Synthesis and comparative assessment of a labeled RGD peptide bearing two different ⁹⁹mTc-tricarbonyl chelators for potential use as targeted radiopharmaceutical

During the past decade radiolabeled RGD-peptides have been extensively studied to develop site-directed targeting vectors for integrins. Integrins are heterodimeric cell-surface adhesion receptors, which are upregulated in cancer cells and neovasculature during tumor angiogenesis and recognize the RGD aminoacid sequence. In the present study, we report the synthesis and development of two derivatives of the Nε-Lys derivatized cyclic Arg-Gly-Asp-D-Phe-Lys peptide, namely of cRGDfKHis and cRGDfK-CPA (CPA: 3-L-Cysteine Propionic Acid), radiolabeled via the [(99m)Tc(H(2)O)(3)(CO)(3)](+) metal aquaion at a high yield even at low concentrations of 10-5M (>87%) for cRGDfK-10-5M (>93%) for cRGDfK-CPA. Radiolabeled peptides were characterized with regard to their stability in saline, in His/Cys solutions, as well as in plasma, serum and tissue homogenates and were found to be practically stable. Internalization and efflux assays using αvβ3-receptor-positive MDA-MB 435 breast cancer cells showed a good percentage of quick internalization (29.1 ± 9.8% for (99m)Tc-HiscRGDfK and 37.0 ± 0.7% for (99m)Tc-CPA-cRGDfK at 15 min) and no retention of radioactivity for both derivatives. Their in vivo behavior was assessed in normal mice and pathological SCID mice bearing MDA-MB 435 ανβ3 positive breast tumors. Both presented fast blood clearance and elimination via both the urinary and hepatobiliary systems, with (99m)Tc-His-cRGDfK remaining for a longer time than (99m)Tc-CPA-cRGDfK in all organs examined. Tumor uptake 30 min pi was higher for (99m)Tc-CPAcRGDfK (4.2 ± 1.5% ID/g) than for (99m)Tc-His-cRGDfK (2.8 ± 1.5% ID/g). Dynamic scintigraphic studies showed that the tumor could be visualized better between 15 and 45 min pi for both radiolabeled compounds but low delineation occurred due to high abdominal background. It was finally noticed that the accumulated activity on the tumor site was depended on the size of the experimental tumor; the smaller the size, the higher was the radioactivity concentration.     

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

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

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

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

Dynamic Near-Infrared Optical Imaging of 2-Deoxyglucose Uptake by Intracranial Glioma of Athymic Mice

Background

It is recognized that cancer cells exhibit highly elevated glucose metabolism compared to non-tumor cells. We have applied in vivo optical imaging to study dynamic uptake of a near-infrared dye-labeled glucose analogue, 2-deoxyglucose (2-DG) by orthotopic glioma in a mouse model.

Methodology and Principal Findings

The orthotopic glioma model was established by surgically implanting U87-luc glioma cells into the right caudal nuclear area of nude mice. Intracranial tumor growth was monitored longitudinally by bioluminescence imaging and MRI. When tumor size reached >4 mm diameter, dynamic fluorescence imaging was performed after an injection of the NIR labeled 2-DG, IRDye800CW 2-DG. Real-time whole body images acquired immediately after i.v. infusion clearly visualized the near-infrared dye circulating into various internal organs sequentially. Dynamic fluorescence imaging revealed significantly higher signal intensity in the tumor side of the brain than the contralateral normal brain 24 h after injection (tumor/normal ratio, TNR  = 2.8±0.7). Even stronger contrast was achieved by removing the scalp (TNR  = 3.7±1.1) and skull (TNR  = 4.2±1.1) of the mice. In contrast, a control dye, IRDye800CW carboxylate, showed little difference (1.1±0.2). Ex vivo fluorescence imaging performed on ultrathin cryosections (20 µm) of tumor bearing whole brain revealed distinct tumor margins. Microscopic imaging identified cytoplasmic locations of the 2-DG dye in tumor cells.

Conclusion and Significance

Our results suggest that the near-infrared dye labeled 2-DG may serve as a useful fluorescence imaging probe to noninvasively assess intracranial tumor burden in preclinical animal models.     

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.     

Biological evaluation of glucose and deoxyglucose derivatives radiolabeled with [99mTc(CO)3(H2O)3]+ core as potential melanoma imaging agents

Glucose 9 and 2-deoxyglucose 10 were successfully synthesized and radiolabeled with [(99m)Tc(CO)(3)(H(2)0)(3)](+) intermediate in high yield. The complexes were characterized by HPLC and its stability with histidine over time was challenged. Cell uptake and biodistribution studies in melanoma-bearing C57BL/6 mice were performed. Both compounds showed accumulation in tumor tissue with high tumor-to-muscle ratios. Thus, D-glucose- and D-2-deoxyglucose-(99m)Tc complex could be considered as agents for melanoma diagnosis.     

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

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

Detection of 3-deoxyfructose and 3-deoxyglucosone in human urine and plasma: evidence for intermediate stages of the Maillard reaction in vivo.

3-Deoxyglucose (3-deoxy-D-erythro-hexos-2-ulose) (3-DG) is a reactive dicarbonyl intermediate involved in the polymerization and browning of proteins by glucose in vitro. Damage to protein by formation of 3-DG in vivo is thought to be limited by enzymes which convert 3-DG to less reactive species, such as 3-deoxyfructose (3-DF). We have developed a sensitive and specific assay for measuring 3-DG and 3-DF in human urine and plasma. In this assay, 3-DG and 3-DF are reduced to 3-deoxy-hexitols (3-DH), using either NaBH4 or NaBD4, and then analyzed by selected ion monitoring gas chromatography-mass spectrometry. Based on comparative analysis of samples reduced with NaBD4 versus NaBD4, 3-DH in urine was derived exclusively (greater than 99%) from 3-DF, while 3-DG accounted for approximately 15% of 3-DH in plasma. The concentrations of 3-DH in fasting human urine and plasma were 5.3 +/- 1.5 micrograms/mg creatinine (n = 18) and 7.2 +/- 1.7 micrograms/dl (n = 18), respectively. The concentrations of 3-DG and 3-DF in plasma (n = 7) were 1.0 +/- 0.2 and 6.7 +/- 1.6 micrograms/dl, respectively. These results suggest that several milligrams of 3-DG are formed in the body per day and detoxified by reduction to 3-DF and support the role of 3-DG as an intermediate in the browning of protein via the Maillard reaction in vivo.     

On the isolation and evaluation of a novel unsubstituted 5-nitroimidazole derivative as an agent to target tumor hypoxia

The presence and extent of hypoxic regions in cancerous tissue bears a negative influence on the effectiveness of radiation therapy and chemotherapy of the cancer hence estimation of hypoxia is an important problem. Several (99m)Tc-labeled nitroimidazole-based non-invasive agents have been tried for this purpose but none had optimal characteristics and the search continues. Herein we report, for the first time to the best of our knowledge, the isolation, (99m)Tc(CO)(3) labeling and evaluation of an unsubstituted 5-nitroimidazole derivative obtained as a side product during the synthesis of 4-nitroimidazole derivative. The (99m)Tc(CO)(3)-labeled complex of 5-nitroimidazole derivative could be prepared in excellent yield under mild conditions. Its evaluation in fibrosarcoma tumor bearing Swiss mice showed uptake and slow clearance of injected activity from tumor. The tumor-to-muscle ratio was found to be very high but tumor-to-blood ratio greater than 1 could not be obtained throughout the limited time point study. The study revealed that complex under investigation has features similar to other 2-nitroimidazole complexes so far as the retention of injected activity in tumor is concerned.     

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

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

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

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

Radiolabeling of folic acid-modified chitosan with (99m)Tc as potential agents for folate-receptor-mediated targeting

The feasibility of chitosan (CS) as a backbone for the design of (99m)Tc-labeled targeting agent was evaluated in this study. Chitosan-folate conjugate (CSFA) and chitosan-folate dithiocarbamate (CSFADTC) were synthesized, characterized and radiolabeled with (99m)Tc as model compounds for folate-receptor (FR) targeting. (99m)Tc-complexes were prepared with high radiochemical purity and high stability. The hydrophilicities of these (99m)Tc-complexes were determined by partition coefficient experiments. The results of biodistribution in normal mice showed that the folic-acid modified agents ((99m)Tc-CSFA and (99m)TcN-CSFADTC) had obviously higher uptake in FR-positive kidney and much lower liver and spleen uptakes than that of non-folic-acid modified (99m)Tc-agent, and the kidney uptakes of FA-modified agents could be blocked significantly by the corresponding cold ligand. Furthermore in vitro and in vivo specific studies will be done in cell line and tumor bearing mice to confirm the usefulness of this chitosan backbone for FR targeting agent design.     

Synthesis and evaluation of a novel (99m)Tc-labeled bioreductive probe for tumor hypoxia imaging

Tumor hypoxia is closely associated with the malignant progression and/or the high metastatic ability of tumors and often induces resistance to chemo- and/or radiotherapy. Thus, the detection and evaluation of hypoxia is important for the optimization of cancer therapy. We designed a novel (99m)Tc-labeled probe for tumor hypoxia imaging that utilizes bioreductive reactions in hypoxic cells. This probe, which contains a 4-nitrobenzyl ester group, is reduced in hypoxic cells to produce a corresponding carboxylate anion that cannot penetrate cell membranes because of its hydrophilicity and negative charge; therefore, it is expected to be trapped inside hypoxic cells. Based on this unique strategy, we synthesized the Technetium-99m ((99m)Tc)-labeled probe (99m)Tc-SD32. The uptake of (99m)Tc-SD32 in tumor cells was investigated under normoxic and hypoxic conditions. (99m)Tc-SD32 showed sufficient accumulation and good retention in hypoxic cells. In addition, we demonstrated that (99m)Tc-SD32 was subjected to bioreduction in hypoxic cells and was trapped as the corresponding carboxylate anion. These results indicated that (99m)Tc-SD32 would be a promising agent for in vivo hypoxia imaging.     

(99m)Tc-maEEE-Z(HER2:342), an Affibody molecule-based tracer for the detection of HER2 expression in malignant tumors

Detection of HER2-overexpression in tumors and metastases is important for the selection of patients who will benefit from trastuzumab treatment. Earlier investigations showed successful imaging of HER2-positive tumors in patients using indium- or gallium-labeled Affibody molecules. The goal of this study was to evaluate the use of (99m)Tc-labeled Affibody molecules for the detection of HER2 expression. The Affibody molecule Z(HER2:342) with the chelator sequences mercaptoacetyl-Gly-Glu-Gly (maGEG) and mercaptoacetyl-Glu-Glu-Glu (maEEE) was synthesized by peptide synthesis and labeled with technetium-99m. Binding specificity, cellular retention, and in vitro stability were investigated. The biodistribution of (99m)Tc-maGEG-Z(HER2:342) and (99m)Tc-maEEE-Z(HER2:342) was compared with (99m)Tc-maGGG-Z(HER2:342) in normal mice, and the tumor targeting properties of (99m)Tc-maEEE-Z(HER2:342) were determined in SKOV-3 xenografted nude mice. The results showed that the Affibody molecules were efficiently labeled with technetium-99m. The labeled conjugates were highly stable in vitro with preserved HER2-binding capacity. The use of glutamic acid in the chelator sequences for (99m)Tc-labeling of Z(HER2:342) reduced the hepatobiliary excretion 3-fold with a single Gly-to-Glu substitution and 10-fold with three Gly-to-Glu substitutions. (99m)Tc-maEEE-Z(HER2:342) showed a receptor-specific tumor uptake of 7.9 +/- 1.0 %IA/g and a tumor-to-blood ratio of 38 at 4 h pi. Gamma-camera imaging with (99m)Tc-maEEE-Z(HER2:342) could detect HER2-expressing tumors in xenografts already at 1 h pi. It was concluded that peptide synthesis for the coupling of chelator sequences to Affibody molecules for (99m)Tc labeling is an efficient way to modify the in vivo kinetics. Increased hydrophilicity, combined with improved stability of the mercaptoacetyl-triglutamyl chelator, resulted in favorable biodistribution, making (99m)Tc-maEEE-Z(HER2:342) a promising tracer for clinical imaging of HER2 overexpression in tumors.     

A Tc-99m-labeled long chain fatty acid derivative for myocardial imaging

C-11- and I-123-labeled long chain fatty acid derivatives have been reported as useful radiopharmaceuticals for the estimation of myocardial fatty acid metabolism. We have reported that Tc-99m-labeled N-[[[(2-mercaptoethyl)amino]carbonyl]methyl]-N-(2-mercaptoethyl)-6-aminohexanoic acid ([(99m)Tc]MAMA-HA), a medium chain fatty acid derivative, is metabolized by beta-oxidation in the liver and that the MAMA ligand is useful for attaching to the omega-position of fatty acid derivatives as a chelating group for Tc-99m. On the basis of these findings, we focused on developing a Tc-99m-labeled long chain fatty acid derivative that reflected fatty acid metabolism in the myocardium. In this study, we synthesized a dodecanoic acid derivative, MAMA-DA, and a hexadecanoic acid derivative, MAMA-HDA, and performed radiolabeling and biodistribution studies. [(99m)Tc]MAMA-DA and [(99m)Tc]MAMA-HDA were prepared using a ligand-exchange reaction. Biodistribution studies were carried out in normal mice and rats. Then, a high initial uptake of Tc-99m was observed, followed by a rapid clearance from the heart. The maximum heart/blood ratio was 3.6 at 2 min postinjection of [(99m)Tc]MAMA-HDA. These kinetics were similar to those with postinjection of p-[(125)I]iodophenylpentadecanoic acid. Metabolite analysis showed [(99m)Tc]MAMA-HDA was metabolized by beta-oxidation in the body. In conclusion, [(99m)Tc]MAMA-HDA is a promising compound as a long chain fatty acid analogue for estimating beta-oxidation of fatty acid in the heart.     

Immunochemical detection of 3-deoxyglucosone in serum

3-Deoxyglucosone (3-DG) is a metabolite of glucose that is thought to lead to the production of advanced glycation end products in diabetes. The previous assay for 3-DG in serum was based on a multi-step protocol, including derivatization, extraction, HPLC separation, and detection. In the current studies, we established a monoclonal antibody that recognizes the 3-DG-derivative, which is generated by the reaction of 3-DG and a 2,3-diamino-benzene derivative. Attachment of a biotin moiety to the 2,3-diamino-benzene ring via a linker allowed development of a highly sensitive chemiluminescent enzyme immunoassay for 3-DG equivalents. Unlike the previous assay, this method does not require extraction of 3-DG derivatives from serum. Treatment of 3-DG in serum with the DAB-link-biotin produced a quinoxaline derivative, which was specifically recognized by the monoclonal antibody. Using this assay, we found that serum 3-DG was higher in streptozotocin-induced diabetic rats than in normal control rats (25+/-5.6 vs. 9.8+/-1.1 microg/L). This simple assay may allow the monitoring of conditions leading to the accumulation of advanced glycation end products and evaluation of the risk of complications in diabetic patients.     

Multimeric system of 99mTc-labeled gold nanoparticles conjugated to c[RGDfK(C)] for molecular imaging of tumor α(v)β(3) expression

Integrin α(V)β(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic RGD peptides can be used for noninvasive imaging of α(V)β(3) expression. The aim of this research was to prepare a multimeric system of technetium-99m-labeled gold nanoparticles conjugated to c[RGDfK(C)] and to evaluate its biological behavior as a potential radiopharmaceutical for molecular imaging of tumor angiogenesis. Hydrazinonicotinamide-GGC (HYNIC-GGC) and c[RGDfK(C)] peptides were synthesized and conjugated to gold nanoparticles (AuNP, 20 nm) by means of spontaneous reaction of the thiol groups of cysteine. The nanoconjugate was characterized by TEM, FT-IR, UV-vis, XPS, and Raman spectroscopy. To obtain (99m)Tc-HYNIC-GGC-AuNP-c[RGDfK(C)] ((99m)Tc-AuNP-RGD), the (99m)Tc-HYNIC-GGC radiopeptide was first prepared and added to 1.5 mL of AuNP solution (1 nM) followed by c[RGDfK(C)] (10 μL, 50 μM) at 18 °C with stirring for 15 min. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in α(V)β(3) receptor-positive C6 glioma cancer cells. Biodistribution studies were accomplished in athymic mice with C6-induced tumors with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/CT. TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with peptides. RP was 96 ± 2% without postlabeling purification. (99m)Tc-AuNP-RGD showed specific recognition for α(V)β(3) integrins expressed in C6 cells, and 3 h after i.p. administration in mice, the tumor uptake was 8.18 ± 0.57% ID/g. Micro-SPECT/CT images showed evident tumor uptake. (99m)Tc-AuNP-RGD demonstrates properties suitable for use as a target-specific agent for molecular imaging of tumor α(V)β(3) expression.     

FTS and 2-DG induce pancreatic cancer cell death and tumor shrinkage in mice

The Ras inhibitor S-trans-trans farnesylthiosalicylic acid (FTS) inhibits active Ras, which controls cell proliferation, differentiation, survival, and metabolism. FTS also inhibits HIF1α expression in cancer cells, leading to an energy crisis. The synthetic glucose analog 2-deoxy-D-glucose (2-DG), which inhibits glycolysis, is selectively directed to tumor cells that exhibit increased glucose consumption. The 2-DG enters tumor cells, where it competes with glucose for glycolytic enzymes. In cancer models, as well as in human phase 1 trials, 2-DG inhibits tumor growth without toxicity. We postulated that under normoxic conditions, tumor cells treated with FTS would be more sensitive than normal cells to 2-DG. We show here that combined treatment with FTS and 2-DG inhibited cancer cell proliferation additively, yet induced apoptotic cell death synergistically both in vitro and in vivo. The induced apoptosis was inferred from QVD-OPH inhibition, an increase in cleaved caspase 3, and loss of survivin. FTS and 2-DG when combined, but not separately, also induced an increase in fibrosis of the tumor tissue, chronic inflammation, and tumor shrinkage. Overall, these results suggest a possible new treatment of pancreatic tumors by the combined administration of FTS and 2-DG, which together induce pancreatic tumor cell death and tumor shrinkage under non-toxic conditions.     

Nepsilon-(Carboxymethyl)lysine and 3-DG-imidazolone are major AGE structures in protein modification by 3-deoxyglucosone

The levels of plasma 3-deoxyglucosone (3-DG) increase under hyperglycemic conditions and are associated with the pathogenesis of diabetic complications because of the high reactivity of 3-DG with proteins to form advanced glycation end products (AGE). To investigate potential markers for 3-DG-mediated protein modification in vitro and in vivo, we compared the yield of several 3-DG-derived AGE structures by immunochemical analysis and HPLC and measured their localization in human atherosclerotic lesions. When BSA was incubated with 3-DG at 37 degrees C for up to 4 wk, the amounts of N(epsilon)-(carboxymethyl)lysine (CML) and 3-DG-imidazolone steeply increased with incubation time, whereas the levels of pyrraline and pentosidine increased slightly by day 28. In contrast, significant amounts of pyrraline and pentosidine were also observed when BSA was incubated with 3-DG at 60 degrees C to enhance AGE-formation. In atherosclerotic lesions, CML and 3-DG-imidazolone were found intracellularly in the cytoplasm of most foam cells and extracellularly in the atheromatous core. A weak-positive immunoreaction with pyrraline was found in the extracellular matrix and a few foam cells in aortic intima with atherosclerotic lesions. Our results provide the first evidence that CML and 3-DG-imidazolone are major AGE structures in 3-DG-modified proteins, and that 3-DG-imidazolone provides a better marker for protein modification by 3-DG than pyrraline.     

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

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