99mTc-labeling of colchicine using [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ core for the preparation of potential tumor-targeting agents

Multidrug resistance (MDR) mediated by over-expression of P-glycoprotein (Pgp) is one of the major causes of failure of chemotherapy in cancer treatment. Colchicine, a naturally occurring alkaloid, is a Pgp substrate and acts as an antimitotic agent by binding to microtubules. Hence, Colchicine and its analogues radiolabeled with 99mTc may have potential for visualization of MDR in tumors. Here we report 99mTc-labeling of colchicine derivatives using [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ cores. Trimethylcolchicinic acid synthesized from colchicine was used as the precursor to prepare iminodiacetic acid and dithiocarbamate derivatives which were then radiolabeled with [99mTc(CO)3(H2O)3]+ and [99mTc triple bond N]2+ cores, respectively. Radiolabeling yield for both the complexes was > 98% as observed by HPLC and TLC patterns. In vitro studies in tumor cell lines showed significant uptake for 99mTc-carbonyl as well as for 99mTc-nitrido colchicine complexes. Biodistribution studies in Swiss mice bearing fibrosarcoma tumor showed 4.1 +/- 1.2% ID/g of uptake at 30 min pi for 99mTc(CO)3-complex as against 0.42 +/- 0.24% ID/g for the 99mTcN-complex. 99mTc(CO)3-colchicine complex exhibited better pharmacokinetics with lower liver accumulation as compared to the 99mTcN-complex. Thus, colchicine radiolabeled with [99mTc(CO)3(H2O)3]+ core is more promising with respect to in vivo distribution characteristics in tumor model.     

Pyridine-containing 6-hydrazinonicotinamide derivatives as potential bifunctional chelators for 99mTc-labeling of small biomolecules

As a continuation of our interest in novel 99mTc chelating systems, several pyridine-containing HYNIC (6-hydrazinonicotinamide) derivatives (L1-L5) have been synthesized and characterized by NMR (1H and 13C) and LC-MS. 99mTc complexes of L1-L5 were prepared by the reaction of the HYNIC derivative with 99mTcO4- in the presence of excess tricine and stannous chloride. Results from this study show that the attachment site of the linker is critical for the formation of macrocyclic 99mTc complexes. For example, the pyridine-N in L3 is not able to bond to the Tc, because the lysine linker is attached to the 4-position. When the linker is at the 2-position, L1 forms the macrocyclic complex [99mTc(L1)(tricine)], but the radiochemical purity is relatively low. If the linker is attached to the 3-position of the pyridine ring, the HYNIC derivatives form macrocyclic complexes [99mTc(L)(tricine)] (L2, L4, and L5) in high yield (>95%). The HPLC data suggest that the macrocyclic complex [(99m)Tc(L2)(tricine)] exists in solution as four isomers: two diastereomers and two conformational isomers. Diastereomers are due to a combination of the chirality of the lysine linker and of the Tc chelate. Replacing lysine with a pentamethylenediamine linker results in the macrocyclic complex [99mTc(L4)(tricine)] with two conformational isomers, which interconvert rapidly at room temperature. Changing the linker from pentamethylenediamine to hexamethylenediamine did not eliminate the minor isomer; but the percentage of the minor isomer was reduced from approximately 10% for [99mTc(L4)(tricine)] to only 6% for [99mTc(L5)(tricine)]. The linker length is an important parameter to minimize the minor isomer. LC-MS data of complexes [99mTc(L)(tricine)] (L2, L4, and L5) are completely consistent with their proposed compositions. On the basis of these data, it is concluded that pyridine-containing HYNIC derivatives have the potential as bifunctional chelators for 99mTc-labeling of small biomolecules if the linker is attached to the 3-position of the pyridine ring.     

Preparation of no-carrier-added technetium-99m complexes via metal-assisted cleavage from a solid phase

A novel method for the preparation of no-carrier-added (nca) complexes [99mTc(CO)3L] (L = diethylenetriamine or picolylamine-N-acetic acid) is described. The ligands were covalently bound to a solid support of organic polymers via formation of a tertiary amine from the chelating unit. This C-N bond to the solid phase is selectively cleaved during the formation of the technetium complexes by intramolecular nucleophilic attack of a remaining hydroxy ligand to the alpha-carbon. The complex [99mTc(CO)3L] is released into solution while uncomplexed ligand and uncleaved complex remain solid-phase bound. High specific activity technetium complexes can then be isolated by simple filtration. Cleavage yield depends on temperature, pH, and ligand. Up to 50% release from the solid phase could be achieved under optimized conditions. Corresponding to the 99mTc concentration, free ligand is present in concentrations lower than 10(-7) M. If a targeting vector is conjugated to these ligands, no-carrier-added radiopharmaceuticals can be prepared in that way.     

Isostructural Re and 99mTc complexes of biotin derivatives for fluorescence and radioimaging studies

The reaction of biotinamine with two equivalents of 2-quinoline aldehyde in the presence of Na(OAc)3BH in dichloroethane provides N,N-bis(methylquinoline)biotinamine (L1), a molecule displaying a tridentate donor terminus which has proven effective in coordinating to the {M(CO)3}+ core (M = Tc, Re). Reaction of L1 with (NEt4)2[Re(CO)3Br3] yields [Re(CO)3(L1)]Br, a compound with an absorbance at 350 nm and luminescence emission maxima at 425 and 580 nm. The luminescence lifetime of 11.4 mus, which is associated with the 580 nm emission, is sufficiently prolonged to enable time-gating techniques to be used during in vitro imaging studies and to overcome interference from endogenous fluorescence. Exposure of avidin beads to {Re(CO)3(L1)]Br resulted in binding, which was qualitatively imaged using fluorescence microscopy. The 99mTc analogue [99mTc(CO)3(L1)]+1 was prepared by reacting L1 with [99mTc(CO)3(H2O)3]+1 and purified by HPLC. The 99mTc complex is chemically robust and resistant to cysteine and histidine challenges. This study demonstrates that complementary fluorescent and radioactive biotin-derived probes may be readily prepared to allow direct correlation of in vitro and in vivo molecular imaging studies.     

Evaluation of a (99m)Tc-labeled cyclic RGD tetramer for noninvasive imaging integrin alpha(v)beta3-positive breast cancer

Integrin alphavbeta3 plays a critical role in tumor angiogenesis and metastasis. Radiolabeled RGD peptides that are integrin alphavbeta3-specific are very useful for noninvasive imaging of integrin expression in rapidly growing and metastatic tumors. In this study, we determined the binding affinity of E{E[c(RGDfK)]2}2 (tetramer) and its 6-hydrazinonicotinamide conjugate (HYNIC-tetramer) against the binding of 125I-echistatin to the integrin alphavbeta3-positive MDA-MB-435 breast cancer cells. The athymic nude mice bearing MDA-MB-435 xenografts were used to evaluate the potential of ternary ligand complex [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3' '-trisulfonate) as a new radiotracer for imaging breast cancer integrin alphavbeta3 expression by single photon emission computed tomography (SPECT). It was found that the binding affinity of tetramer (IC50 = 51 +/- 11 nM) was slightly higher than that of its dimeric analogue (IC50 = 78 +/- 27 nM) and is comparable to that of the HYNIC-tetramer conjugate (IC50 = 55 +/- 11 nM) within the experimental error. Biodistribution data showed that [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had a rapid blood clearance (4.61 +/- 0.81 %ID/g at 5 min postinjection (p.i.) and 0.56 +/- 0.12 %ID/g at 120 min p.i.) and was excreted mainly via the renal route. [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] had high tumor uptake with a long tumor retention (5.60 +/- 0.87 %ID/g and 7.30 +/- 1.32 %ID/g at 5 and 120 min p.i., respectively). The integrin alphavbeta3-specificity was demonstrated by co-injection of excess E[c(RGDfK)]2, which resulted in a significant reduction in tumor uptake of the radiotracer. The metabolic stability of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] was determined by analyzing urine and feces samples from the tumor-bearing mice at 120 min p.i. In the urine, about 20% of [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] remained intact while only approximately 15% metabolized species was detected in feces. SPECT images displayed significant radiotracer localization in tumor with good contrast as early as 1 h p.i. The high tumor uptake and fast renal excretion make [99mTc(HYNIC-tetramer)(tricine)(TPPTS)] a promising radiotracer for noninvasive imaging of the integrin alphavbeta3-positive tumors by SPECT.     

99mTc(CO)3-15-[N-(Acetyloxy)-2-picolylamino]pentadecanoic acid: a potential radiotracer for evaluation of fatty acid metabolism

99mTc(CO)3-15-[N-(Acetyloxy)-2-picolylamino]pentadecanoic acid (1a) was prepared by incorporating [99mTc(CO)3]+ into 15-[N-(hydroxycarbonylmethyl)-2-picolylamino]pentadecanoic acid (2a). The overall radiochemical yield of 1a after HPLC purification was 60-63%. Radiotracer 1a was found to be chemically stable when incubated in human plasma for 4 h at 37 degrees C. Tissue distribution studies showed that high radioactivity accumulated in the heart with rapid clearance. The maximum heart-to-blood uptake ratio was 1.87 at 5 min after a tail-vein injection. Radioactive metabolites were analyzed in urine samples of mice and corresponded to a 9.3:1 ratio of 99mTc(CO)3-5-[N-(acetyloxy)-2-picolylamino]pentanoic acid (1b) to 99mTc(CO)3-3-[N-(acetyloxy)-2-picolylamino]propionic acid (1c), indicating that 1a is mainly metabolized to 1b via beta-oxidation in the body. These results suggest that 1a is a promising radiotracer for evaluation of fatty acid metabolism in myocardium.     

99mTc-labeled neuropeptide Y analogues as potential tumor imaging agents.

The possible use of neuropeptide Y (NPY) as a novel radiopeptide has been investigated. NPY is a 36-amino acid peptide of the pancreatic polypeptide family, which is expressed in the peripheral and central nervous system, and is one of the most abundant neuropeptides in the brain. Its receptors are produced in a number of neuroblastoma and the thereof derived cell lines. As structure-activity relationships of NPY are well-known, we could assume where a radionuclide might be introduced without affecting receptor affinity. We applied the novel [99mTc(OH2)3(CO)3]+ aqua complex and PADA (2-picolylamine-N,N-diacetic acid) as bifunctional chelating agent. The peptides were synthesized by solid-phase peptide synthesis, and PADA was coupled to the side chain of Lys4 of the resin-bound peptide. Upon postlabeling of [K4(PADA)]-NPY, 99mTc(CO)3 did not only bind to the desired PADA, but presumably as well to the His in position 26. Since the replacement of His26 by Ala only slightly decreased binding affinity, [K4(PADA),A26]-NPY was specifically postlabeled, and the 185Re surrogate maintained high binding affinity. Furthermore, the prelabeling approach has been applied for the centrally truncated analogue [Ahx5-24]-NPY, which is highly selective for the Y2 receptor. The resulting Ac-[Ahx5-24,K4(99mTc(CO)3-PADA)]-NPY was produced with a yield of only 16%. Therefore, postlabeling was applied for the short analogue as well, again substituting His26 by Ala. Competitive binding assays using (185)Re as a surrogate for 99mTc showed high binding affinity of Ac-[Ahx5-24,K4(185Re(CO)3-PADA),A26]-NPY. Internalization studies with the corresponding 99mTc-labeled analogue revealed receptor-mediated internalization. Furthermore, biodistribution studies were performed in mice, and stability was tested in human plasma. Our centrally truncated analogue revealed a 6-fold increased stability compared to the natural peptide NPY. We conclude that Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY has promising characteristics for future applications in nuclear medicine.     

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.     

Synthesis and in vitro characterization of organometallic rhenium and technetium glucose complexes against Glut 1 and hexokinase

A series of nine organometallic technetium-99m and rhenium complexes of glucose are presented and characterized in vitro regarding their potential as surrogates of [18F]-2-fluoro-desoxy glucose ([18F]-FDG). The glucose derivatives are functionalized at positions C-1, C-2, C-3, and C-6. Different spacer lengths and chelating systems have been introduced at these sites. For the (radio)labeling, the organometallic precursors [99mTc(H2O)3(CO)3]+ and [ReBr3(CO)3](2-) respectively have been used. The resulting complexes have been characterized chemically and radiochemically. The formation of uniform products has been observed on the macroscopic (Re) and no-carrier-added level (99mTc). The Tc-99m complexes revealed good inertness against ligand exchange (Cys and His) and excellent stability in physiological buffered saline as well as in human plasma over a period of 24 h at 37 degrees C. The rhenium complexes have been tested for competitive inhibition of the (yeast) hexokinase. Only for C-2 derivatized glucose complexes with extended spacer functionalities Ki values in the millimolar and sub-millimolar range have been observed. In silico molecular docking experiments supported these experimental findings. However, the competitive inhibitors are not recognized as a pseudosubstrate of hexokinase. The cellular uptake of all 99mTc-complexes into HT-29 colon carcinoma cells via Glut1 was generally low and unspecific independent of the position at the hexose ring, the chelating systems, or the overall charge of the corresponding metal complexes. The current results seem to preclude the use of these compounds as [18F]-FDG surrogates primarily due to the low cellular uptake via Glut1.     

Technetium labeling of monoclonal antibodies with functionalized BATOs. 1. TcCl(DMG)3PITC

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive m-phenyl isothiocyanate (PITC). The 99TcCl(dioxime)3PITC complexes [dioxime = dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO)] were prepared from 99Tc(dioxime)3(mu-OH)SnCl3 and characterized. The X-ray crystal structure of 99TcCl(DMG)3PITC was determined. The 99mTc complexes were prepared from 99mTcO4- in a process using a freeze-dried kit, either in a one-step procedure or via 99mTcCl(dioxime)3. Initial labeling studies with 99mTcCl(dioxime)3PITC were performed on glycine and polylysine and, subsequently, on mouse IgG and the B72.3 monoclonal antibody. Covalent attachment of 99mTcCl(DMG)3PITC to B72.3 was demonstrated by SDS-PAGE electrophoresis. B72.3 labeled with 99mTcCl(DMG)3PITC displayed high binding to a TAG 72 affinity column and had a distribution in normal mice similar to that reported for iodine-labeled B72.3.     

Control of radioactivity pharmacokinetics of 99mTc-HYNIC-labeled polypeptides derivatized with ternary ligand complexes

An enhancement of the target/nontarget ratio of radioactivity levels enables reliable diagnosis and therapy using polypeptide radiopharmaceuticals in nuclear medicine. In the present study, we investigated the effects of the physicochemical properties of radiometabolites on the radioactivity pharmacokinetics after administration of 99mTc-labeled polypeptides using 6-hydrazinopyridine-3-carboxylic acid (HYNIC). Four ternary ligands (L) [3-benzoylpyridine (BP), 3-acetylpyridine (AP), 3-nicotinic acid (NIC), pyridine (PY)] with different lipophilicity were selected as coligands for the preparation of 99mTc-HYNIC-polypeptides. Each of the ternary ligands tested provided 99mTc-HYNIC-labeled galactosyl-neoalbumin (NGA) and Fab fragments of high stability with high radiochemical purity. Moreover, after administration of each 99mTc-HYNIC-labeled NGA into normal mice, the respective ternary ligand [99mTc](HYNIC-lysine)(tricine)(L) complexes were generated as final radiometabolites in the hepatic lysosome. The partition coefficients of [99mTc](HYNIC-lysine)(tricine)(BP), [99mTc](HYNIC-lysine)(tricine)(AP), [99mTc](HYNIC-lysine)(tricine)(NIC), and [99mTc](HYNIC-lysine)(tricine)(PY) were determined to be -2.21, -2.37, -2.93, and -2.73, respectively. Elimination rates of these radiometabolites from the lysosome were enhanced in the order of increasing lipophilicity of the radiometabolites. After injection of the four 99mTc-HYNIC-labeled Fab fragments into normal mice, blood clearances of radioactivity were similar while radioactivity elimination rates from the kidney were enhanced in the order of increasing lipophilicity of the radiometabolites. The present study indicated that the lipophilicity of the radiometabolites constitutes one important factor affecting their elimination rates from the tissues. Thus, as ternary ligands facilitate alteration of the physicochemical properties of radiometabolites, the use of ternary ligand complexes might be applicable for controlling the pharmacokinetics of 99mTc-labeled polypeptides.     

Carbohydrate conjugates for molecular imaging and radiotherapy: 99mTc(I) and 186Re(I) tricarbonyl complexes of N-(2'-Hydroxybenzyl)-2-amino-2-deoxy-D-glucose

An approach to a new class of potential radiopharmaceuticals is demonstrated by the labeling of a glucosamine derivative with the tricarbonyls of 99mTc and 186Re. The proligand HL2 (N-(2'-hydroxybenzyl)-2-amino-2-deoxy-D-glucose) was produced by hydrogenation of the corresponding Schiff base and reacted with [NEt4]2[Re(CO)3Br3] to form the neutral complex [(L2)Re(CO)3] in 40% yield. 1H and 13C NMR spectra indicate that the [Re(CO)3] core is bound in a tridentate fashion via the amino N, phenolato O, and C-3 hydroxyl O atoms of the ligand. At the tracer-level, labeling of HL2 with [99mTc(CO)3(H2O)3]+ and [186Re(CO)3(H2O)3]+ was achieved in aqueous conditions in 95 +/- 2% and 94 +/- 3% average radiochemical yields, respectively.     

99mTc-labeling of HYNIC-conjugated cyclic RGDfK dimer and tetramer using EDDA as coligand

In this study, EDDA (ethylenediamine- N, N'-diacetic acid) was used as the coligand for 99mTc-labeling of cyclic RGDfK conjugates: HYNIC-dimer (HYNIC = 6-hydrazinonicotinamide; dimer = E[c(RGDfK)]2) and HYNIC-tetramer (tetramer = E{E[c(RGDfK)]2}2). First, HYNIC-dimer was allowed to react with 99mTcO4 (-) in the presence of excess tricine and stannous chloride to form the intermediate complex [99mTc(HYNIC-dimer)(tricine)2], which was then allowed to react with EDDA to afford [99mTc(HYNIC-dimer)(EDDA)] with high yield (>90%) and high specific activity ( approximately 8.0 Ci/micromol). Under the same radiolabeling conditions, the yield for [99mTc(HYNIC-tetramer)(EDDA)] was always <65%. The results from a mixed-ligand experiment show that there is only one EDDA bonding to the 99mTc-HYNIC core in [99mTc(HYNIC-dimer)(EDDA)]. The athymic nude mice bearing subcutaneous U87MG human glioma xenografts were used to evaluate the impact of EDDA coligand on the biodistribution characteristics and excretion kinetics of the 99mTc-labeled HYNIC-dimer and HYNIC-tetramer. Surprisingly, [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] had almost identical tumor uptake over the 2 h period. The use of EDDA as coligand to replace tricine/TPPTS (TPPTS = trisodium triphenylphosphine-3,3',3'-trisulfonate) did not significantly change the uptake of the 99mTc-labeled HYNIC-dimer in noncancerous organs, such as the liver, kidneys, and lungs; but it did result in a significantly lower kidney uptake for the 99mTc-labeled HYNIC-tetramer due to faster renal excretion. It was also found that the radiotracer tumor uptake decreases in a linear fashion as the tumor size increases. The smaller the tumors are, the higher the tumor uptake is regardless of the identity of radiotracer.     

Impact of PKM linkers on biodistribution characteristics of the 99mTc-labeled cyclic RGDfK dimer

This report describes synthesis of three new cyclic RGDfK peptide conjugates, HYNIC-PKM-SU016 (PKM = E, K and PEG4) and in vivo evaluation of the impact of PKM linkers on biodistribution characteristics of their ternary ligand complexes [99mTc(HYNIC-PKM-SU016)1(tricine)(TPPTS)] in athymic nude mice bearing the MDA-MB-435 human breast cancer xenografts. Results from biodistribution studies show that PKM linkers have minimal impact on the integrin alphavbeta3 binding capability of radiotracers. Even though they have different charges under physiological conditions, all three linkers (E, K, and PEG4) are able to reduce the uptake of 99mTc-labeled E[c(RGDfK)]2 in blood, kidneys, liver, and lungs, and increase target-to-background (T/B) ratios at >30 min postinjection. E and K may have advantages over PEG4 due to a combination of relatively low liver uptake and high tumor/liver and tumor/lung ratios of ternary ligand complexes [99mTc(HYNIC-PKM-SU016)(tricine)(TPPTS)] (PKM = E and K).     

A novel approach to the high-specific-activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP = diphosphine ligand).

A new labeling approach for incorporating bioactive peptides into a technetium-99m coordination complex is described. This method exploits the chemical properties of the novel metal-nitrido fragment [99mTc(N)(PXP)]2+, composed of a terminal Tc[triple bond] N multiple bond bound to an ancillary diphosphine ligand (PXP). It will be shown that this basic, molecular building block easily forms in solution as the dichloride derivative [99mTc(N)(PXP)Cl2], and that this latter complex selectively reacts with monoanionic and dianionic, bidentate ligands (YZ) having soft, pi-donor coordinating atoms to afford asymmetrical nitrido heterocomplexes of the type [99mTc(N)(PXP)(YZ)]0/+ without removal of the basic motif [99mTc(N)(PXP)]2+. The reactions of the amino acid cysteine was studied in detail. It was found that cysteine readily coordinates to the metal fragment [99mTc(N)(PXP)]2+ either through the [NH2, S-] pair of donor atoms or, alternatively, through the [O-, S-] pair, to yield the corresponding asymmetrical complexes in very high specific activity. Thus, these results were conveniently employed to devise a new, efficient procedure for labeling short peptide sequences having a terminal cysteine group available for coordination to the [99mTc(N)(PXP)]2+ fragment. Examples of the application of this novel approach to the labeling of the short peptide ligand H-Arg-Gly-Asp-Cys-OH (H(2)1) and of the peptidomimetic derivative H-Cys-Val-2-Nal-Met-OH (H2) will be discussed.     

Synthesis, metal complexation and biological evaluation of a novel semi-rigid bifunctional chelating agent for 99mTc labelling

A novel bifunctional chelating agent bearing an aromatic ring has been synthesised and characterised. This ligand formed well-defined oxorhenium complexes. The analogous 99mTcO-complex was obtained in an excellent yield with high radiochemical purity (>95%). The biodistribution of the 99mTc-complex after intravenous injection studied in normal rats showed that the activity was excreted mainly via renal-urinary pathway indicating its use for labelling peptides with 99mTc.     

Development of novel mixed-ligand oxotechnetium [SNS/S] complexes as potential 5-HT1A receptor imaging agents

The "3 + 1" ligand system [SN(R)S/S combination] was applied in order to synthesize neutral mixed-ligand oxotechnetium complexes of the general formula 99mTcO[SN(R)S]/[S] as potential 5-HT1A receptor imaging agents. The complexes are carrying the 1-(2-methoxyphenyl)piperazine moiety, a fragment of the true 5-HT1A antagonist WAY 100635, either on the monodentate ligand [S] or on the tridentate ligand [SN(R)S]. The complexes MO[EtN(CH2CH2S)2] [o-MeOC6H4N(CH2CH2)2NCH2CH2S] (3), MO[o- MeOC6H4N(CH2CH2)2N(CH2)3N(CH2CH2S)2][PhS] (6) and MO[o-MeOC6H4N(CH2CH2)2N(CH2)3N(CH2CH2S)2] [PhCH2CH2S] (9), where M = 99mTc, were prepared at tracer level using 99mTc glucoheptonate as precursor. For structural characterization, the analogous oxorhenium (M = Re, 1, 4 and 7, respectively) and oxotechnetium (M = 99gTc, 2, 5 and 8, respectively) complexes were prepared by ligand exchange reactions. All products were characterized by elemental analysis and spectroscopic methods. Complexes 1, 4 and 7 were further characterized by crystallographic analysis. For 1, the coordination geometry about rhenium can be described as trigonally distorted square pyramidal (tau = 0.36), while for 4 and 7, as distorted trigonal bipyramidal (tau = 0.66 and tau = 0.61, respectively). The coordination sphere about oxorhenium in all complexes is defined by the SNS donor atom set of the tridentate ligand and the sulfur atom of the monodentate coligand. The structure of the 99mTc complexes 3, 6 and 9 was established by comparative HPLC using authentic oxorhenium and oxotechnetium samples. The binding affinity of oxorhenium compounds for the 5-HT1A receptor subtype was determined in rat brain hippocampal preparations (IC50 = 6-31 nM). Preliminary tissue distribution data in healthy mice revealed the ability of all three 99mTc complexes to cross the intact blood-brain barrier (0.49-1.15% ID at 1 min p.i.). In addition, complexes 6 and 9 showed significant brain retention. These promising results have demonstrated that the SNS/S mixed-ligand system can be used in the development of 99mTc complexes as potential 5-HT1A receptor imaging agents.     

Technetium-99m-labeled medium-chain fatty acid analogues metabolized by beta-oxidation: radiopharmaceutical for assessing liver function.

External imaging of energy production activity of living cells with 99mTc-labeled compounds is a challenging task requiring good design of 99mTc-radiopharmaceuticals. On the basis of our recent findings that 11C- and 123I-labeled medium-chain fatty acids are useful for measuring beta-oxidation activity of hepatocytes, we focused on development of 99mTc-labeled medium-chain fatty acid analogues that reflect beta-oxidation activity of the liver. In the present study, monoamine-monoamide dithiol (MAMA) ligand and triamido thiol (MAG) ligand were chosen as chelating groups because of the stability and size of their complexes with 99mTc and their ease of synthesis. Each ligand was attached to the omega-position of hexanoic acid (MAMA-HA and MAG-HA, respectively). In biodistribution studies, [99mTc]MAMA-HA showed high initial accumulation in the liver followed by clearance of the radioactivity in the urine. Analysis of the urine revealed [99mTc]MAMA-BA as the sole radiometabolite. Furthermore, when [99mTc]MAMA-HA was incubated with living liver slices, generation of [99mTc]MAMA-BA was observed. However, [99mTc]MAMA-HA remained intact when the compound was incubated with liver slices in the presence of 2-bromooctanoate, an inhibitor of beta-oxidation. The findings in this study indicated that [99mTc]MAMA-HA was metabolized by beta-oxidation after incorporation into the liver. On the other hand, poor hepatic accumulation was observed after administration of [99mTc]MAG-HA.     

99mTc-labeled cyclic RGD peptides for noninvasive monitoring of tumor integrin αVβ3 expression

This report describes the biologic evaluations of [99mTc(HYNIC-3P-RGD2)(tricine)(TPPTS)] (99mTc-3P-RGD2: 6-hydrazinonicotinyl; 3P-RGD2 = PEG4-E[PEG4-c(RGDfK)]2; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid; and TPPTS = trisodium triphenylphosphine-3,3',3'-trisulfonate), [99mTc(HYNIC-3G-RGD2)(tricine)(TPPTS)] (99mTc-3G-RGD2: 3G-RGD2 = G3-E[G3-c(RGDfK)]2 and G3 = Gly-Gly-Gly), and 99mTcO(MAG2-3G-RGD2) (MAG2 = mercaptoacetylglycylglycyl) as radiotracers for noninvasive imaging of tumor integrin αvβ3 expression in five xenografted tumor-bearing models. Biodistribution and imaging studies were performed in athymic nude mice bearing U87MG, MDA-MB-435, A549, HT29, or PC-3 tumor xenografts. Immunochemistry was performed using the cultured primary tumor cells and xenografted tumor tissues. It was found that the radiotracer tumor uptake followed the trend U87MG > MDA-MB-435 ≈ HT29 ≈ A549 > PC-3. The total integrin β3 expression levels followed the general trend: U87MG > MDA-MB-435 ≈ A549～HT29 > PC-3. There is a linear relationship between the radiotracer injected dose per gram tumor uptake and the total integrin β3 expression levels. On the basis of these, it was concluded that radiotracer tumor uptake is contributed by integrin αvβ3 expressed on tumor cells and activated endothelial cells of the tumor neovasculature. 99mTc-3P-RGD2 has the capability to monitor integrin αvβ3 expression in a noninvasive fashion.     

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.