ABSTRACT
It is evident that 99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known γ-emission of 99mTc. We have synthesized a series of new 99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or double-strand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when 99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the 99mTc nuclide. However, a critical distance between the 99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the 99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tc-complexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding 99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding 99mTcO4-, since nearly all DNA damage caused by 99mTcO4- was prevented by incubating with DMSO.
Subject(s)
DNA Damage , DNA/chemistry , Organotechnetium Compounds/toxicity , Plasmids , Pyrenes/chemistry , ElectronsABSTRACT
Sunitinib (SU11248) is a highly potent tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR). Radiolabeled inhibitors of receptor tyrosine kinases (RTKs) might be useful tools for monitoring RTKs levels in tumor tissue giving valuable information for anti-angiogenic therapy. Herein we report the synthesis of 5-methoxy-sunitinib 5 and its (11)C-radiolabeled analog [(11)C]-5. The non-radioactive reference compound 5 was prepared by Knoevenagel condensation of 5-methoxy-2-oxindole with the corresponding substituted 5-formyl-1H-pyrrole. A binding constant (K(d)) of 20 nM for 5 was determined by competition binding assay against VEGFR-2. In addition, the binding mode of sunitinib and its 5-methoxy substituted derivative was studied by flexible docking simulations. These studies revealed that the substitution of the fluorine at position 5 of the oxindole scaffold by a methoxy group did not affect the inhibitor orientation, but affected the electrostatic and van der Waals interactions of the ligand with residues near the DFG motif of VEGFR-2. 5-[(11)C]methoxy-sunitinib ([(11)C]-5) was synthesized by reaction of the desmethyl precursor with [(11)C]CH(3)I in the presence of DMF and NaOH in 17 ± 3% decay-corrected radiochemical yield at a specific activity of 162-205 GBq/µmol (EOS). In vivo stability studies of [(11)C]-5 in rat blood showed that more than 70% of the injected compound was in blood stream, 60 min after administration.
Subject(s)
Indoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrroles/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Carbon Isotopes , Cell Line , Dose-Response Relationship, Drug , Humans , Indoles/chemical synthesis , Indoles/chemistry , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrroles/chemical synthesis , Pyrroles/chemistry , Structure-Activity Relationship , Sunitinib , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
INTRODUCTION: Phosphopeptides represent interesting compounds to study and elucidate cellular protein phosphorylation/dephosphorylation processes underlying various signal transduction pathways. However, studies of phosphopeptide action in cells are severely constrained by the negatively charged phosphate moiety of the phosphopeptide resulting in poor transport through the cell membrane. The following study describes the synthesis and radiopharmacological evaluation of two (18)F-labeled phosphopeptide-cell-penetrating peptide dimers. The polo-like kinase-1-binding hexaphosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH was coupled to cell-penetrating peptides (CPPs), either sC18, a cathelicidin-derived peptide, or the human calcitonin derivative hCT(18-32)-k7. METHODS: Radiolabeling was accomplished with the prosthetic group N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) using both, conventional and microfluidic-based bioconjugation of [(18)F]SFB to N-terminal end of phosphopeptide part of the peptide dimers. Cellular uptake studies in human cancer cell lines HT-29 and FaDu cells at 4 °C and 37 °C and small animal PET in BALB/c mice were utilized for radiopharmacological characterization. RESULTS: Isolated radiochemical yields ranged from 2% to 4% for conventional bioconjugation with [(18)F]SFB. Significantly improved isolated radiochemical yields of up to 26% were achieved using microfluidic technology. Cellular uptake studies of radiolabeled phosphopeptide and phosphopeptide-CPP dimers indicate enhanced internalization of 50% ID/mg protein after 2 h for both phosphopeptide dimers compared to the phosphopeptide alone (<1% ID/mg protein). In vivo biodistribution of (18)F-labeled peptide dimers was determined with small animal PET revealing a superior biodistribution pattern of sC18-containing peptide dimer MQSpTPL-sC18 [(18)F]4. CONCLUSION: ([18)F]SFB labeling of the phosphopeptide-CPP dimers using a microfluidic system leads to an improved chemoselectivity towards the N-terminal NH(2) group compared to the conventional labeling approach. Cell-penetrating peptide sC18 can be considered as an ideal molecular shuttle for intracellular delivery of the Plk1-PBD-binding hexaphosphopeptide as demonstrated by its favourable radiopharmacological profile.
Subject(s)
Cell-Penetrating Peptides/chemistry , Dimerization , Fluorine Radioisotopes/chemistry , Phosphopeptides/chemistry , Phosphopeptides/metabolism , Acylation , Amino Acid Sequence , Animals , Benzoates/chemistry , HT29 Cells , Humans , Isotope Labeling , Mice , Molecular Sequence Data , Positron-Emission Tomography , Protein Transport , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/metabolism , Succinimides/chemistryABSTRACT
The radiosynthesis of 3-(4-[(18)F]fluorophenyl)-2-(4-methylsulfonylphenyl)-1H-indole [(18)F]-3 as potential PET radiotracer for functional characterization of cyclooxygenase-2 (COX-2) in vitro and in vivo is described. [(18)F]-3 was prepared by McMurry cyclization of a (18)F-labeled intermediate with low valent titanium and zinc via a two-step procedure in a remote controlled synthesizer unit including HPLC purification and solid phase extraction. In this way [(18)F]-3 was synthesized in 80 min synthesis time in 10% total decay corrected yield from [(18)F]fluoride in radiochemical purity >98% and a specific activity of 74-91 GBq/µmol (EOS). [(18)F]-3 was evaluated in vitro using pro-inflammatory stimulated THP-1 and COX-2 expressing tumor cell lines (FaDu, A2058, HT-29), where the radiotracer uptake was shown to be consistent with up regulated COX-2 expression. The stability of [(18)F]-3 was determined by incubation in rat whole blood and plasma in vitro and by metabolite analysis of arterial blood samples in vivo, showing with 75% of original compound after 60 min an acceptable high metabolic stability. However, no substantial tumor accumulation of [(18)F]-3 could be observed by dynamic small animal PET studies on HT-29 tumor-bearing mice in vivo. This may be due to the only moderate COX-1/COX-2 selectivity of 3 as demonstrated by both cellular and enzymatic cyclooxygenase inhibition assay in vitro. Nevertheless, the new approach first using McMurry cyclization in (18)F-chemistry gives access to (18)F-labeled diarylsubstituted heterocycles that hold promise as radiolabeled COX-2 inhibitors.
Subject(s)
Cyclooxygenase 2 Inhibitors/pharmacology , Cyclooxygenase 2/analysis , Cyclooxygenase 2/metabolism , Indoles/chemical synthesis , Indoles/pharmacokinetics , Positron-Emission Tomography/methods , Sulfones/chemical synthesis , Sulfones/pharmacokinetics , Animals , Cell Line, Tumor , Cyclization , Cyclooxygenase 2 Inhibitors/chemistry , Cyclooxygenase 2 Inhibitors/metabolism , Fluorine Radioisotopes , HT29 Cells , Humans , Indoles/blood , Isotope Labeling/methods , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology , Male , Mice , Mice, Nude , Radiochemistry/methods , Rats , Rats, Wistar , Tissue Distribution , Xenograft Model Antitumor AssaysABSTRACT
Human polo-like kinase 1 (Plk1) is involved in cell proliferation and overexpressed in a broad variety of different cancer types. Due to its crucial role in cancerogenesis Plk1 is a potential target for diagnostic and therapeutic applications. Peptidic ligands can specifically interact with the polo-box domain (PBD) of Plk1, a C-terminal located phosphoepitope binding motif. Recently, phosphopeptide MQSpTPL has been identified as ligand with high binding affinity. However, a radiolabeled version of this peptide showed only insufficient cellular uptake. The present study investigated peptide dimers consisting of PBD-targeting phosphopeptide MQSpTPL and a cell-penetrating peptide (CPP) moiety. The new constructs demonstrate superior uptake in different cancer cell-lines compared to the phosphopeptide alone. Furthermore, we could demonstrate binding of phosphopeptide-CPP dimers to PBD of Plk1 making the compounds interesting leads for the development of molecular probes for imaging Plk1 in cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Phosphopeptides/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HeLa Cells , Humans , Ligands , Phosphopeptides/chemical synthesis , Phosphopeptides/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Tissue Distribution , Polo-Like Kinase 1ABSTRACT
Data concerning the pathophysiological role of extracellular S100A4, a member of the multigenic family of Ca(2+)-modulated S100 proteins, and its interaction with the receptor for advanced glycation endproducts (RAGE) or other putative receptors in tumorigenesis, metastasis, and inflammatory processes in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A4 as potential probe for molecular imaging and functional characterization of this interaction. Therefore, human S100A4 was cloned as GST fusion protein in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100A4 was radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radioligand [(18)F]fluorobenzoyl-S100A4 ((18)F-S100A4) was used in cell binding experiments in RAGE-bearing human melanoma cells and endothelial cells in vitro, and in both biodistribution experiments and small animal positron emission tomography (PET) studies in normal rats in vivo. The cellular association and tissue-specific distribution of (18)F-S100A4 in vitro and in vivo correlated well with the protein expression and anatomical localization of RAGE, e.g., in the vascular system and in lung. Compared to other S100 RAGE radioligands, the overall findings of this study indicate that extracellular S100A4 in vivo shows only a moderate interaction with RAGE and, furthermore, exhibits a substantially faster metabolic degradation. On the other hand, the approach allows the use of quantitative small animal PET and provides a novel probe to both delineate functional expression and differentiate multiligand interaction of RAGE under normal and pathophysiological conditions in rodent models of disease.
Subject(s)
Receptors, Immunologic/analysis , Receptors, Immunologic/metabolism , S100 Proteins/isolation & purification , S100 Proteins/metabolism , Animals , Benzoates/chemistry , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Fluorine Radioisotopes/chemistry , Glycation End Products, Advanced , Humans , Male , Melanoma/metabolism , Positron-Emission Tomography/methods , Rats , Rats, Wistar , Receptor for Advanced Glycation End Products , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , S100 Calcium-Binding Protein A4 , S100 Proteins/genetics , Succinimides/chemistryABSTRACT
Two neurotensin(8-13)-containing peptide heterodimers were prepared via copper(I)-mediated click chemistry. The resulting peptide dimers could be obtained in 28-31% yield after HPLC purification. Neurotensin(8-13)-containing peptide dimers were used in an in vitro binding assay to determine binding affinity towards the neurotensin receptor-1 (NTR1). The determined IC(50) values of 8.3 microM and 0.7 microM indicate only very low binding affinity of the neurotensin(8-13)-containing peptide heterodimers towards the NTR1.
Subject(s)
Neurotensin/chemistry , Peptide Fragments/chemistry , Phosphopeptides/chemical synthesis , Chromatography, High Pressure Liquid , Dimerization , Phosphopeptides/chemistryABSTRACT
Increased plasma levels of S100 proteins and interaction of S100 proteins with receptor for advanced glycation end products (RAGE) have been associated with a number of disease states, including chronic inflammatory processes and atherosclerosis. However, data concerning the role of circulating S100 proteins in these pathologies in vivo are scarce and, furthermore, it is currently not known whether RAGE is the sole receptor for extracellular S100 proteins in vivo. We report a novel methodology using recombinant human S100 proteins radiolabelled with fluorine-18, particularly, (18)F-S100A12, in receptor binding studies and cellular association studies in vitro, and in dynamic small animal positron emission tomography (PET) studies in rats in vivo. Association to both human aortic endothelial cells and macrophages revealed specific binding of (18)F-S100A12 to RAGE, but, furthermore, provides evidence for interaction of (18)F-S100A12 to various scavenger receptors (SR). PET data showed temporary association of (18)F-S100A12 with tissues overexpressing RAGE (e.g., lung), and, moreover, accumulation of (18)F-S100A12 in tissues enriched in cells overexpressing SR (e.g., liver and spleen). Blockade of overall SR interaction by maleylated BSA (malBSA) clearly shows diminished in vivo association of (18)F-S100A12 to these tissues as well as a significant increment of the mean plasma residence time of (18)F-S100A12 (4.8+/-0.4 h vs. 2.3+/-0.3 h). The present approach first demonstrates that besides RAGE also scavenger receptors contribute to distribution, tissue association and elimination of circulating proinflammatory S100A12.
Subject(s)
Receptors, Immunologic/metabolism , Receptors, Scavenger/metabolism , S100 Proteins/metabolism , Animals , Cells, Cultured , Cricetinae , Cricetulus , Fluorine Radioisotopes/blood , Fluorine Radioisotopes/metabolism , Glycation End Products, Advanced , Half-Life , Humans , Lipoproteins, LDL , Male , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Organ Specificity , Positron-Emission Tomography , Protein Binding , RNA, Messenger/metabolism , Rats , Rats, Wistar , Receptor for Advanced Glycation End Products , Receptors, Immunologic/antagonists & inhibitors , Receptors, Immunologic/genetics , Receptors, Scavenger/antagonists & inhibitors , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/blood , Recombinant Proteins/metabolism , S100 Calcium Binding Protein beta Subunit , S100 Proteins/antagonists & inhibitors , S100 Proteins/blood , S100 Proteins/genetics , S100A12 Protein , Serum Albumin, Bovine , Tissue DistributionABSTRACT
PURPOSE: To analyse short term and long term X-ray irradiation effects on proliferation, viability, glucose and amino acid uptake of murine melanoma cells in vitro and metastasis in vivo. MATERIALS AND METHODS: B16-F10 melanoma cells were irradiated with different doses of X-ray irradiation (200 kV) in the range from 1-20 Gy. One, two and three days respectively 7, 14 and 21 days after treatment cells were analysed concerning cell growth, viability, proliferation, cell cycle distribution, glucose and amino acid transport. Moreover the capability of the cells for in vivo metastasis was examined. RESULTS: As short term response on irradiation we detected decreased cell growth, viability and arrest in the G2/M phase of the cell cycle. Long term response involves re-start of proliferation, increased cell growth and glucose uptake but still decreased viability and amino acid transport. In vivo metastasis is lost immediately after irradiation and regained to a low extent beyond two weeks time for recurrence of cells before injection. CONCLUSIONS: In vitro data suggest that surviving melanoma cells compensate the initial irradiation-dependent damage of proliferation within three weeks possibly by increase in glucose uptake. For metastasis in vivo the role of additional mechanisms is strongly suggested.
Subject(s)
Melanoma, Experimental/radiotherapy , Amino Acids/metabolism , Animals , Base Sequence , Cell Cycle/radiation effects , Cell Line, Tumor , Cell Proliferation/radiation effects , Cell Survival/radiation effects , Cyclin B1/genetics , Cyclin B1/metabolism , DNA Primers/genetics , Glucose/metabolism , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Melanoma, Experimental/secondary , Mice , Neoplasm Metastasis/prevention & control , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Radiopharmaceuticals/pharmacokinetics , Tumor Stem Cell AssayABSTRACT
We describe the radiosynthesis of two new [(90)Y]-DOTA-based maleimide reagents, suitable for the mild radiolabeling of L-RNAs and peptides modified with thiol-bearing linkers. The synthesis procedure of both maleimide-bearing (90)Y complexes, [{(2S)-2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl}tetraacetato][(90)Y]yttrate(1-)([(90)Y]3) and [{(2S)-2-(4-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}benzyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetrayl]tetraacetato}[(90)Y]yttrate(1-)([(90)Y]4), was optimized in terms of an easy purification method via solid-phase extraction (SPE). Application as well as reactivity of both maleimide reagents were initially evaluated by the prelabeling of glutathione (GSH) and a thiol-modified 12mer L-RNA as model substances. In comparison to the N-aryl maleimide-bearing complex [(90)Y]3, N-alkyl maleimide-bearing complex [(90)Y]4 showed an increased hydrolytic stability at pH > or = 7. A slightly higher reactivity was found for [(90)Y]3 by prelabeling of 0.1 and 1 microg glutathione, respectively, in phosphate buffer (pH 7.2) at room temperature. In terms of very high radiochemical yields, the direct radiolabeling of DOTA-L-RNA conjugate with [(90)Y]YCl(3) proved to be more suitable than the prelabeling of the thiol-modified 12mer L-RNA derivative with [(90)Y]4.
Subject(s)
Heterocyclic Compounds/chemistry , Isotope Labeling/methods , Maleimides/chemistry , Oligonucleotides/chemistry , Organometallic Compounds/chemistry , Peptides/chemistry , Glutathione/analysis , Glutathione/chemistry , Heterocyclic Compounds/chemical synthesis , Maleimides/chemical synthesis , Molecular Structure , Oligonucleotides/analysis , Organometallic Compounds/chemical synthesis , Peptides/analysis , RNA/analysis , RNA/chemistry , Solid Phase Extraction , Sulfhydryl Compounds/chemistry , Yttrium Radioisotopes/chemistryABSTRACT
A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.
Subject(s)
Heterocyclic Compounds/chemistry , Oligonucleotides/chemistry , Oligonucleotides/pharmacokinetics , Organometallic Compounds/chemistry , Animals , Autoradiography , Benzene/chemistry , Heterocyclic Compounds/metabolism , Heterocyclic Compounds/pharmacokinetics , Isomerism , Magnetic Resonance Spectroscopy , Male , Oligonucleotides/metabolism , Organometallic Compounds/metabolism , Organometallic Compounds/pharmacokinetics , RNA/chemistry , RNA/metabolism , RNA/pharmacokinetics , Rats , Rats, Wistar , Tissue Distribution , Yttrium RadioisotopesABSTRACT
UNLABELLED: In patients with pulmonary hypertension (PH) the right ventricular (RV)-to-left ventricular (LV) ratio of fatty acid uptake is reduced. In animal studies, such a finding was combined with an increased glucose uptake in RV myocardium. The aim of this study was to measure the metabolic rates of glucose uptake for the RV and LV myocardium in patients in relationship to parameters of RV and LV function. METHODS: Thirty patients with PH underwent PET with (18)F-FDG and SPECT with (99m)Tc-tetrofosmine. The metabolic rate of glucose uptake was determined for RV and LV myocardium using the method of Patlak. A right heart catheter, thermodilution, and Doppler sonography were used to characterize RV and LV function. From these methods, the stroke work of both ventricles and the RV Tei index were calculated. RESULTS: RV-to-LV ratios of (18)F-FDG-uptake increased with rising pulmonary arteriolar resistance (PAR). With increasing PAR, the metabolic rate of glucose uptake of the left ventricle decreased (r = -0.547; P < 0.01) together with LV stroke work (r = -0.838; P < 0.001). The metabolic rate of glucose uptake of the right ventricle, however, correlated neither with RV stroke work (r = 0.124) nor with PAR (r = 0.189) but with the Tei index (r = 0.78; P < 0.001). CONCLUSION: Increasing right-to-left ratios of glucose uptake with an increasing pressure load in the right ventricle in PH are caused mainly by a significant reduction in the LV metabolic rate of glucose uptake. This is obviously due to a reduced energy demand of the LV myocardium caused by reduced stroke work. An increased metabolic rate of glucose uptake in the right ventricle presumably indicates RV impairment, correlating with the Tei index, which is an established prognostic parameter for cardiac dysfunction and poor survival.