Design, synthesis, and initial evaluation of a high affinity positron emission tomography probe for imaging matrix metalloproteinases 2 and 9.

The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions. Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor for imaging active MMP2/MMP9 in vivo. We synthesized a series of N-sulfonylamino acid derivatives with low to high nanomolar inhibitory potencies. (R)-2-(4-(4-Fluorobenzamido)phenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (7) exhibited the best in vitro binding properties: MMP2 IC50 = 1.8 nM, MMP9 IC50 = 7.2 nM. Radiolabeling of 7 with no carrier added (18)F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly depended on the iodonium counteranion (ClO4(-) > Br(-) > TFA(-) > tosylate). (18)F-7 was obtained in up to 20% radiochemical yield (decay corrected), high radiochemical purity, and >90 GBq/µmol specific radioactivity. The radiolabeled compound showed excellent stability in vitro and in mice in vivo.

Radiofluorinated histamine H3 receptor antagonist as a potential probe for in vivo PET imaging: radiosynthesis and pharmacological evaluation.

The histamine H(3) receptor (H(3)R) plays a role in cognition and memory processes and is implicated in different neurological disorders, including Alzheimer's disease, schizophrenia, and narcolepsy. In vivo studies of the H(3)R occupancy using a radiolabeled PET tracer would be very useful for CNS drug discovery and development. We report here the radiosynthesis, in vitro and in vivo evaluation of a novel (18)F-labeled high-affinity H(3)R antagonist (18)F-ST889. The radiosynthesis was accomplished via nucleophilic substitution of the mesylate leaving group with a radiochemical yield of 8-20%, radiochemical purity >99%, and specific radioactivity > 65 GBq/µmol. (18)F-ST889 exhibited high in vivo stability and rather low lipophilicity (logD(7.4)=0.35 ± 0.09). In vitro autoradiography showed specific binding in H(3)R-rich brain regions such as striatum and cortex. However, in vivo PET imaging of the rat brain with (18)F-ST889 was not successful. Possible reasons are discussed.

4-[18F]fluoroglutamic acid (BAY 85-8050), a new amino acid radiotracer for PET imaging of tumors: synthesis and in vitro characterization.

There is a high demand for tumor specific PET tracers in oncology imaging. Besides glucose, certain amino acids also serve as energy sources and anabolic precursors for tumors. Therefore, (18)F-labeled amino acids are interesting probes for tumor specific PET imaging. As glutamine and glutamate play a key role in the adapted intermediary metabolism of tumors, the radiosynthesis of 4-[(18)F]fluoro l-glutamic acid (BAY 85-8050) as a new specific PET tracer was established. Cell-uptake studies revealed specific tumor cell accumulation.

Radioligands for the PET imaging of metabotropic glutamate receptor subtype 5 (mGluR5).

Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCR), which activate intracellular secondary messenger systems when bound by the physiological ligand glutamate. Modulation of mGluR5s has potential for the treatment of variety of psychiatric and neurological diseases such as depression, anxiety, schizophrenia and Parkinson's disease. Positron emission tomography (PET) might offer the possibility to visualize the mGluR5 and present an interesting tool for studying this receptor-subtype under physiologic and pathologic conditions. In this review paper, emphasis is given to the radiosynthesis, in vitro and in vivo characterization of recently published mGluR5 PET tracers.

Synthesis, radiolabeling, in vitro and in vivo evaluation of [18F]-FPECMO as a positron emission tomography radioligand for imaging the metabotropic glutamate receptor subtype 5.

INTRODUCTION: [18F]-(E)-3-((6-Fluoropyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl oxime ([18F]-FPECMO) is a novel derivative of [11C]-ABP688. [18F]-FPECMO was characterized as a PET imaging agent for the metabotropic glutamate receptor subtype 5 (mGluR5). METHODS: [18F]-FPECMO was synthesized in a one-step reaction sequence by reacting [(18)F]-KF-K(222) complex with (E)-3-((6-bromopyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl oxime in dry DMSO. The in vitro affinity of FPECMO was determined by displacement assays using rat whole brain homogenates (without cerebellum) and the mGluR5-specific radioligand [(3)H]-M-MPEP. Further in vitro characterization involved metabolite studies, lipophilicity determination and autoradiographical analyses of brain slices. In vivo evaluation was performed by postmortem biodistribution studies and PET experiments using Sprague-Dawley rats. RESULTS: The radiochemical yield after semipreparative HPLC was 35+/-7% and specific activity was >240 GBq/micromol. [(18)F]-FPECMO exhibited optimal lipophilicity (logD=2.1) and high metabolic stability in vitro. Displacement studies revealed a K(i) value of 3.6+/-0.7 nM for FPECMO. Biodistribution studies and ex vivo autoradiography showed highest radioactivity accumulation in mGluR5-rich brain regions such as the striatum and hippocampus. Co-injection of [18F]-FPECMO and ABP688 (1 mg/kg body weight), an mGluR5 antagonist, showed 40% specific binding in the striatum, hippocampus and cortex, regions known to contain high densities of the mGluR5. PET imaging, however, did not allow the visualization of mGluR5-rich brain regions in the rat brain due to a fast washout of [18F]-FPECMO from mGluR5-expressing tissues and rapid defluorination. CONCLUSIONS: [18F]-FPECMO showed significant potential for the detection of mGluR5 in vitro; however, its in vivo characteristics are not optimal for a clear-cut visualization of the mGluR5 in rats.

In-line radiolabeling: a novel continuous-flow system for commercial-scale protein labeling.

UNLABELLED: A key limitation in developing radiotherapeutic proteins is the expense of manufacturing the drug in small batches using traditional reaction vessels. Removing limitations on the quantity of protein labeled at any one time significantly decreases the cost of production, and nowhere is the need for cost-effective radiotherapeutics more acute than in the treatment of cancer. METHODS: We describe a novel method that can rapidly radiolabel, theoretically, unlimited amounts of protein, without causing significant damage to binding potency or structural integrity. Our process controls the reaction rate for the isotope and reactants as they simultaneously flow through a reaction tube. RESULTS: We have demonstrated proof of principle by labeling nearly a gram of antibody with 481 GBq (13 Ci) of (131)I during a single 30-min reaction run. CONCLUSION: Simple to construct, our system is already used to manufacture a radiolabeled antibody, both in the United States and in India, as part of clinical trials to treat glioblastoma multiforme. Modified, this system may be also applicable for nonradioactive labeling.

The soluble form of the cancer-associated L1 cell adhesion molecule is a pro-angiogenic factor.

A soluble form of the L1 cell adhesion molecule (sL1) is released from various tumor cells and can be found in serum and ascites fluid of uterine and ovarian carcinoma patients. sL1 is a ligand for several Arg-Gly-Asp (RGD)-binding integrins and can be deposited in the extracellular matrix. In this study we describe a novel function of this physiologically relevant form of L1 as a pro-angiogenic factor. We demonstrated that the anti-L1 monoclonal antibody (mAb) chCE7 binds near or to the sixth Ig-like domain of human L1 which contains a single RGD sequence. mAb chCE7 inhibited the RGD-dependent adhesion of ovarian carcinoma cells to sL1 and reversed the sL1-induced proliferation, matrigel invasion and tube formation of bovine aortic endothelial (BAE) cells. A combination of sL1 with vascular endothelial growth factor-A (VEGF-A(165)), which is an important angiogenic inducer in tumors, strongly potentiated VEGF receptor-2 tyrosine phosphorylation in BAE cells. Chick chorioallantoic membrane (CAM) assays revealed the pro-angiogenic potency of sL1 in vivo which could be abolished by chCE7. These results indicate an important role of released L1 in tumor angiogenesis and represent a novel function of antibody chCE7 in tumor therapy.

Organofluorosilanes as model compounds for 18F-labeled silicon-based PET tracers and their hydrolytic stability: experimental data and theoretical calculations (PET = positron emission tomography).

Silicon chemistry has only recently been discovered by radiochemists as a straightforward tool for the introduction of (18)F into biomolecules for positron emission tomography (PET) imaging. (18)F-labeled PET tracers must be stable towards defluorination under physiological conditions, but it is known that the hydrolytic stability of the silicon-fluorine bond is determined by the nature of the substituents on silicon. In the presented study we performed an extensive investigation on the hydrolytic stability of various synthesized organofluorosilane model compounds. By means of density functional theory (DFT) methods a theoretical model of organofluorosilane hydrolysis, which correlates with the experimentally determined hydrolytic half-lives, is developed. The calculation of the difference of Si-F bond lengths between the optimized structures of the starting material A and the intermediate structure C allows the estimation of the hydrolytic stability of newly designed compounds. This model permits the facilitated development of improved building blocks for the synthesis of novel (18)F-silyl-modified biomolecules for PET imaging.

A stable neurotensin-based radiopharmaceutical for targeted imaging and therapy of neurotensin receptor-positive tumours.

PURPOSE: Neurotensin (NT) and its high affinity receptor (NTR1) are involved in several neoplastic processes. Thus, NT-based radiopharmaceuticals are potential tracers for targeted diagnosis and therapy of NTR-positive tumours. A new analogue based on NT(8-13), NT-XIX, with the three enzymatic cleavage sites stabilised, was synthesised and tested. METHODS: The synthesis was performed by Boc strategy. Labelling with (99m)Tc/(188)Re was performed using the tricarbonyl technique. Metabolic stability was tested in vitro and in vivo. NT-XIX was further characterised in vitro in HT-29 cells and in vivo in nude mice with HT-29 xenografts. RESULTS: NT-XIX showed much longer half-lives than non-stabilised analogues. Binding to NTR1 was highly specific, although the affinity was lower than that of natural NT. Bound activity rapidly internalised into HT-29 cells and 50% remained trapped after 24 h. In the time-course biodistribution, the highest uptake was found in the tumour at all p.i. times. In vivo uptake was specific, and accumulation of activity in the kidneys was low. Radioactivity clearance from healthy organs was faster than that from the tumour, resulting in improved tumour-to-tissue ratios and good SPECT/CT imaging. Treatment with (188)Re-NT-XIX (30 MBq, in three or four fractions) decreased tumour growth by 50% after 3 weeks. CONCLUSION: The high in vivo stability and the favourable in vivo behaviour makes NT-XIX an excellent candidate for the imaging and therapy of NTR1-positive tumours.

Fluorine-18 click radiosynthesis and preclinical evaluation of a new 18F-labeled folic acid derivative.

The folate receptor (FR) is highly expressed on most epithelial cancer cells, while normal cells show only restricted expression of FR. As a result, the FR is an ideal target for receptor-based molecular imaging and therapy of cancer and has become a promising target in oncology. To date, several folate-based chemotherapeutics and imaging probes such as radiopharmaceuticals for single photon emission computed tomography (SPECT) have been developed. However, an (18)F-labeled folic acid derivative suitable for positron emission tomography (PET) imaging that can be routinely applied is still lacking. In this study, a new fluorinated and radiofluorinated folic acid derivative, (18/19)F-click folate, was synthesized using click chemistry. In a convenient and very efficient two-step radiosynthesis, the isolated (18)F-click folate was obtained in good radiochemical yields of 25-35% with a specific activity of 160+/-70 GBq/micromol after

Novel glycated [99mTc(CO)3]-labeled bombesin analogues for improved targeting of gastrin-releasing peptide receptor-positive tumors.

Radiolabeled bombesin (BBS) analogues are promising pharmaceuticals for imaging of cancer cells expressing gastrin-releasing peptide receptors (GRPR). However, most of the radiolabeled BBS derivatives show a high accumulation of activity in the liver and a strong hepatobiliary excretion, both unfavorable for imaging and therapy of abdominal lesions. For this reason, we introduced hydrophilic carbohydrated linker moieties into our BBS analogues to reduce the abdominal accumulation and to improve the tumor-to-background ratios. A stabilized BBS(7-14) sequence bearing the (NalphaHis)Ac-chelator was modified with amino acid linkers containing a lysine or propargylglycine residue. The epsilon-amino group of a lysine was either coupled to shikimic acid or reacted with glucose to form the Amadori conjugate. Alternatively, a glucose was attached to the peptide via "click" chemistry with the propargylglycine side chain. The peptides were synthesized on Rink amide resin using solid-phase peptide synthesis and labeled with 99mTc using the tricarbonyl technique. Binding and degradation were tested in Vitro in GRPR-expressing PC-3 cells. Biodistribution and SPECT/CT imaging studies were performed in nude mice bearing PC-3 tumor xenografts. The new peptides showed a log D between -0.2 and -0.5 and kept the high affinity for GRPR with Kd values of <0.5 nM. In Vitro, they were rapidly internalized into the tumor cells and showed an increased cellular retention and stability (t(1/2 )>35 min). In ViVo, all new compounds exhibited higher tumor-to-background ratios compared to the nonglycated reference. Thus, the best results were obtained with the triazole coupled glucose with a 4-fold increased uptake and retention in tumor tissue (3.6 and 2.5%ID/g at 1.5 h and 5 h p.i, respectively) and a significantly reduced accumulation in the liver (0.6 vs 2.4%ID/g, 1.5 h p.i., respectively). Apart from higher tumor-to-liver ratios (17-fold, 1.5 h p.i.), both tumor-to-kidney and tumor-to-blood ratios could be significantly improved by a factor of 1.5 and 2.7, respectively (1.5 h p.i., P<0.05). The imaging studies proved the reduction of abdominal background, and tumor xenografts could clearly be visualized. In conclusion, the introduction of a carbohydrated linker substantially improved the biodistribution properties of BBS analogues labeled with the 99mTc-tricarbonyl core.

Glycation methods for bombesin analogs containing the (NalphaHis)Ac chelator for 99mTc(CO)3 radiolabeling.

The overexpression of peptide receptors in a variety of human carcinomas has generated considerable interest in peptide-based radiopharmaceuticals for peptide receptor imaging and peptide receptor radiotherapy. The gastrin-releasing peptide receptor is overexpressed in human prostate-, breast-, colon- and small cell lung carcinoma cells. We have developed metabolically stable (99m)Tc-radiolabeled bombesin ([Cha(13), Nle(14)]BBS(7-14)) analogs, which bind with high affinity to the gastrin-releasing peptide receptors. However, because of their lipophilicity, they showed unfavorable biodistribution with high hepatic accumulation and hepatobiliary excretion. We now report a study of different glycation methods for [Cha(13), Nle(14)]BBS(7-14) analogs to improve their biodistribution profile. Whereas the glycation using the Maillard reaction was problematic, resulting in low yields, selective introduction of the glycomimetic shikimic acid to the side chain of a Lys residue was possible. A chemoselective ligation of alpha-D-glucose to an amino-oxyacetylated [Cha(13), Nle(14)]BBS(7-14) analog could be achieved, but was complicated by the co-elution of starting peptide and glycopeptide. The best procedure consisted of the [1,3]-cycloaddition of N(3)-beta-D-glucose to a propargylglycine-containing [Cha(13), Nle(14)]BBS(7-14) analog, using a catalytic amount of Cu(I)I. All glycated [Cha(13), Nle(14)]BBS(7-14) analogs showed high affinity for the gastrin-releasing peptide receptor and rapid accumulation into PC-3 tumor cells.

Influence of the molecular charge on the biodistribution of bombesin analogues labeled with the [99mTc(CO)3]-core.

The overexpression of Bombesin (BBS) receptors on a variety of human cancers make them interesting targets for tumor imaging and therapy. Analogues of the neuropeptide BBS have been functionalized with the (NalphaHis)- chelator for labeling with the 99mTc-tricarbonyl core. The introduction of a betaAla-betaAla linker between the stabilized BBS binding sequence and the chelator led to increased tumor uptake but still rather unfavorable in ViVo properties. Novel polar linkers, with different charge, have been introduced in the molecule and tested for their influence on the biodistribution. The new analogues showed a shift in hydrophilicity from a Log D=0.9 to Log D values between 0.4 and -2.2. All compounds kept the increased stability in both human plasma (t(1/2)>16 h) and in tumor cells (t(1/2)=30-40 min). The compounds with Log D values between +1 and -1 showed the highest binding affinities with Kd values of <0.5 nM, as well as the highest cellular uptake. However, higher hydrophilicity (Log D < -1.8) led to lower affinity and a substantial decrease of internalization. The introduction of a positive charge (beta3hLys) resulted in unfavorable biodistribution, with increased kidney uptake. The introduction of an uncharged hydroxyl group (beta3hSer) improved the biodistribution, resulting in significantly better tumor-to-tissue ratios. The compound with one single negative charge (beta3hGlu) showed a significant increase in the tumor uptake (2.1+/-0.6% vs 0.80+/-0.35% ID/g in comparison to the betaAla-betaAla analogue) and also significantly higher tumor-to-tissue ratios. The specificity of the in ViVo uptake was confirmed by coinjection with natural BBS. Moreover, the analogue provided a much clearer image of the tumor xenografts in the SPECT/CT studies. The introduction of a single negative charge may be useful in the development of new BBS analogues to obtain an improved biodistribution profile, with increased tumor uptake and better imaging.

Synthesis, 18F-labeling, and in vitro and in vivo studies of bombesin peptides modified with silicon-based building blocks.

The gastrin-releasing peptide receptor (GRPr) is overexpressed on various human tumors. The goal of our study was the synthesis of new 18F-labeled bombesin analogues for the PET imaging of GRPr expression in prostate tumor using a silicon-based one-step n. c. a. radiolabeling method. The silicon-containing building blocks were efficiently coupled to the N-terminus of the peptides via solid-phase synthesis. Radiolabeling of the obtained peptide precursors proceeded smoothly under acidic conditions (34-85% conversion). Using the di-tert-butyl silyl building block as labeling moiety, products containing a hydrolytically stable 18F-label were obtained. In in vitro receptor binding experiments 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH 2 ( 4b, IC50 = 22.9 nM) displayed a 12-fold higher binding affinity than 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2 ( 3b, IC50 = 276.6 nM), and 4b was therefore chosen for further evaluation. In vitro and ex vivo metabolite studies of [18F]4b showed no significant degradation. In biodistribution experiments, tumor uptake of [18F]4b was low and unspecific, whereas the GRPr-rich pancreas revealed a high and specific accumulation of the radiotracer. This study demonstrates the applicability of our silicon-based one-step n. c. a. radiolabeling method for the synthesis of new 18F-labeled bombesin derivatives. This innovative approach represents a general, straightforward access to radiolabeled peptides as PET imaging probes.

Antibodies directed against L1-CAM synergize with Genistein in inhibiting growth and survival pathways in SKOV3ip human ovarian cancer cells.

Antibodies directed against the L1 cell adhesion protein inhibit growth of SKOV3ip human ovarian cancer cells in vitro and in vivo. Responses of SKOV3ip cells in vitro to anti-L1 mAb chCE7 and Genistein were investigated. Genistein potentiated the anti-proliferative and pro-apoptotic effects of chCE7 in SKOV3ip cells. A combination of mAb chCE7 and Genistein strongly reduced the sensitivity of p44/42 (Erk1,2) kinase, Src kinase and Akt kinase to extracellular stimulation with serum, Epidermal Growth Factor and Hepatocyte Growth Factor. The observed synergy of antibodies directed against L1 with Genistein could lead to a new therapeutic option for ovarian cancer.

Radiolabeling and in vitro and in vivo evaluation of [18F]-FE-DABP688 as a PET radioligand for the metabotropic glutamate receptor subtype 5.

INTRODUCTION: Fluoroethyl-desmethyl-ABP688 (FE-DABP688) is a novel derivative of the previously described positron emission tomography (PET) ligand 3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-[11C]-methyl-oxime. FE-DABP688 was radiolabeled with fluorine-18 and characterized as a PET imaging agent for the metabotropic glutamate receptor subtype 5 (mGluR5). METHODS: FE-DABP688 was radiolabeled by reacting 2-[18F]-fluoroethyl tosylate with the sodium salt of 3-(pyridin-2-ylethynyl)-cyclohex-2-enone-oxime in dry DMF. The in vitro affinity of [18F]-FE-DABP688 for mGluR5 was determined by Scatchard analysis of saturation binding data using rat whole-brain membranes (without cerebellum). Further in vitro characterization of the tracer involved plasma stability and lipophilicity testing. In vivo evaluation of [18F]-FE-DABP688 was performed by postmortem biodistribution experiments and PET studies in rats using the dedicated small-animal PET tomograph quad-HIDAC. RESULTS: The radiotracer was obtained in good radiochemical yields in an overall synthesis time of 150 min. The radiochemical yield after semipreparative HPLC was 25+/-8% (n>7, decay corrected), and specific activity was 30+/-5 GBq/micromol (n>7). [18F]-FE-DABP688 exhibited optimal lipophilicity with a logD value of 2.1+/-0.1 and high plasma stability. Saturation assays of [(18)F]-FE-DABP688 revealed a single high-affinity binding site with a dissociation constant (Kd) of 1.6+/-0.4 nM and a Bmax value of 119+/-24 fmol/mg protein. PET scanning indicated radioactivity uptake in mGluR5-rich regions such as the hippocampus, striatum and cortex, while radioactivity accumulation in the cerebellum, a region with negligible mGluR5 density, was significantly lower. Biodistribution studies showed a similar distribution pattern of [18F]-FE-DABP688 binding in the brain. The hippocampus-to-cerebellum and striatum-to-cerebellum ratios were 1.81+/-0.16 and 1.93+/-0.36, respectively. Blocking studies using coinjection of [18F]-FE-DABP688 and unlabeled 2-methyl-6-((3-methoxyphenyl)ethynyl)-pyridine (1 mg/kg) revealed more than 45% specific binding in the hippocampus and striatum, thus demonstrating the in vivo specificity of tracer binding. CONCLUSIONS: [18F]-FE-DABP688 may be a useful PET tracer for imaging mGluR5 in rodents.