolated rat's cerebral membranes using the corresponding Tc-99g complexes.