Synthesis of 99mTc-labeled 2-Mercaptobenzimidazole as a novel radiotracer to diagnose tumor hypoxia

Discovery of ^99mTc-labeled imidazole derivatives as a potential radiotracer for hypoxic tumor imaging is considered to be of great interest because of non-invasive detection capabilities. 2-Mercaptobenzimidazole (2-MBI) was successfully synthesized, characterized and radiolabeled with [^99mTc (CO)₃(H₂O)₃]⁺ intermediate to form ^99mTc-2-MBI complex with radiochemical purity of ≥95% yield as observed by instant-thin layer chromatography (ITLC) and radio-high performance liquid chromatography (radio-HPLC). The ^99mTc-2-MBI complex was observed to be stable in saline and serum with no noticeable decomposition at room temperature and 37 °C, respectively, over a time period of 24 h. Biodistribution results in Balb/c mice bearing S180 tumor show that ^99mTc-2-MBI highly internalized in tumor tissue, also possess preferably high tumor/muscle and tumor/blood ratios 4.14 ± 0.77 and 3.91 ± 0.63, respectively at 24 h incubation. Scintigraphic imaging study shows ^99mTc-2-MBI is visibly accumulated in hypoxic tumor tissue, suggesting it would be a promising radiotracer for early stage diagnosis of tumor hypoxia.     

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.     

A novel 99mTc-labeled testosterone derivative as a potential agent for targeting androgen receptors

With an insight that ligands possessing a N2S2 tetradentate array of donor atoms serve as ideal bifunctional chelating agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized conditions with 99mTc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radiolabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety with the testosterone receptor.     

Water-soluble 99mTc-labeled dendritic novel porphyrins tumor imaging and diagnosis

We have synthesized two water soluble dendritic porphyrins, termed DP1 and DP2 and have successfully radiolabeled them with 99mTc. These 99mTc-labeled porphyrins were administered to C6-glioma tumor bearing Wistar rats and scintiimaging and biodistribution studies were carried out. Tumor to muscle ratios of DP1 and DP2 were 8.0 and 9.7, respectively. These molecules may have potential for tumor imaging and diagnosis and may even prove useful as photosensitizers in photodynamic therapy applications.     

Evaluation of 99mTc-labeled cyclic RGD peptide with a PEG4 linker for thrombosis imaging: comparison with DMP444

DMP444 is a (99m)Tc-labeled cyclic RGD peptide, which has been evaluated in preclinical canine deep vein thrombosis (DVT) and pulmonary embolism (PE) models, and in patients with DVT and PE by SPECT (single photon emission computed tomography). Clinical data indicated that DMP444 is useful for imaging DVT, but it had limited utility for imaging PE in patients. To understand its clinical findings, we prepared a new radiotracer P4-DMP444 by replacing the lipophilic 6-aminocaproic acid (CA) in DMP444 with a highly water-soluble PEG(4) (15-amino-4,7,10,13-tetraoxapentadecanoic acid) linker. The objective of this study was to explore the impact of PEG(4) on biological properties (biodistribution, excretion kinetics, and capability to image thrombi) of (99m)Tc radiotracer. We also used canine DVT and PE models to perform imaging studies with/without the heparin pretreatment. These studies were specifically designed to explore the impact of heparin treatment on thrombosis uptake of P4-DMP444. It was found that replacing the CA linker with PEG(4) could enhance the radiotracer clearance kinetics from blood and normal organs in both rats and dogs. The fact that P4-DMP444 and DMP444 share very similar thrombosis uptake in both DVT and PE models suggests that the PEG(4) linker has little effect on GPIIb/IIIa binding affinity of cyclic RGD peptide. Even though P4-DMP444 had less accumulation than DMP444 in the blood, heart, lungs, and muscle over the 2 h study period in both rats and dogs, the difference in PE/lung and DVT/muscle ratios is marginal, suggesting that one PEG(4) linker is not sufficient to dramatically change the contrast between thrombus and background. It is very important to note that the heparin treatment of dogs with DVT and PE resulted in dramatic decrease in accumulation of P4-DMP444 in fresh thrombi. On the basis of these results, we believe that DMP444 and P4-DMP444 are excellent radiotracers for imaging both DVT and PE, and should be used in patients without antithrombosis treatment at the time of imaging.     

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.     

Preliminary in vivo evaluation of a novel 99mTc-labeled HYNIC-cys-annexin A5 as an apoptosis imaging agent

A novel cys-annexin A5 with a single cysteine-residue at its concave side has been developed by site-directed mutagenesis to allow conjugation through thiol-chemistry without affecting its apoptotic cell binding properties and was derivatized with HYNIC in a 1:1 stoichiometry. Similar to that of the 1st generation 99mTc-HYNIC-annexin A5, the novel 99mTc-HYNIC-cys-annexin A5 derivative shows in normal mice mainly renal and, to a lesser extent, hepatobiliary excretion. In murine models of hepatic apoptosis there was 257% increase in hepatic uptake of 99mTc-HYNIC-cys-annexin A5 as compared to normal mice. Using the novel tracer agent, acute reperfused myocardial infarction in rabbits was unequivocally delineated at 7h post-injection by muSPECT. The results indicate that the novel 99mTc-HYNIC-cys-annexin A5 shows similar apoptosis avidity as the 1st generation 99mTc-HYNIC-annexin A5.     

N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression

RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90?min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15?min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.     

99mTc-labeled monomeric and dimeric NGR peptides for SPECT imaging of CD13 receptor in tumor-bearing mice

CD13 receptor plays a critical role in tumor angiogenesis and metastasis. We therefore aimed to develop ^99mTc-labeled monomeric and dimeric NGR-containing peptides, namely, NGR1 and NGR2, for SPECT imaging of CD13 expression in HepG2 hepatoma xenografts. Both NGR-containing monomer and dimer were synthesized and labeled with ^99mTc. In vivo receptor specificity was demonstrated by successful blocking of tumor uptake of ^99mTc-NGR dimer in the presence of 20 mg/kg NGR2 peptide. Western blot and immunofluorescence staining confirmed the CD13 expression in HepG2 cells. The NGR dimer showed higher binding affinity and cell uptake in vitro than the NGR-containing monomer, presumably due to a multivalency effect. ^99mTc-Labeled monomeric and dimeric NGR-containing peptides were subjected to SPECT imaging and biodistribution studies. SPECT scans were performed in HepG2 tumor-bearing mice at 1, 4, 12, and 24 h post-injection of ~7.4 MBq tracers. The metabolism of tracers was determined in major organs at different time points after injection which demonstrated rapid, significant tumor uptake and slow tumor washout for both traces. Predominant clearance from renal and hepatic system was also observed in ^99mTc-NGR1 and ^99mTc-NGR2. In conclusion, monomeric and dimeric NGR peptide were developed and labeled with ^99mTc successfully, while the high integrin avidity and long retention in tumor make ^99mTc-NGR dimer a promising agent for tumor angiogenesis imaging.     

Micro-PET imaging of alphavbeta3-integrin expression with 18F-labeled dimeric RGD peptide

The alphav integrins, which act as cell adhesion molecules, are closely involved with tumor invasion and angiogenesis. In particular, alphavbeta3 integrin, which is specifically expressed on proliferating endothelial cells and tumor cells, is a logical target for development of a radiotracer method to assess angiogenesis and anti-angiogenic therapy. In this study, a dimeric cyclic RGD peptide E[c(RGDyK)]2 was labeled with 18F (t(1/2) = 109.7 min) by using a prosthetic 4-[18F]fluorobenzoyl moiety to the amino group of the glutamate. The resulting [18F]FB-E[c(RGDyK)]2, with high specific activity (200-250 GBq/micromol at the end of synthesis), was administered to subcutaneous U87MG glioblastoma xenograft models for micro-PET and autoradiographic imaging as well as direct tissue sampling to assess tumor targeting efficacy and in vivo kinetics of this PET tracer. The dimeric RGD peptide demonstrated significantly higher tumor uptake and prolonged tumor retention in comparison with a monomeric RGD peptide analog [18F]FB-c(RGDyK). The dimeric RGD peptide had predominant renal excretion, whereas the monomeric analog was excreted primarily through the biliary route. Micro-PET imaging 1 hr after injection of the dimeric RGD peptide exhibited tumor to contralateral background ratio of 9.5 +/- 0.8. The synergistic effect of polyvalency and improved pharmacokinetics may be responsible for the superior imaging characteristics of [18F]FB-E[c(RGDyK)]2.     

Structure-activity relationships of 111In- and 99mTc-labeled quinolin-4-one peptidomimetics as ligands for the vitronectin receptor: potential tumor imaging agents

The integrin receptor alpha(v)beta(3) is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. Radiolabeled alpha(v)beta(3) antagonists have demonstrated potential application as tumor imaging agents and as radiotherapeutic agents. This report describes the total synthesis of eight new HYNIC and DOTA conjugates of receptor alpha(v)beta(3) antagonists belonging to the quinolin-4-one class of peptidomimetics, and their radiolabeling with (99m)Tc (for HYNIC) and (111)In (for DOTA). Tethering of the radionuclide-chelator complexes was achieved at two different sites on the quinolin-4-one molecule. All such derivatives maintained high affinity for receptor alpha(v)beta(3) and high selectivity versus receptors alpha(IIb)beta(3), alpha(v)beta(5), alpha(5)beta(1). Biodistribution of the radiolabeled compounds was evaluated in the c-neu Oncomouse mammary adenocarcinoma model. DOTA conjugate (111)In-TA138 presented the best biodistribution profile. Tumor uptake at 2 h postinjection was 9.39% of injected dose/g of tissue (%ID/g). Activity levels in selected organs was as follows: blood, 0.54% ID/g; liver, 1.94% ID/g; kidney, 2.33% ID/g; lung, 2.74% ID/g; bone, 1.56% ID/g. A complete biodistribution analysis of (111)In-TA138 and the other radiolabeled compounds of this study are presented and discussed. A scintigraphic imaging study with (111)In-TA138 showed a clear delineation of the tumors and rapid clearance of activity from nontarget tissues.     

Synthesis and biological evaluation of a novel 99mTc labeled 2-nitroimidazole derivative as a potential agent for imaging tumor hypoxia

Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with ^99mTc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue. The biodistribution and scintigraphic image of the corresponding ^99mTc-complex showed accumulation in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.     

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.     

Comparative evaluation of 99mTc-labeled aminothiols as possible brain perfusion imaging agents

Several new ^99mTc aminodithiols were prepared and evaluated comparatively in experimental animals. The ligands were diamine, triamine or tetramine dithiols. Substituents were either attached on one of the nitrogens or introduced in between the two nitrogens of diamino dithiol (DADT) backbone. ^99mTc-derivatives prepared by coupling DADT to secondary amines via ethylene group showed in mice high initial brain uptake and significant retention in brain tissue. These preparations were mixtures of more than one ^99mTc-complex differing in brain uptake and clearance from the brain. The highest brain retention (brain to blood ratio 2.53, 15 min p.i.) was achieved with the ^99mTc-complex prepared by coupling DADT with ethylene pyrrolidine. Lengthening the chain between the nitrogens of DADT moiety by introducing methyl or amino alkyl groups resulted in ^99mTc-complexes with poor brain accumulation.     

18F Labeled benzimidazole derivatives as potential radiotracer for positron emission tomography (PET) tumor imaging

This article reported the synthesis and bioevaluation of two [¹⁸F] labeled benzimidazole derivatives, 4-(5-(2-[¹⁸F] fluoro-4-nitrobenzamido)-1-methyl-1H-benzimidazol-2-yl) butanoic acid ([¹⁸F] FNBMBBA, [¹⁸F]a1) and 3-(2-fluoroethyl)-7-methyl-2-propyl-3H-benzimidazole-5-carboxylic acid ([¹⁸F] FEMPBBA, [¹⁸F]b1) for PET tumor imaging. The preparation [¹⁸F] FEMPBBA was completed in 1 h with overall radiochemical yield of 50–60% (without decay corrected). Biodistribution assay in S180 tumor bearing mice of both compounds were carried out, and the results are both meaningful. [¹⁸F] FEMPBBA which can be taken as a revision of [¹⁸F] FNBMBBA got an excellent result, and has significant advantages in some aspects compared with L-[¹⁸F] FET and [¹⁸F]-FDG in the same animal model, especially in tumor/brain uptake ratio. The tumor/brain uptake ratio of [¹⁸F] FEMPBBA gets to 4.81, 7.15, and 9.8 at 30 min, 60 min and 120 min, and is much higher than that of L-[¹⁸F] FET (2.54, 2.92 and 2.95) and [¹⁸F]-FDG (0.61, 1.02, 1.33) at the same time point. The tumor/muscle and tumor/blood uptake ratio of [¹⁸F] FEMPBBA is also higher than that of L-[¹⁸F] FET at 30 min and 60 min. This result indicates compound [¹⁸F] FEMPBBA is a promising radiotracer for PET tumor imaging.     

A 99mTc(I)-postlabeled high affinity bombesin analogue as a potential tumor imaging agent

The overexpression of neuropeptide receptors observed in many cancers provides an attractive target for tumor imaging and therapy. Bombesin is a peptide exhibiting a high affinity for the gastrin releasing peptide (GRP) receptor, which is overexpressed by a variety of tumors such as breast or prostate cancer. In the present study, we have evaluated if the bombesin analogue [N(alpha)-histidinyl acetate]bombesin(7-14), radiolabeled with the novel [99mTc(OH(2))(3)(CO)(3)]+, has the potential to be used as a diagnostic radiopharmaceutical. Receptor saturation studies, carried out on the GRP receptor-expressing PC-3 human prostate cancer cell line, revealed for [99mTc(CO)(3)-N(alpha)-histidinyl acetate]bombesin(7-14) K(d) values in the subnanomolar range. Competitive binding assays, using the cold rhenium(I)-labeled analogue as a surrogate for the 99mTc-conjugate, also showed high affinity binding. Incubation of the radioconjugate with PC-3 cells resulted in a rapid temperature- and time-dependent specific internalization. At 37 degrees C more than 70% was internalized within the first 15 min and remained constant up to 2 h. Despite the weak proteolytic stability of [99mTc(CO)(3)-N(alpha)-histidinyl acetate]bombesin(7-14) in vitro, biodistribution studies, performed in PC-3 tumor-bearing mice, showed low uptake in the tumor (0.89 +/- 0.27% ID/g 30 min pi) but high uptake into the pancreas (7.11 +/- 3.93% ID/g 30 min pi), a GRP receptor-positive organ. Blockade experiment (coinjection of 300 microg bombesin/mouse with the radioligand) showed specificity of the uptake. Despite the low tumor uptake, tumor-to-blood ratios of 2.0 and 2.7 and tumor-to-muscle ratios of 8.9 and 8.0 were obtained at 30 min and 1.5 h postinjection, respectively. The promising results merit the future in vivo investigation of 99mTc/188Re-tricarbonyl-labeled bombesin analogues.     

In vitro and in vivo evaluation of a Technetium-99m-labeled cyclic RGD peptide as a specific marker of alpha(V)beta(3) integrin for tumor imaging

Three amino acids residues, Arg-Gly-Asp (RGD), in vitronectin and fibronectin show affinity for alpha(V)beta(3) integrins expressed in vascular endothelial cells. That tumor growth can upregulate the expression of these integrins on tumor cells for invasion and metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD peptides as antagonists of alpha(V)beta(3) integrins for broad spectrum tumor specific imaging. The polypeptide RGD-4C, which contains four cysteine residues for cyclization, has shown preferential localization on integrins at sites of tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for 99mTc radiolabeling. After purification of the conjugated peptides by a C18 Sep-Pak cartridge with 20% methanol, both peptides were radiolabeled using tricine. For cell binding studies, both 99mTc peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/micromol was achieved. Both 99mTc complexes were stable in the labeling solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degrees C and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of 99mTc-labeled RGD to cells. The binding percentage of 99mTc-labeled RGD to purified alpha(V)beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the integrin while 99mTc-labeled RGE showed no binding. The association constant for 99mTc-RGD was modest at 7 x 10(6) M(-)(1). In both human renal adenocarcinoma (ACHN) and human colon cancer cell line (LS174T) nude mouse tumor models, the accumulation of 99mTc-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers of alpha(V)beta(3) integrin receptors per tumor cell and low binding affinity, radiolabeled RGD peptides may have limitations as tumor imaging agents.     

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin α_vβ₃ is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the α_vβ₃ binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to α_vβ₃ dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging.