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.     

Assessment of the effect of antiseizure drugs on the labeling process of red blood cells and plasma proteins with technetium-99m.

It is estimated that about 2.5 million people only in the United States are affected by epilepsy. Labelled red blood cells (RBC) and plasma proteins (PP) are used for several evaluations in nuclear medicine and drugs affecting those labelings have previously been described. The aim of this study was to evaluate whether the most popular antiseizure drugs interfere with the 99mTc labeling process of RBC and PP. Heparinized blood withdrawn from Wistar rats was incubated with phenobarbital (0.2, 2, 20, 200, 2,000 microg/ml), phenytoin (0.15, 1.5, 15, 150, 1,500 microg/ml), carbamazepine (0.7, 7, 70 microg/ml), clonazepam (0.5, 5, 50, 500 microg/ml) or valproic acid (0.5, 5, 50, 500 microg/ml) for I hr. Stannous chloride (SnCl2), in two different concentrations (0.012 or 1.2 microg/ml) and 99mTc were added. Plasma and cellular fractions were isolated by centrifugation, soluble and insoluble fractions were separated by trichloroacetic acid precipitation. The percentage of radioactivity was calculated for each fraction. Statistical analysis was performed with ANOVA and Dunnet tests. The analysis of the results has shown that phenobarbital (2,000 microg/ml) and clonazepam (50 microg/ml) significantly have reduced the RBC labeling efficiency when it was used the optimal SnCl2 concentration (1.2 microg/ml) and clonazepam (5, 50 microg/ml) has significantly decreased the PP labeling efficiency with 99mTc. Phenytoin (1,500 microg/ml) has decreased the RBC labeling efficiency when the experiments were carried out with a small SnCl2 concentration (0.012 microg/ml). We can suggest that with this in vitro assay, at the therapeutic level of phenytoin, phenobarbital, carbamazepine and valproic acid will not interfere on the 99mTc labeling process of RBC. Interference is displayed at higher phenobarbital concentrations (2,000 microg/ml). However, humans do not tolerate this concentration. On the other hand, a decreased RBC and PP labeling efficiency with 99mTc may be expected for clonazepam at therapeutic levels.     

Evaluation of 99mTc(CO)3 complex of 2-methyl-5-nitroimidazole as an agent for targeting tumor hypoxia

An iminodiacetic acid (IDA) derivative of 2-methyl-5-nitroimidazole was synthesized as a carrier molecule for radiolabeling with the gamma emitting radioisotope, 99mTc, for imaging hypoxic regions of tumors. The ligand was synthesized in excellent yield and labeled using freshly prepared [99mTc(CO)3(H2O)3]+ intermediate. A complexation yield of over 95% could be achieved under mild conditions using a ligand concentration of 1 mg/mL [ approximately 3 x 10(-3)M]. The complex was characterized by HPLC and its stability in human serum was studied. Biodistribution studies performed in Swiss mice bearing fibrosarcoma tumor showed maximum accumulation in the tumor to the extent of approximately 0.52 % ID/g at 30 min post-injection (pi). The major clearance of the complex was through the hepatobiliary route. The complex showed tumor/muscle ratio of 1.75 at 30 min pi, which significantly increased to 17 at 180 min pi. However, the tumor/blood ratio was below one throughout the period of study, which could be due to slow clearance of the complex from blood.     

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.     

Organometallic 99mTc(III) '4 + 1' bombesin(7-14) conjugates: synthesis, radiolabeling, and in vitro/in vivo studies

Bombesin (BBN) peptide exhibits high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). The GRPr is overexpressed on many human cancer cell types, thus making BBN a potent delivery vehicle for radionuclide targeting. In this study, the biologically active minimal sequence BBN(7-14) was labeled using the novel Tc '4 + 1' mixed-ligand system, [Tc(NS3)(CN-R)], in which Tc(III) is coordinated by a monodentate isocyanide linker bearing the peptide and the tetradentate, tripodal chelator, 2,2',2'-nitrilotriethanethiol (NS3). BBN(7-14) was N-terminally modified with Gly-Gly-Gly, betaAla, and Ser-Ser-Ser spacer groups (X) and functionalized with 4-(isocyanomethyl)benzoic acid (L1) or 4-isocyanobutanoic acid (L2), resulting in a series of [M(NS3)(L-X-BBN(7-14))] conjugates (M = 99mTc, Re). The isocyanide ligand frameworks were introduced using novel bifunctional coupling agents. The spacer groups (X), the monodentate isocyanide units, and a tetradentate NS3 chelator bearing a pendant carboxylic acid (NS3COOH) were proposed as pharmacological modifiers. 99mTc-labeling was performed in a two-step procedure by first preparing 99mTc-EDTA/mannitol followed by reactions with the isocyanides and NS3 or NS3COOH ligand frameworks. The 99mTc complexes were obtained with a radiochemical yield of 30-80% depending on the amount of the isocyanide (20-100 nmol) used. These new conjugates were purified by reversed-phased high-performance liquid chromatography (RP-HPLC) to give a radiochemical purity of >or=95%. The 99mTc conjugates exhibited high in vitro stability (>90%, 24 h). Analogous nonradioactive Re conjugates were synthesized and characterized by electrospray ionization mass spectrometry (ESI-MS). RP-HPLC analyses of the Re conjugates indicated that they exhibited identical retention times to the corresponding 99mTc conjugates under identical HPLC conditions, demonstrating structural similarity between the two metalated species. The [Re(NS3)(L-X-BBN(7-14))] conjugates exhibited GRPr affinity in the nanomolar range as demonstrated by in vitro competitive binding assays using PC-3 human prostate cancer cells. In vitro internalization/externalization assays indicated that approximately 65% of [99mTc(NS3)(L2-betaAla-BBN(7-14))] conjugate was either surface-bound or internalized in PC-3 cells. Cell-associated activity for all other 99mTc conjugates was below 20%. Biodistribution studies of [99mTc(NS3)(L-betaAla-BBN(7-14))], L = L1 or L2, in normal, CF-1 mice showed minimal accumulation in normal pancreas (a tissue expressing the GRPr in high density in rodent models) and rapid hepatobiliary elimination. Introduction of a carboxyl group onto the NS3 ligand framework had only minimal effects to increase renal excretion. Activity distribution and accumulation was highly dominated by the relatively lipophilic '4 + 1' complex unit.     

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.     

Novel procedures for preparing 99mTc(III) complexes with tetradentate/monodentate coordination of varying lipophilicity and adaptation to 188Re analogues

Improved methods are presented for the preparation of 99mTc and 188Re mixed-ligand complexes with tetradentate and monodentate ligands of the general formula [MIII(Lm)(Ln)] (M = Tc, Re; Lm = NS3 or NS3COOH; Ln = isocyanide or phosphine). To avoid the undesired formation of reduced-hydrolyzed species of both metals, the preparation of complexes is performed in a two-step procedure. At first the Tc(III)- or Re(III)-EDTA complex is formed which reacts in a second step with the tripodal ligand 2,2',2' '-nitrilotris(ethanethiol) (NS3) or its carboxyl derivative NS3COOH (a) and the monodentate phosphine ligands (triphenylphosphine L1, dimethylphenylphosphine L2) or isocyanides (tert-butyl isonitrile L3, methoxyisobutyl isonitrile L4, 4-isocyanomethylbenzoic acid-L-arginine L5, 4-isocyanomethylbenzoic acid-L-arginyl-L-arginine L6, 4-isocyanomethylbenzoic acid-neurotensin(8-13) L7) to the so-called '4+1' complex. Copper(I) isocyanide complexes are used for preparing the '4+1' complexes. That facilitates storage stability and allows kit formulations, and, moreover, enables the formation of 188Re complexes in acidic solution. Only micromolar amounts of the monodentate ligand are needed, and that results in high specific activity labeling of interesting molecules. The lipophilicity of complexes can be controlled by introducing a carboxyl group into the tetradentate ligand and/or derivatization of the monodentate ligands. Furthermore, the carboxyl group enables the conjugation of biomolecules. As an example, the neurotensin derivative CN-NT(8-13) was prepared and labeled with 99mTc according to the '4+1' approach, and its behavior in vivo was studied.     

Synthesis, characterization, and labeling with 99mTc/188Re of peptide conjugates containing a dithia-bisphosphine chelating agent

Radiolabeling of small receptor-avid peptides at specific predetermined chelation sites with radioactive metals has been an effective approach for production of target-specific radiopharmaceuticals for diagnosis and therapy of diseases. Among various electron-donating groups found on chelator frameworks, phosphines are unique because they display versatile coordination chemistry with a wide range of transition metals. We have recently reported the utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to form 99mTc-labeled receptor-avid peptides by the preconjugation approach. Here we report a novel strategy for labeling small peptides with both 99mTc and 188Re using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)propylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approach. The first step in this approach involves the coupling of the corresponding (PH2)2S2-COOH intermediate to the N-terminus of the peptide(s). Formylation of P-H bonds with aqueous formaldehyde in the presence of HCl in ethanol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjugates in the form of an oxidatively stable phosphonium salt. The P2S2-peptide conjugates are generated (where the PH2 groups are converted to P(CH2OH)2 groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M sodium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with 99mTc is achieved by direct reduction with Sn(II) tartarate to yield the 99mTc-P2S2-peptide conjugate in near quantitative yields. Complexation of the BFCA conjugates with 188Re is achieved by transchelation with 188Re citrate in yields of >/=90%. In this study, (PH2)2S2-COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)2S2-COOH BFCA conjugate was converted to the hydroxymethylene phosphine form and complexed with 99mTc to produce the 99mTcO2-P2S2-GlyGlyOEt conjugate 8 in RCPs of >/=95%. This singular 99mTc product is stable over 24 h in aqueous solution as confirmed by HPLC. Identical retention times of the 99mTcO2-P2S2-GlyGlyOEt complex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The utility of this postconjugation strategy was further demonstrated by synthesizing a P2S2-D-Lys6-LHRH conjugate and producing its corresponding 99mTc complex in RCPs of >/=88%. Finally, the P2S2-5-Ava-BBN[7-14]NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converted to P(CH2OH)2 groups and subsequently labeled with 188Re to yield a 188Re-labeled bombesin analogue with a RCP of >/=90%. The biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)2S2-COOH BFCA can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (99mTc) and therapeutic (188Re) radionuclides via the postconjugation approach in high yields.     

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.     

Synthesis of novel peptide nucleic acid-peptide chimera for non-invasive imaging of cancer

A chelator-peptide-PNA-peptide chimera specific for KRAS has been prepared by continuous solid phase coupling with a C-terminal insulin-like growth factor 1 (IGF1) ligand, D(cys-ser-lys-cys), and N-terminal bis(S-benzoyl thioglycoloyl) diaminopropanoate chelator for radionuclide labeling. The probe was purified by RP-HPLC and characterized by MALDI-TOF mass spectroscopy. The probe was labeled with 99mTc and 64Cu. Both labeled probes accumulated in human pancreatic cancer xenografts in immunocompromised mice. Control experiments with mismatch chimeras and control xenografts will be necessary to determine the specificity of this molecular diagnostic strategy.     

99mTc-labeled MIBG derivatives: novel 99mTc complexes as myocardial imaging agents for sympathetic neurons

Radioactive-iodine-labeled meta-iodobenzylguanidine (MIBG) is currently being used as an in vivo imaging agent to evaluate neuroendocrine tumors as well as the myocardial sympathetic nervous system in patients with myocardial infarct and cardiomyopathy. It is generally accepted that MIBG is an analogue of norepinephrine and its uptake in the heart corresponds to the distribution of norepinephrine and the density of sympathetic neurons. A series of MIBG derivatives containing suitable chelating functional groups N2S2 for the formation of [TcvO]3+N2S2 complex was successfully synthesized, and the 99mTc-labeled complexes were prepared and tested in rats. One of the compounds, [99mTc]2, tested showed significant, albeit lower, heart uptakes post iv injection in rats (0.21% dose/g at 4 h) as compared to [125I]MIBG (1.7% dose/g at 4 h). The heart uptake of the 99mTc-labeled complex appears to be specific and can be reduced by co-injection with nonradioactive MIBG or by pretreatment with desipramine, a selective norepinephrine transporter inhibitor. Further evaluation of the in vitro uptake of [99mTc]2 in cultured neuroblastoma cells displayed consistently lower, but measurable uptake (approximately 10% of that for [125I]MIBG). These preliminary results suggested that the mechanisms of heart uptake of [99mTc]2 may be related to those for [125I]MIBG uptake. If suitable 99mTc-labeled MIBG derivatives can be further developed, the prevalent availability of 99mTc in nuclear medicine clinics will allow them to be readily available for widespread application.     

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.     

A simple two-step approach for introducing a protected diaminedithiol chelator during solid-phase assembly of peptides

A simple synthetic strategy is described to incorporate a protected diaminedithiol (N(2)S(2)) chelator during Fmoc solid-phase synthesis of short peptides. The resulting constructs could be efficiently labeled with technetium-99m (99mTc). The chelator was assembled at the N-terminus of peptides in a two-step procedure where the deprotected terminal amino group was first reacted with di-Fmoc-diaminopropionic acid (Fmoc-DAP-[Fmoc]-OH). The two protected amino groups were then simultaneously deprotected and subsequently reacted with S-benzoylthiolglycolic acid (TGA) to generate a protected N(2)S(2) chelator. This metal binding site was introduced into di- and tripeptides. Each peptide construct was composed of a C-terminal lysine residue and an N-terminal diaminopropionic moiety modified to create the chelator site. The epsilon-amino group at the C-terminal lysine was further derivatized with a nitroimidazole group to facilitate cellular retention. The resulting constructs were then cleaved from the resin support, purified, and labeled with [99mTc]pertechnetate. Six constructs were prepared differing by a single amino acid inserted between the diaminopropionic acid and lysine residues. Optimal labeling yields of >70% were achieved around neutral pH and heating at 75 degrees C for 10 min. Purified 99mTc-labeled constructs were found to accumulate in Chinese hamster ovary (CHO) cells in vitro as a function of charge and hydrophobicity.     

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.     

Detection of myocardial ischemia-reperfusion injury using a fluorescent near-infrared zinc(II)-dipicolylamine probe and 99mTc glucarate

A fluorescent zinc 2,2'-dipicolylamine coordination complex PSVue?794 (probe 1) is known to selectively bind to phosphatidylserine exposed on the surface of apoptotic and necrotic cells. In this study, we investigated the cell death targeting properties of probe 1 in myocardial ischemia-reperfusion injury. A rat heart model of ischemia-reperfusion was used. Probe 1, control dye, or 99mTc glucarate was intravenously injected in rats subjected to 30-minute and 5-minute myocardial ischemia followed by 2-hour reperfusion. At 90 minutes or 20 hours postinjection, myocardial uptake was evaluated ex vivo by fluorescence imaging and autoradiography. Hematoxylin-eosin and cleaved caspase-3 staining was performed on myocardial sections to demonstrate the presence of ischemia-reperfusion injury and apoptosis. Selective accumulation of probe 1 could be detected in the area at risk up to 20 hours postinjection. Similar topography and extent of uptake of probe 1 and 99mTc glucarate were observed at 90 minutes postinjection. Histologic analysis demonstrated the presence of necrosis, but only a few apoptotic cells could be detected. Probe 1 selectively accumulates in myocardial ischemia-reperfusion injury and is a promising cell death imaging tool.     

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.     

Noninvasive molecular imaging of MYC mRNA expression in human breast cancer xenografts with a [99mTc]peptide-peptide nucleic acid-peptide chimera

Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a [99mTc]chelator peptide on the N-terminus, could measure levels of MYC mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in nude mice. We prepared the chelator-MYC PNA-IGF1 peptide, as well as a 4-nt mismatch PNA control, by solid-phase synthesis. We imaged MCF7 xenografts scintigraphically and measured the distribution of [99mTc]probes by scintillation counting of dissected tissues. MCF7 xenografts in nude mice were visualized at 4 and 24 h after tail vein administration of the [99mTc]PNA probe specific for MYC mRNA, but not with the mismatch control. The [99mTc]probes distributed normally to the kidneys, livers, tumors, and other tissues. Molecular imaging of oncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might provide additional genetic characterization of preinvasive and invasive breast cancers.     

Distamycin paradoxically stimulates the copying of oligo(dA).poly(dT) by DNA polymerases

Distamycin A, a polypeptide antibiotic, binds to dA.dT-rich regions in the minor groove of B-DNA. By virtue of its nonintercalating binding, distamycin acts as a potent inhibitor of the synthesis of DNA both in vivo and in vitro. Here we report that distamycin paradoxically stimulates Escherichia coli DNA polymerase I (pol I), its large (Klenow) fragment, and bacteriophage T4 DNA polymerase to copy oligo(dA).poly(dT) in vitro. It is found that distamycin increases the maximum velocity (Vmax) of the extension of the oligo(dA) primer by pol I without affecting the Michaelis constant (Km) of the primer. Gel electrophoresis of the extended primer indicates that the antibiotic specifically increases the rate of addition of the first three dAMP residues. Lastly, in the presence of both distamycin and the oligo(dT)-binding protein factor D, which increases the processivity of pol I, a synergistic stimulation of polymerization is attained. Taken together, these results suggest that distamycin stimulates synthesis by increasing the rate of initiation of oligo(dA) extension. The stimulatory effect of distamycin is inversely related to the stability of the primer-template complex. Thus, maximum stimulation is exerted at elevated temperatures and with shorter oligo(dA) primers. That distamycin increases the thermal stability of [32P](dA)9.poly(dT) is directly demonstrated by electrophoretic separation of the hybrid from dissociated [32P](dA)9 primer. It is proposed that by binding to the short primer-template duplex, distamycin stabilizes the oligo(dA).poly(dT) complex and, therefore, increases the rate of productive initiations of synthesis at the primer terminus.     

Imidazoles as well as thiolates in proteins bind technetium-99m.

99mTc is widely thought to directly bind proteins through thiolate groups of cysteine residues, resulting in Tc-cysteinyl-protein bonds. Chemical reduction of disulfide bonds in proteins is widely used to generate thiolates with the goal of increasing 99mTc binding. This strategy is used because most proteins contain no thiolates, but many do contain disulfide bonds. In this study, we have evaluated the hypothesis that imidazole groups of histidine are also involved in direct 99mTc binding to proteins. Human gamma-globulin was used as the model protein in these studies. The immunoglobulin was used (a) without reduction or was (b) treated with stannous ions to reduce disulfide bonds thereby increasing thiolate concentration. These proteins were used to evaluate the hypothesis that imidazole as well as thiolate groups bind Tc. The proteins were evaluated by (a) using free amino acids to compete with proteins for 99mTc and (b) by chemical modification of amino acid side chains. In addition, peptides known to contain either cysteine or histidine, but not both, were also successfully directly labeled with 99mTc. These results indicate that in proteins (and peptides) imidazole-containing groups as well as thiolate-containing groups bind Tc.