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ABSTRACT: Peptides are excellent alternatives to small molecules and proteinaceous drugs. Their high medicinal potential for diagnostic and therapeutic applications has prompted the development of tumor targeting peptides. Despite its excellent tumor binding capacity, FROP-DOTA (H-Glu-Asn-Tyr-Glu-Leu-Met-Asp-Leu-Leu-Ala-Tyr-Leu-Lys(DOTA)-NH2), a peptide that we had identified in phage display libraries, revealed slow binding kinetics. Consequently, biodistribution studies showed that its excretion forestalled a significant tumor accumulation. The aim of this study was to investigate whether the conjugation of PEG to FROP-DOTA resulted in a derivative with a prolonged residence time in the blood. A synthetic method for the PEGylation of the tumor specific peptide FROP-DOTA was developed. Thereafter, binding studies were done in vitro and a biodistribution was performed in tumor bearing animals. These were compared to the data obtained with FROP-DOTA. The binding kinetics of the PEGylated FROP-DOTA was even slower than that of FROP-DOTA. Biodistribution studies of the labeled conjugate in mice bearing human FRO82-2 tumors showed a time dependent increased uptake of the PEGylated peptide with a high retention (at 24 h p.i. 76% of the maximal activity concentration persisted in the tumor). The highest uptake values were determined at 120 min p.i. reaching 2.3%ID/g tumor as compared to 0.06%ID/g observed for the non-PEGylated derivative at 135 min p.i. Apparently, PEGylation provides a substantially improved stabilization in the circulation which allowed a stable tumor accumulation.
Organic & Biomolecular Chemistry 03/2013; · 3.70 Impact Factor
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European Journal of Nuclear Medicine 03/2013; · 4.53 Impact Factor
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ABSTRACT: The ubiquitin-proteasome pathway has been identified as a potential molecular target for cancer therapy. In this study, we investigated the effect of the proteasome inhibitor bortezomib on anaplastic thyroid carcinoma (ATC) characterized by complete refractoriness to multimodal therapeutic approaches. METHODS: The ATC cell lines C643 and SW1736 were treated with bortezomib (1 nM to 1 μM) for 12-72 h. Thereafter, growth inhibition was analyzed by thymidine uptake experiments and determination of the viable cell number. Apoptosis was measured and a cell cycle analysis was done. Using gene chip analysis and the real-time quantitative PCR system, we measured transcriptional changes. The activity of the nuclear factor (NF)-κB and p53 signal transduction pathways was monitored using the reporter constructs pNF-κB-TA-Luc and pp53-TA-Luc in the luciferase activity assay. Uptake measurements using (3)H-FDG, (14)C-aminoisobutyric acid, and Na(125)iodide were performed to investigate metabolic changes and iodide symporter activity in vitro. Moreover, the (18)F-FDG uptake was evaluated in ATC tumor-bearing nude mice 1 or 2 d after treatment with bortezomib. RESULTS: Bortezomib induced growth inhibition, apoptosis, and G(2)-M cell cycle arrest associated with upregulation of p21(CIP1/WAF1) expression in SW1736 and C643 cells. Moreover, the glucose metabolism and aminoisobutyric acid uptake significantly decreased in vitro in both of the ATC cell lines in vivo only in SW1736 tumors at 2 d after the bortezomib treatment. The transcriptional profile in bortezomib-treated SW1736 and C643 cells revealed increased expression of genes involved in stress response, apoptosis, regulation of the cell cycle, and differentiation. Using real-time quantitative PCR for the quantification of gene expression, we additionally noticed upregulation of the tumor necrosis factor-related apoptosis-inducing ligand and the thyroid-specific transcription factors Pax8 and TTF-1, leading to expression of the thyroid-specific target genes thyroglobulin, sodium iodide symporter, thyroperoxidase, and thyroid-stimulating hormone receptor and to a moderate accumulation of iodide in ATC cells. CONCLUSION: On the basis of our data, bortezomib represents a promising antineoplastic agent for the treatment of ATC. To improve the clinical outcome, further investigation into the potential of bortezomib therapy of thyroid cancer is clearly warranted.
Journal of Nuclear Medicine 10/2012; · 6.38 Impact Factor
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ABSTRACT: BACKGROUND: Alternative positron-emission tomography (PET) probes like labeled inhibitors of the prostate-specific membrane antigen (PSMA) are of emerging clinical impact as they show the ability to image small lesions of recurrent prostate cancer. Here, the dimerization of the pharmacophore GluureidoLys via the 68Ga chelator N,N' -bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N' -diacetic acid (HBED-CC) was investigated to further improve the binding characteristics and pharmacokinetics. METHODS: The peptidomimetic structures were synthesized by solid-phase chemistry, and the resulting products were coupled with the respective 2,3,5,6-tetrafluorophenol esters of HBED-CC to form the monomeric reference and the dimeric GluureidoLys derivative. The binding properties were analyzed in competitive binding, internalization, and cell surface retention experiments. PET images and biodistribution data were obtained 1 h after injection in BALB/c nu/nu mice bearing LNCaP tumor xenografts. RESULTS: Cell binding data revealed significant better binding properties of the dimer (IC50 = 3.9 +/- 1.8 nM; IC50 (monomer) = 12.1 +/- 2.1 nM). The inhibition potency investigated by the enzyme-based NAALADase assay confirmed these results. Specific internalization in LNCaP cells was demonstrated for both, the monomer and dimer. As shown by efflux measurements, the dimeric compound was more effectively retained on the cell surface, resulting in advanced in vivo properties (T/BMonomer = 9.2; T/BDimer = 26.5). CONCLUSIONS: The dimeric [68Ga]7 is a promising imaging agent for PSMA-expressing tumors as it shows higher tumor uptake while observing more favorable background clearance. As compared to the respective monomer, the higher affinity and prolonged tumor retention additionally represent promising features and warrant further evaluation regarding 68Ga-PET imaging of PSMA expression.
EJNMMI research. 06/2012; 2(1):23.
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ABSTRACT: PET imaging with somatostatin receptor ligands, such as (68)Ga-DOTATOC, is a well-established method for detection and target volume definition of meningiomas prior to radiotherapy. Since DOTATOC PET delivers a higher contrast between meningiomas and surrounding tissues than MRI, we conducted a retrospective analysis to compare the diagnostic accuracy of contrast-enhanced MRI (CE-MRI) with (68)Ga-DOTATOC PET/CT in patients with cranial meningiomas prior to radiotherapy.
Over a period of 6 years, 134 patients (20-82 years of age, 107 women and 27 men) underwent cranial CE-MRI and (68)Ga-DOTATOC PET/CT. To compare the two methods, the lesions considered typical of meningiomas visually were counted and analysed with respect to their location and SUVmax.
In the 134 patients investigated by both modalities, 190 meningiomas were detected by (68)Ga-DOTATOC PET/CT and 171 by CE-MRI. With knowledge of the PET/CT data, the MRI scans were reinvestigated, which led to the detection of 4 of the 19 incidental meningiomas, resulting in an overall detection rate of 92 % of the meningioma lesions that were found by PET/CT.
Ga-DOTATOC PET/CT demonstrated an improved sensitivity in meningioma detection when compared to CE-MRI. Tumours adjacent to the falx cerebri, located at the skull base or obscured by imaging artefacts or calcification are particularly difficult to detect by MRI. Therefore (68)Ga-DOTATOC PET/CT may provide additional information in patients with uncertain or equivocal results on MRI or could help to confirm a diagnosis of meningioma based on MRI or could help to confirm MRI-based diagnosis of meningiomas in cases of biopsy limitations. It is possible that not only radiotherapy and surgical planning, but also follow-up strategies would benefit from this imaging modality.
European Journal of Nuclear Medicine 06/2012; 39(9):1409-15. · 4.53 Impact Factor
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ABSTRACT: PurposeDynamic PET studies with68Ga-DOTATOC were performed in patients with non-small cell lung cancer (NSCLC) to assess the somatostatin receptor 2 (SSTR2)
expression. Furthermore, dynamic18F-fluorodeoxyglucose (FDG) studies were performed in the same patients to compare the SSTR2 expression with the tumour viability.
MethodsThe study population comprised nine patients, examined with both tracers on two different days within 1 week. Standardised
uptake values (SUVs) were calculated and a two-tissue compartment model was applied to the data. Furthermore, a non-compartment
model based on the fractal dimension (FD) was applied to the data.
ResultsThe DOTATOC uptake was generally lower than the FDG uptake. Moderately enhanced DOTATOC uptake was noted in seven of the nine
tumours. All kinetic parameters exceptk
4 were lower for DOTATOC than for FDG. The mean SUV was 2.018 for DOTATOC, in comparison to 5.683 for FDG. In particular,k
3 was highly variable for DOTATOC and showed an overlap with the normal lung tissue. The fractional blood volumeV
B was relatively low for both tracers, not exceeding 0.3. The highest significant logarithmic correlation was found for the
FD of the two tracers (r=0.764,p=0.017). The logarithmic correlation for SUVs was also significant (r=0.646,p=0.060), as was that forV
B (r=0.629,p=0.069). In contrast, none of the eight metastases which were positive on FDG PET showed any DOTATOC uptake.
ConclusionThe results demonstrated moderate68Ga-DOTATOC uptake in primary NSCLC but did not provide any evidence for SSTR2 expression in metastases. This may be caused
by loss of the gene expression in metastases as compared with the primary tumours.
European journal of nuclear medicine and molecular imaging 04/2012; 33(7):823-830. · 4.99 Impact Factor
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ABSTRACT: PurposePeptides with restricted conformation provide increased affinity and stability against degradation as compared to linear peptides.
This study investigates the characteristics of derivatives of the sunflower trypsin inhibitor 1 (SFTI-1), a 14 amino acid
peptide with high intrinsic stability.
MethodsThree SFTI-1 derivatives (cyclic cSFTI, acyclic oSFTI, and DOTA-SFTI) were generated by Fmoc-based automated synthesis. Thereafter,
the inhibitory activity for trypsin was determined. After radiolabeling, kinetic and competition studies were done in a variety
of tumor cell lines including prostate carcinoma, colon carcinoma, mammary carcinoma, and hepatoma to characterize the binding
affinity of the peptides. The stability was determined by incubating the molecules in human serum for increasing time periods.
Furthermore, the biodistribution was measured in nude mice bearing human prostate carcinomas.
ResultsThe inhibitory constants for trypsin inhibition were 0.08nM (cSFTI), 0.15nM (oSFTI), and 0.3nM (DOTA-SFTI). Among the different
tumor cell lines evaluated, the prostate cancer cell lines PC-3 and DU-145 showed the highest accumulation of the radiolabeled
peptides. The open-chain derivatives generally bound better than the cyclic one. Binding was constant during 4h and could
be competed by addition of the cold peptide up to 75%. The stability in serum revealed half-lives of 75.8h for cSFTI, 34.5h
for oSFTI, and 41.7h for DOTA-SFTI. The biodistribution showed a rapid renal clearance for all three compounds and tumor
uptake values up to 3%ID/g.
ConclusionsSFTI derivatives are small stable molecules readily accessible by solid-phase synthesis. The trypsin inhibition was not influenced
by the cyclization, and addition of a chelator had no significant influence. The exceptional rigidity and stability allow
the use of SFTI derivatives as scaffolds for the introduction of tumor-specific peptide motifs which could be used to increase
cell-binding affinities and thus their use as diagnostic and/or therapeutic tools.
Key wordsPeptides-Scaffold proteins-Sunflower trypsin inhibitor I-Peptide stability-Tracer development-Prostate carcinoma
Molecular Imaging & Biology 04/2012; 12(4):377-385. · 3.84 Impact Factor
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ABSTRACT: Radiopharmaceuticals constitute diagnostic and therapeutic tools for both clinical and preclinical applications. They are a blend of a tracer moiety that mediates a site specific accumulation and an effector: a radioisotope whose decay enables either molecular imaging or exhibits cytotoxic effects. Radioactive halogens and lanthanides are the most commonly used isotopes for radiopharmaceuticals. Due to their ready availability and the facile labeling metallic radionuclides offer ideal characteristics for applications in nuclear medicine. A stable link between the radionuclide and the carrier molecule is the primary prerequisite for in vivo applications. The radionuclide is selected according to its physical and chemical properties i.e. half-life, the type of decay, the energy emitted and its availability. Bifunctional chelating agents are used to stably link the radiometal to the carrier moiety of the radiopharmaceutical. The design of the bifunctional chelator has to consider the impact of the radiometal chelate on the biological properties of the target-specific pharmaceutical. Here, with an emphasis on oncology, we review applications of radiopharmaceuticals that contain bifunctional chelators, while highlighting successes and identifying the key challenges that need to be addressed for the successful translation of target binding molecules into tracers for molecular imaging and endoradiotherapy.
Current Medicinal Chemistry 03/2012; 19(17):2667-88. · 4.86 Impact Factor
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ABSTRACT: Urea-based inhibitors of the prostate-specific membrane antigen (PSMA) represent low-molecular-weight pepidomimetics showing the ability to image PSMA-expressing prostate tumors. The highly efficient, acyclic Ga(III) chelator N,N'-bis [2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-N,N'- diacetic acid (HBED-CC) was introduced as a lipophilic side chain into the hydrophilic pharmacophore Glu-NH-CO-NH-Lys which was found favorable to interact with the PSMA "active binding site". This report describes the syntheses, in vitro binding analyses, and biodistribution data of the radiogallium labeled PSMA inhibitor Glu-NH-CO-NH-Lys(Ahx)-HBED-CC in comparison to the corresponding DOTA conjugate. The binding properties were analyzed using competitive cell binding and enzyme-based assays followed by internalization experiments. Compared to the DOTA-conjugate, the HBED-CC derivative showed reduced unspecific binding and considerable higher specific internalization in LNCaP cells. The (68)Ga complex of the HBED-CC ligand exhibited higher specificity for PSMA expressing tumor cells resulting in improved in vivo properties. (68)Ga labeled Glu-NH-CO-NH-Lys(Ahx)-HBED-CC showed fast blood and organ clearances, low liver accumulation, and high specific uptake in PSMA expressing organs and tumor. It could be demonstrated that the PET-imaging property of a urea-based PSMA inhibitor could significantly be improved with HBED-CC.
Bioconjugate Chemistry 02/2012; 23(4):688-97. · 4.93 Impact Factor
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ABSTRACT: The expression of the chemokine receptor CXCR4 in tumors is associated with tumor aggressiveness and poor prognosis for the patient and contributes to metastatic seeding. Therefore it is of high interest to find a specific PET tracer for the imaging of CXCR4 expression in tumors. The aim of this study was the synthesis, (68)Ga labeling and first evaluation of DOTA-4-FBn-TN14003 as a potential PET tracer for this purpose. DOTA-4-FBn-TN14003 was synthesized using solid phase peptide synthesis and radiolabeling of this versatile precursor was performed with (68)Ga, which was obtained from a (68)Ge/(68)Ga generator. (68)Ga-DOTA-4-FBn-TN14003 was reproducibly obtained in isolated radiochemical yields of 72.5±4.9% with an excellent radiochemical purity of >99.5%. Specific activities of up to 29.8±3.1 GBq/μmol were achieved. In competition binding assays with SDF-1α, human T cell lymphoma Jurkat cells expressed high levels of CXCR4 whereas human breast cancer MDA-MB-231 cells expressed significantly lower levels of this chemokine receptor. The inhibition constants (IC(50)) of Ga-DOTA-4-FBn-TN14003 and 4-FBn-TN14003 to CXCR4 were determined in a competition assay against (125)I-SDF-1α using Jurkat as well as MDA-MB-231 cells. The IC(50) values of Ga-DOTA-4-FBn-TN14003 (1.99±0.31 nM) and 4-FBn-TN14003 (4.07±1.00 nM) proved to be comparable, indicating negligible influence of the metal complex. These results suggest (68)Ga-DOTA-4-FBn-TN14003 as a promising agent for the imaging of CXCR4 expression in tumors and metastases.
Bioorganic & medicinal chemistry 02/2012; 20(4):1502-10. · 2.82 Impact Factor
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ABSTRACT: In vivo cell-death imaging is still a challenging issue. Until now, only (99m)Tc-labeled HYNIC-rh-annexin A5 has been extensively studied in clinical trials. In the ongoing search for an alternative imaging agent, we synthesized a series of fluorescent zinc-cyclen complexes as annexin A5 mimics and studied structural variations on the uptake behavior of cells undergoing apoptosis/necrosis. The number of cyclen chelators was varied and the spacer separating cyclen from the central scaffold was modified. Five zinc-cyclen complexes were labeled with fluorescein for flow cytometric studies and one was labeled with (18)F for in vivo applications. Jurkat cells were treated with staurosporine to induce apoptosis/necrosis, incubated with the fluorescein-labeled zinc complexes and analyzed them by flow cytometry. Fluorescent annexin A5 and propidium iodide were applied as reference dyes. Flow cytometry revealed greater accumulation of zinc-cyclen complexes in staurosporine treated cells. The uptake was contingent on the presence of zinc and the fluorescence intensity was dependent on the number of zinc-cyclen groups. Confocal laser scanning microscopy showed the {bis[Zn(cyclen)]}(4+) complex distributed throughout the cytosol different to annexin A5. Owing to the structural similarity of the bis-cyclen ligands with CXCR4 binding bis-cyclam derivatives the zinc-cyclen complex uptake was challenged with the meta derivative of AMD3100. Lack of uptake depletion in staurosporine treated cells ruled out measurable CXCR4 interaction. PET imaging using the (18)F labeled zinc-cyclen complex revealed significantly higher uptake in an irradiated Dunning R3327-AT1 prostate tumor as compared to the contralateral control tumor. PET imaging of a HelaMatu tumor model additionally showed an increased uptake after taxol treatment. It could be demonstrated that the fluorescent zinc-cyclen complexes offer potential as new agents for flow cytometry and microscopic imaging of cell death. In addition, the (18)F labeled analogue holds promise for in vivo applications providing informations about cell death after radiation therapy and cytostatic drug treatment.
Bioconjugate Chemistry 12/2011; 22(12):2611-24. · 4.93 Impact Factor
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ABSTRACT: Dynamic PET studies with a ⁶⁸Ga-Bombesin analog, the ⁶⁸Ga-BZH₃, were performed in patients with highly suspected recurrent gliomas to investigate the effect of the receptor scintigraphy on tumor grading. Furthermore, dynamic F-18 Fluorodeoxyglucose (FDG) studies were performed for comparison.
The study consisted of 15 patients with histologically confirmed recurrent gliomas. Dynamic PET scans using ⁶⁸Ga-BZH₃ and FDG were obtained on 2 different days within 1 week. Multivariate analysis was used for the evaluation of the kinetic data. Standardized uptake values were calculated and a compartment (2-tissue) as well as a noncompartment model was used for data evaluation of both tracers.
The evaluation includes 6 patients with a WHO II, 6 patients with a WHO III, and 3 patients with a WHO IV recurrent gliomas. Of the 15 patients, 10 patients demonstrated an increased ⁶⁸Ga-BZH₃ uptake visually, 3 of them with a WHO II, 4 with a WHO III, and 3 with a WHO IV tumor. Of the 15 patients, 6 patients revealed an enhanced FDG metabolism visually, 3 of them with a WHO II, and 3 with a WHO III. None of the 3 patients with WHO IV tumor demonstrated an enhanced FDG-uptake. Discriminant analysis based on a combination of FDG influx and binding potential of ⁶⁸Ga-BZH₃ best discriminated between low- and high-grade gliomas with a correct classification rate of 100%.
⁶⁸Ga-BZH₃ seems to be helpful in patients with recurrent gliomas for the differentiation between low- and high-grade gliomas. Overall, the quantitative evaluation was superior to the visual analysis and the parameters of the ⁶⁸Ga-BZH₃ kinetics were more helpful than those of FDG for the differentiation between low- and high-grade gliomas.
Clinical nuclear medicine 02/2011; 36(2):101-8. · 3.92 Impact Factor
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ABSTRACT: Cell-penetrating peptides (CPPs) are able to penetrate the cell membrane carrying cargoes such as peptides, proteins, oligonucleotides, siRNAs, radioisotopes, liposomes, and nanoparticles. Consequently, many delivery approaches have been developed to use CPPs as tools for drug delivery. However, until now a systematic analysis of their in vivo properties including potential tumor binding specificity for drug targeting purposes has not been conducted. Ten of the most commonly applied CPPs were obtained by solid phase peptide synthesis and labeled with (111)In or (68)Ga. Uptake studies were conducted using a panel of six tumor cell lines of different origin. The stability of the peptides was examined in human serum. Biodistribution experiments were conducted in nude mice bearing human prostate carcinoma. Finally, positron emission tomography (PET) measurements were performed in male Wistar rats. The in vitro uptake studies revealed high cellular uptake values, but no specificity toward any of the cell lines. The biodistribution in PC-3 tumor-bearing nude mice showed a high transient accumulation in well-perfused organs and a rapid clearance from the blood. All of the CPPs revealed a relatively low accumulation rate in the brain. The highest uptake values were observed in the liver (with a maximal uptake of 51 %ID/g observed for oligoarginine (R(9))) and the kidneys (with a maximal uptake of 94 %ID/g observed for NLS). The uptake values in the PC-3 tumor were low at all time points, indicating a lack of tumor specific accumulation for all peptides studied. A micro-PET imaging study with (68)Ga-labeled penetratin, Tat and transportan(10) (TP(10)) confirmed the organ distribution data. These data reveal that CPPs do not show evidence for application in tumor targeting purposes in vivo. However, CPPs readily penetrate into most organs and show rapid clearance from the circulation. The high uptake rates observed in vitro and the relatively low specificity in vivo imply that CPPs would be better suited for topical application in combination with cargoes which show passive targeting and dominate the pharmacokinetic behavior. In conclusion, CPPs are suitable as drug carriers for in vivo application provided that their pharmacokinetic properties are also considered in design of CPP drug delivery systems.
Molecular Pharmaceutics 12/2010; 7(6):2224-31. · 4.78 Impact Factor
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ABSTRACT: Dysregulation of histone acetylation associated with an up-regulation of histone deacetylase (HDAC) activity is common in malignant tumours. Therefore, HDAC inhibitors were developed whose effects on proliferation and apoptosis have been shown in different tumour entities. Since non-iodide-concentrating thyroid carcinomas represent a therapeutic problem, this study addressed the effects of the HDAC inhibitor MS-275 on thyroid carcinoma cells.
After the antiproliferative effect of MS-275 had been proven in different human and rat thyroid carcinoma cell lines, FRO82-2, SW1736 and FTC133 cells were further investigated with respect to changes in apoptosis, cell cycle and metabolism by the annexin V/propidium iodide assay, FACS analysis and uptake experiments employing 3-O-methyl-D-(3H)glucose, fluoro-2-deoxy-D-glucose2 [5,6-(3)H] and 14C-aminoisobutyric acid (AIB). The induction of iodide transport and gene expression were investigated in 125iodide uptake experiments and real-time polymerase chain reaction (PCR).
MS-275 induced a concentration- and time-dependent inhibition of proliferation in the thyroid carcinoma cell lines with varying IC50 values. In FRO82-2, SW1736 and FTC133 cells characterized by low, moderate and high sensitivity an up-regulation of p21CIP/WAF1 expression and G1 and/or G2 phase arrest were observed upon MS-275 exposure corresponding to the sensitivity of individual cell lines. In addition, high MS-275 concentrations increased the apoptotic cell fraction of FTC133 and SW1736 cells, whereas resistance to apoptosis and simultaneous up-regulation of Bcl-2 gene expression were observed in FRO82-2 cells. MS-275 treatment also mediated a concentration-dependent decrease of 3H-FDG uptake and an increased 3-O-methyl-D-(3H)glucose uptake in all thyroid carcinoma cell lines after 24 h, an increased uptake of both tracers in FTC133 cells after 48 h, and restored the functional activity of the sodium-iodide symporter in SW1736 and FTC133 cells up to 20- and 45-fold.
MS-275 exerts dose-dependent antiproliferative effects including growth arrest, differentiation and apoptosis in some thyroid carcinoma cell lines and might, therefore, be considered for the treatment of anaplastic and non-iodide-concentrating thyroid carcinomas.
European Journal of Nuclear Medicine 12/2010; 37(12):2286-97. · 4.53 Impact Factor
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ABSTRACT: Peptides involving the RGD motive (arginine-glycine-aspartic acid) recognize members of the integrin receptor family. Since the receptors are located mainly on the surface of endothelial cells, structural modifications including multimers of c(RGDfE) were recently found to improve the binding avidity for α(v)β(3) integrin significantly. The multivalent RGD peptides exhibited rather loose linkages partly including oligo(ethylene glycol) spacers (EG(n)) with different chain lengths. Therefore, the dependence of multivalent RGD systems with and without EG(n) linkers were investigated on their binding properties to cultured α(v)β(3) integrin-expressing U87MG cells.
We synthesized a series of di-, tri- and tetravalent rigid scaffolds (terephthalic acid, trimesic acid and adamantane-1,3,5,7-tetracarboxylic acid) conjugated to c(RGDyK) ligands, which were linked contiguously or separated by the oligo(ethylene glycol) spacers. The inhibition constants of these c(RGDyK) derivatives were determined by competition assays with (125)I-labeled echistatin.
While c(RGDyK) function is a relative weak competitor against [(125)I]echistatin (K(i), 329 ± 18 nM) for α(v)β(3) integrin-expressing U87MG cells, RGD dimers improved the competition potency considerably (K(i), 64 ± 23 nM). This effect was even more pronounced with the RGD trimers (K(i), 40 ± 7 nM) and tetramers (K(i), 26±9 nM). The introduction of EG(n) spacers and the increase of linker lengths proved to be detrimental since more competitors were needed to compete with [(125)I]echistatin. The EG(6) group, for example, reduced the inhibition constants by 29% (dimer), 57% (trimer) and 97% (tetramer).
The binding experiments performed with the three forms of multivalent RGD ligands indicate the weakening of competitive potency against [(125)I]echistatin with the introduction of EG(n) spacers. This effect may be related to the decrease of the effective RGD molarity, which becomes most prominent within the tetravalent series.
Nuclear Medicine and Biology 11/2010; 37(8):885-91. · 3.02 Impact Factor
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ABSTRACT: Integrin α(ν)β(3) is overexpressed on endothelial cells of growing vessels as well as on several tumor types, and so integrin-binding radiolabeled cyclic RGD pentapeptides have attracted increasing interest for in vivo imaging of α(ν)β(3) integrin expression by positron emission tomography (PET). Of the cRGD derivatives available for imaging applications, systems comprising multiple cRGD moieties have recently been shown to exhibit highly favorable properties in relation to monomers. To assess the synthetic limits of the cRGD-multimerization approach and thus the maximum multimer size achievable by using different efficient conjugation reactions, we prepared a variety of multimers that were further investigated in vitro with regard to their avidities to integrin α(ν)β(3.) The synthesized peptide multimers containing increasing numbers of cRGD moieties on PAMAM dendrimer scaffolds were prepared by different click chemistry coupling strategies. A cRGD hexadecimer was the largest construct that could be synthesized under optimized reaction conditions, thus identifying the current synthetic limitations for cRGD multimerization. The obtained multimeric systems were conjugated to a new DOTA-based chelator developed for the derivatization of sterically demanding structures and successfully labeled with (68)Ga for a potential in vivo application. The evaluated multimers showed very high avidities-increasing with the number of cRGD moieties-in in vitro studies on immobilized α(ν)β(3) integrin and U87MG cells, of up to 131- and 124-fold, respectively, relative to the underivatized monomer.
ChemBioChem 10/2010; 11(15):2168-81. · 3.94 Impact Factor
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ABSTRACT: VEGF receptors play a key role in angiogenesis and are important targets for several approved and many experimental drugs. Imaging of VEGF receptor expression in malignant tumors would provide important information, which can influence patient management. The aim of this study was the development of an easy-to-label positron-emitting tracer for imaging VEGF receptors. The tracer is based on engineered single-chain VEGF (scVEGF), expressed with cysteine-containing fusion tag (Cys-tag) for site-specific conjugation of PEGylated bifunctional chelating agents, HBED-CC or NOTA, suitable for labeling with (68)Ga at ambient temperature.
scVEGF-PEG-HBED-CC was synthesized by activating a single carboxyl group of the [Fe(HBED-CC)](-) complex with N-hydroxysuccinimide. Reaction of the activated complex with NH(2)-PEG-maleimide was followed by site-specific conjugation of PEGylated chelator to a thiol group in Cys-tag of scVEGF. The scVEGF-PEG-NOTA conjugate was synthesized using NHS-PEG-maleimide and p-NH(2)-Bn-NOTA. (68)Ga complexation was performed in HEPES buffer (pH 4.2) at room temperature. The functional activity after labeling was tested by radioligand cell binding assays. Biodistribution and PET studies in tumor-bearing mice were performed after 1, 2, 3 and 4 h postinjection.
The radiolabeling of scVEGF-PEG-HBED-CC proved more efficient than scVEGF-PEG-NOTA allowing to stop the reaction after 4 min (>97% radiochemical yield). Radioligand cell binding assays performed on HEK-293 cells overexpressing VEGFR-2 revealed no change in the binding properties of (68)Ga-radiolabeled scVEGF relative to other scVEGF-based tracers. Both tracers showed comparable results in biodistribution, such as tumor accumulation and low liver uptake. The tracers were stable in 50% human serum for at least 72 h.
The conjugates scVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA revealed comparable in vivo characteristics and allowed easy-to-perform labeling with high stability for fast [(68)Ga]PET imaging of VEGF receptors in angiogenic vasculature.
Nuclear Medicine and Biology 05/2010; 37(4):405-12. · 3.02 Impact Factor
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ABSTRACT: Therapy with the somatostatin analogue DOTA-(0)-Phe(1)-Tyr(3)-octreotide (DOTATOC) labeled with a beta-(DOTA-Phe-Tyr-Octreotide) emitter such as 90Y or 177Lu is accepted for the palliative treatment of unresectable neuroendocrine cancer. However, the optimal route of administration has not been determined. Using positron-emission tomography (PET)-labeled 68Ga-DOTATOC, we compared selective tumoral uptake on PET/computed tomography (CT) after arterial or venous administration of the agent in patients with gastroenteropancreatic neuroendocrine tumor.
Fifteen patients with neuroendocrine cancer were examined with 68Ga-DOTATOC PET/CT after intravenous (i.v.) and intraarterial (i.a.) administration within 4 weeks of each other and without any intervening therapy. Eleven patients had multifocal metastases, six were considered to have unresectable primary tumor. The intraarterial catheter was placed in the vessel supplying the main tumor burden. The standard uptake value (SUV) was used to compare intratumoral concentrations of 68Ga-DOTATOC.
Compared with i.v. infusion, the i.a. infusion resulted in an increased SUV in 117 of 122 (96%) liver metastases. The average increase in SUV was 3.75-fold higher with i.a. administration. The increase in uptake for the primary tumors was dependent on the selectivity of the catheter placement, resulting in variable increases in SUV after i.a. injection (1.44- to 7.8-fold higher).
This study showed that uptake of DOTATOC is commonly several fold higher after selective i.a. administration in comparison with i.v. injection in both the primary tumor as well as in liver metastases of neuroendocrine cancer. Therefore, intraarterial DOTATOC is a promising drug for regionally intensified radiopeptide therapy.
Clinical Cancer Research 05/2010; 16(10):2899-905. · 7.74 Impact Factor
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ABSTRACT: Recombinant antibodies isolated from human antibody libraries have excellent affinities and high target specificity. As full-length IgGs are cleared inadequately slowly from the circulation, the aim of this work was to figure out which kind of recombinant antibody fragment proves to be appropriate for imaging epithelial cell adhesion molecule (EpCAM)-expressing tumours with the short-living radioisotope (68)Ga.
In order to combine the promising tumour targeting properties of antibodies with (68)Ga, four antibody variants with the same specificity and origin only differing in molecular weight were constructed for comparison. Therefore, the binding domains of a single-chain fragment variable (scFv) isolated from a human naïve antibody library were modified genetically to construct the respective full-length IgG, the tria- and diabody variants. These molecules were conjugated with the bifunctional chelating agent N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) to enable (68)Ga labelling at ambient temperature and compared in biodistribution and immuno-PET imaging experiments.
The antibody variants with identical specificity proved to have the correct molecular weight, high binding affinity and specificity to their antigen, EpCAM. Radiometal complexation was efficiently performed at room temperature leading to (68)Ga-labelled antibodies with unchanged binding properties compared to the original antibody variants. The best targeting properties were obtained with the scFv and especially with the diabody. The triabody showed higher absolute tumour uptake but only moderate clearance from circulation.
The antibody variants differed considerably in normal organ uptake, clearance from circulation and tumour accumulation. The data demonstrate the feasibility of imaging solid tumours with the (68)Ga-labelled diabody format. This type of recombinant protein might be a promising carrier even for the short-lived radiometal (68)Ga to support e.g. the management of immunotherapy which may provide important information regarding receptor expression of solid tumours.
European Journal of Nuclear Medicine 02/2010; 37(7):1397-407. · 4.53 Impact Factor
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ABSTRACT: Endoradiotherapy represents an alternative therapeutic method in cancer treatment with advantageous features compared to chemotherapy and radiation therapy. Intelligent dose delivery concepts using small drugs, peptides or antibodies as radionuclide carriers enable the verification of a selective accumulation in the tumour lesion and to reduce radiation toxicity for the peripheral organs. The development of endoradiotherapeutic agents, especially chelator-conjugated biomolecules, for example ibritumomab tiuxetan or DOTATOC, gains importance due to the stable complexation of versatile radiometals, such as (90)Y or (177)Lu. The rational design of novel target binding sides and their grafting into a drug scaffold is a highly promising strategy, which may promote further implication in endoradiotherapy. This review highlights the basic concepts of endoradiotherapy and discusses the potential of targeted therapy and the properties of energy-rich particles emitted by radionuclides for tumour therapy.
European journal of pharmacology 10/2009; 625(1-3):55-62. · 2.59 Impact Factor
