Search

Variations in vascular wall shear stress are often presumed to result in the formation of atherosclerotic lesions at specific arterial regions, where continuous laminar flow is disturbed. The influences of altered blood flow dynamics and oscillations on the integrity of endothelial cells and the endothelial layer have been extensively studied in vitro and in vivo. Under pathological conditions, the Arg-Gly-Asp (RGD) motif binding integrin αvβ3 has been identified as a relevant target, as it induces endothelial cell activation. Animal models for in vivo imaging of endothelial dysfunction (ED) mainly rely on genetically modified knockout models that develop endothelial damage and atherosclerotic plaques upon hypercholesterolemia (ApoE−/− and LDLR−/−), thereby depicting late-stage pathophysiology. The visualization of early ED, however, remains a challenge. Therefore, a carotid artery cuff model of low and oscillating shear stress was applied in CD-1 wild-type mice, which should be able to show the effects of altered shear stress on a healthy endothelium, thus revealing alterations in early ED. Multispectral optoacoustic tomography (MSOT) was assessed as a non-invasive and highly sensitive imaging technique for the detection of an intravenously injected RGD-mimetic fluorescent probe in a longitudinal (2–12 weeks) study after surgical cuff intervention of the right common carotid artery (RCCA). Images were analyzed concerning the signal distribution upstream and downstream of the implanted cuff, as well as on the contralateral side as a control. Subsequent histological analysis was applied to delineate the distribution of relevant factors within the carotid vessel walls. Analysis revealed a significantly enhanced fluorescent signal intensity in the RCCA upstream of the cuff compared to the contralateral healthy side and the downstream region at all time points post-surgery. The most obvious differences were recorded at 6 and 8 weeks after implantation. Immunohistochemistry revealed a high degree of αv-positivity in this region of the RCCA, but not in the left common carotid artery (LCCA) or downstream of the cuff. In addition, macrophages could be detected by CD68 immunohistochemistry in the RCCA, showing ongoing inflammatory processes. In conclusion, MSOT is capable of delineating alterations in endothelial cell integrity in vivo in the applied model of early ED, where an elevated expression of integrin αvβ3 was detected within vascular structures.

The endothelin (ET) axis plays a pivotal role in cardiovascular diseases. Enhanced levels of circulating ET-1 have been correlated with an inferior clinical outcome after myocardial infarction (MI) in humans. Thus, the evaluation of endothelin-A receptor (ETAR) expression over time in the course of myocardial injury and healing may offer valuable information toward the understanding of the ET axis involvement in MI. We developed an approach to track the expression of ETAR with a customized molecular imaging probe in a murine model of MI. The small molecular probe based on the ETAR-selective antagonist 3-(1,3-benzodioxol-5-yl)-5-hydroxy-5-(4-methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)methyl]-2(5H)-furanone (PD156707) was labeled with fluorescent dye, IRDye800cw. Mice undergoing permanent ligation of the left anterior descending artery (LAD) were investigated at day 1, 7, and 21 post surgery after receiving an intravenous injection of the ETAR probe. Cryosections of explanted hearts were analyzed by cryotome-based CCD, and fluorescence reflectance imaging (FRI) and fluorescence signal intensities (SI) were extracted. Fluorescence-mediated tomography (FMT) imaging was performed to visualize probe distribution in the target region in vivo. An enhanced fluorescence signal intensity in the infarct area was detected in cryoCCD images as early as day 1 after surgery and intensified up to 21 days post MI. FRI was capable of detecting significantly enhanced SI in infarcted regions of hearts 7 days after surgery. In vivo imaging by FMT localized enhanced SI in the apex region of infarcted mouse hearts. We verified the localization of the probe and ETAR within the infarct area by immunohistochemistry (IHC). In addition, neovascularized areas were found in the affected myocardium by CD31 staining. Our study demonstrates that the applied fluorescent probe is capable of delineating ETAR expression over time in affected murine myocardium after MI in vivo and ex vivo.

Iron oxide nanoparticles (ION) are highly sensitive probes for magnetic resonance imaging (MRI) that have previously been used for in vivo cell tracking and have enabled implementation of several diagnostic tools to detect and monitor disease. However, the in vivo MRI signal of ION can overlap with the signal from endogenous iron, resulting in a lack of detection specificity. Therefore, the long-term fate of administered ION remains largely unknown, and possible tissue deposition of iron cannot be assessed with established methods. Herein, we combine nonradioactive 57Fe-ION MRI with ex vivo laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging, enabling unambiguous differentiation between endogenous iron (56Fe) and iron originating from applied ION in mice. We establish 57Fe-ION as an in vivo MRI sensor for cell tracking in a mouse model of subcutaneous inflammation and for assessing the long-term fate of 57Fe-ION. Our approach resolves the lack of detection specificity in ION imaging by unambiguously recording a 57Fe signature.

Tumor development and metastasis are dependent on tumor infiltrating immune cells which form a characteristic tumor microenvironment (TME). Activated monocytes secrete the protein heterodimer S100A8/A9 promoting TME formation. Monocyte-dependent proteases facilitate local tumor cell invasion by degradation of the extracellular matrix. We aimed for target specific in vivo imaging of S100A8 and proteases to provide differentiating biomarkers for local tumor growth and metastatic potential. Murine breast cancer cells of the 4T1 model with graduated metastatic potential (4T1 and 4T07: both hematogenous metastasis > 168FAR: lymph-node metastasis > 67NR: no metastasis) were orthotopically implanted into female BALB/c mice. At 4 mm size, tumors were investigated by injecting the protease-specific probe ProSense 750EX (PerkinElmer, 4T1 n = 7, 4T07 n = 10, 168FAR n = 16, 67NR n = 15) and anti-S100A8-Cy5.5 (n = 6 each) and performing fluorescence reflectance imaging at 0 and 24 h after injection. In vivo imaging was validated with immunohistochemistry. At 24 h, S100A8-specific signals in 4T1 and 4T07 were significantly higher (1714.05/1683.45 AU) as compared to 168FAR and 67NR (174.85/167.95 AU, p = 0.0012/p = 0.0003), reflecting the capability of hematogenous spread. Protease-specific signals were significantly higher in 4T1 and 4T07 (348.01/409.93 AU) as compared to 168FAR (214.91 AU) and 67NR (129.78 AU p

Background To facilitate onsite decision-making during endoscopy, both accurate detection and in vivo characterization of preneoplasia are prerequisites. However, no endoscopy technique is available that meets both demands satisfactorily. We evaluated endothelin-receptor A (ETAR)-guided fluorescence endoscopy (FE) in vivo and fluorescence reflectance imaging (FRI) ex vivo for detection and characterization of early dysplastic colitis-associated colonic lesions. Methods Colorectal cancerogenesis was investigated in the inflammatory driven AOM-DSS model and spontaneous adenoma development in ApcMin mice. A Cy5.5-labeled nonpeptidic ETAR-specific imaging probe was injected intravenously to assess tumor development in vivo by white light endoscopy (WLE) and FE. Ex vivo tumors were evaluated by FRI, histological examination, and western blot analysis. In addition, tissue samples from patients with colitis-associated malignant and nonmalignant mucosal alterations were analyzed. Specificity experiments were performed using an unspecific Cy3.5-glycine tracer. Results Overall, 62 adenomas were observed. FE was able to detect and quantify ETAR expression targeting the ETAR-specific photoprobe. A significantly higher fluorescent contrast was detected in colonic adenomas compared to adjacent nonmalignant mucosa by FE (64.3 ± 7.9 vs. 56.6. ± 7.0; P < 0.001). These results were confirmed by FRI examination, immunochemistry, and western blot analysis. Additionally, ETAR expression in samples from human patients with colitis-associated cancer was highly elevated compared to nonmalignant alterations. Specificity experiments indicated a high binding-specificity of the applied ETAR photoprobe (1.4 ± 0.3 vs. 2.5 ± 0.7; P < 0.001). Conclusions We introduced ETAR guided FE in mice for successful in vivo detection and characterization of colorectal neoplasia on a molecular level.

Integrins are the main cell adhesion mediators on the cell surface. Especially integrin αvβ3 has gained a lot of attention as a target in cancer therapy because it mediates diverse angiogenic processes during tumor development. The peptide sequence Arg-Gly-Asp (RGD), which is present in a number of endogenous integrin ligands like fibronectin, vitronectin, and related proteins of the extracellular matrix (ECM), has been extensively used as a targeting vector for therapeutic as well as diagnostic purposes, and cilengitide, a cyclic RGD peptide, has entered clinical trials for the treatment of various cancers. However, recent advancements utilizing isoDGR, a sequence that was found in aged fibronectin, already show that RGD-based targeting is not the end of the line. Novel developments and a closer investigation of the binding mode of these peptides now show promising results for the future use of such compounds.

Iron oxide nanoparticles (ION) are highly sensitive probes for magnetic resonance imaging (MRI) that have previously been used for in vivo cell tracking and have enabled implementation of several diagnostic tools to detect and monitor disease. However, the in vivo MRI signal of ION can overlap with the signal from endogenous iron, resulting in a lack of detection specificity. Therefore, the long-term fate of administered ION remains largely unknown, and possible tissue deposition of iron cannot be assessed with established methods. Herein, we combine nonradioactive

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.

SummaryOptical imaging has long been considered a method for histological or microscopic investigations. Over the last 15 years, however, this method was applied for preclinical molecular imaging and, just recently, was also able to show its principal potential for clinical applications (e.g. fluorescence-guided surgery). Reviewing the development and preclinical evaluation of new fluorescent dyes and target-specific dye conjugates, these often show characteristic patterns of their routes of excretion and biodistribution, which could also be interesting for the development and optimization of radiopharmaceuticals. Especially ionic charges show a great influence on biodistribution and netcharge and charge-distribution on a conjugate often determines unspecific binding or background signals in liver, kidney or intestine, and other organs.Learning from fluorescent probe behaviour in vivo and translating this knowledge to radio-pharmaceuticals might be useful to further optimize emerging and existing radiopharmaceuticals with respect to their biodistribution and thereby availability for binding to their targets.

Integrins are heterodimeric transmembrane protein receptors consisting of different α and β subunits. α(v)β(3) integrins are overexpressed on many tumor cells and tumor-associated angiogenic vessels, whereas α(IIb)β(3) is a receptor for, e.g., fibrinogen and mediates platelet aggregation. In this study, a near-infrared fluorescent imaging probe has been designed and synthesized by conjugating fluorescent dyes to a non-peptidic, pharmacophore-based ligand, based on a molecular modeling design approach. Affinity values were determined, and in vitro cell binding assays and preliminary in vivo xenograft studies in nude mice were performed to evaluate target binding. Competition assays revealed excellent binding and selectivity to α(v)β(3) compared to that for α(IIb)β(3). In vitro, the probe showed high target binding on α(v)β(3)-positive M-21 cells and negligible binding to α(v)β(3)-negative MCF-7 cells. In vivo, the tracer is able to image target expression in U-87 xenografts with a maximum signal-to-noise ratio (SNR) of 2.5:1 at 24 h after injection.

Small molecular imaging probes are often found to be rapidly cleared from the circulation. In order to improve signal to noise ratio (SNR) by high probe accumulation in the target tissue we intended to prolong the presence of the probes in the circulation by exploiting inherent transport mechanisms. Human serum albumin (HSA) is playing an increasingly important role as a drug carrier in clinical settings and drugs directly bound to albumin or attached to albumin binding moieties have been successfully developed for treatment approaches. To optimize the bioavailability of existing fluorescent probes, a hydrophobic affinity tag is installed, which enhances albumin binding. In a first experiment an endothelin-A receptor (ETAR) probe is modified by inserting a trivalent linker, attaching an albumin affinity tag and labeling the conjugate with the fluorescent dye Cy 5.5. The spectroscopic properties of the conjugate are examined by photometer- and fluorometer measurements in comparison to a probe without albumin binding tag. Albumin binding was proven by agarose gel electrophoresis. The affinity towards ETAR was confirmed in vitro by cell binding assays on human fibrosarcoma cells (HT-1080) and in vivo by murine xenograft imaging studies. In vitro, the modified probe retains high target binding in the absence and presence of albumin. Binding could be blocked by predosing with ETAR antagonist atrasentan, proving specificity. The in vivo examinations in comparison to the established probe showed a reduced renal elimination and a prolonged circulation of the tracer resulting in significantly higher signal intensity (SI) at the target and a higher signal-to-noise ratio (SNR) between 3h and 96 h after injection. In summary, we designed a small molecular, non-peptidic fluorescent probe which targets ETAR and reversibly binds to serum albumins. The reversible binding to albumin enhances the biological half-life of the probe substantially and enables near infrared optical imaging of subcutaneous tumors for several days. This approach of reversibly attaching probes to serum albumin may serve as a tool to optimize tracer distribution for more precise target characterization in molecular imaging experiments.

We report here the synthesis of a nonpeptide, small-molecule fluorescent imaging agent with high affinity to aminopeptidase N (APN/CD13), a key player in a variety of pathophysiological angiogenic processes. On the basis of a recently described lead structure, we synthesized three putative precursor compounds by introducing polyethylene glycol (PEG) spacers comprising amino groups for dye labeling. Different attachment sites resulted in substantial differences in target affinity, cell toxicity, and target imaging performance. In comparison to bestatin, a natural inhibitor of many aminopeptidases, two of our compounds (22, 23) exhibit comparable inhibition potency, while a third (21) does not show any inhibiting effect. Cell binding assays with APN-positive BT-549 and APN-negative BT-20 cells and the final fluorescent probes Cy 5.5-21 and Cy 5.5-23 confirm these findings. The favorable characteristics of Cy 5.5-23 will now be proven in in vivo experiments with murine models of high APN expression and may serve as a tool to better understand APN pathophysiology.

Endothelin (ET) receptor dysregulation has been described in a number of pathophysiological processes, including cardiovascular disorders, renal failure, and cancer. The aim of this study was to evaluate the expression of the ET-A receptor (ETAR) in murine models of thyroid carcinoma using optical imaging methods. A recently developed near-infrared fluorescent tracer was first assessed in isolated artery preparations for its functional performance in comparison with known ETAR antagonists BQ123 and PD156707. Before evaluation of the tracer in vivo, different thyroid carcinoma cell lines were characterized with respect to their ET receptor expression by RT-PCR and autoradiography. In vivo, sc and orthotopic papillary thyroid tumor xenografts were clearly visualized by fluorescence reflectance imaging and fluorescence-mediated tomography up to 48 h after injection of the tracer. Binding specificity of the probe was demonstrated by predosing with PD156707 as a competing inhibitor. In conclusion, optical imaging with a fluorescent ETAR tracer allows the noninvasive imaging of tumor-associated ETAR expression in vivo. In the future, this technique may help surgeons to evaluate lesion dimensions in intraoperative settings (e.g. thyroidectomy). (Endocrinology 153: 2907-2918, 2012)

(19) F MRI offers high specificity but usually low sensitivity. Here, paramagnetic relaxation enhancement is assessed as a method to improve SNR efficiency in (19) F MRI. Compounds with short relaxation times are used that combine fluorine and a paramagnetic ion within the same molecule. Different molecular designs provide T1 values in the range of 1.4-15 ms and T2*/T1 ratios from 0.3 to 1. Gradient echo, as well as ultrafast radial MR sequences, is optimized to achieve highest SNR efficiency. Compared to nonparamagnetic compounds, ultrafast sequences can yield a gain of up to a factor 27 in sensitivity, whereas the gain with gradient echo is only factor 11. Comparison among the paramagnetic compounds shows that T2*/T1 close to unity is a prerequisite for highest SNR efficiency gain and that best results are obtained for compounds with T1 in the range of 1-5 ms.

The process of angiogenesis, an essential hallmark for tumour development as well as for several inflammatory diseases and physiological phenomena, is of growing interest for diagnosis and therapy in oncology. In the context of biochemical characterisation of key molecules involved in angiogenesis, several targets for imaging and therapy could be identified in the last decade. Optical imaging (OI) relies on the visualisation of near infrared (NIR) light, either its absorption and scattering in tissue (non-enhanced OI) or using fluorescent contrast agents. OI offers excellent signal to noise ratios due to virtually absent background fluorescence in the NIR range and is thus a versatile tool to image specific molecular target structures in vivo. This work intends to provide a survey of the different approaches to imaging of angiogenesis using OI methods in preclinical research as well as first clinical trials. Different imaging modalities as well as various optical contrast agents are briefly discussed.

The endothelin (ET) axis plays a major role in cardiovascular diseases and a number of human cancers. This review summarizes the work that has been published in the past ten years using labeled endothelin receptor ligands for the visualization of endothelin receptor expression in vivo.

The expression and function of endothelin (ET) receptors is abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. In this study, we prepared two [(18)F]-fluorinated derivatives of the non-peptide ET(A) receptor antagonist PD 156707 and evaluated their ET receptor binding potencies. Ex vivo as well as in vivo biodistribution studies in mice were performed, as well as the metabolism of the radiotracer, which was examined by metabolite analysis in mice and rats. All tested derivatives of PD 156707 exhibited potent in vitro pharmacological characteristics with K(i) values comparable to that of the lead compound. The biodistribution studies showed a high accumulation of the tracer in bile and intestine. In vivo we were able to show that the visualization of the heart as a major target organ with high ET(A)R expression is possible.

Purpose Optical imaging would be desirable for cancer diagnostics since it can potentially resolve relevant oncological target structures in vivo. We therefore synthesised an αvβ3 targeted fluorochrome and imaged tumour xenografts with different αvβ3 expression levels using both planar and tomographic optical imaging methods.

Caspases are the unique enzymes responsible for the execution of the cell death program and may represent an exclusive target for the specific molecular imaging of apoptosis in vivo. 5-Pyrrolidinylsulfonyl isatins represent potent nonpeptidyl caspase inhibitors that may be suitable for the development of caspase binding radioligands (CBRs). (S)-5-[1-(2-Methoxymethylpyrrolidinyl)sulfonyl]isatin (7) served as a lead compound for modification of its N-1-position. Corresponding pairs of N-1-substituted 2-methoxymethyl- and 2-phenoxymethylpyrrolidinyl derivatives were examined in vitro by biochemical caspase inhibition assays. All target compounds possess high in vitro caspase inhibition potencies in the nanomolar to subnanomolar range for caspase-3 (Ki=0.2-56.1 nM). As shown for compound (S)-1-(4-(2-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (35), the class of N-1-substituted 5-pyrrolidinylsulfonyl isatins competitively inhibits caspase-3. All caspase inhibitors show selectivity for the effector caspases-3 and -7 in vitro. The 2-methoxymethylpyrrolidinyl versions of the isatins appear to possess superior caspase inhibition potencies in cellular apoptosis inhibition assays compared with the 2-phenoxymethylpyrrolidinyl inhibitors.

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent secreted or membrane anchored endopeptidases. MMPs are involved in many physiological processes but also take part in the pathophysiological mechanisms responsible for a wide range of diseases. Pathological expression and activation of MMPs are associated with cancer, atherosclerosis, stroke, arthritis, periodontal disease, multiple sclerosis and liver fibrosis. Thus, noninvasive visualisation and quantification of MMP activity in vivo are of great interest in basic research and clinical application. This can be achieved by scintigraphic molecular imaging techniques such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) provided suitable radiolabelled tracers exist, e.g. radioactive inhibitors of matrix metalloproteinases (MMPIs). The approach to monitor MMP activity in vivo using radiolabelled small molecule inhibitors suitable for SPECT and PET is summarised in this review. Briefly, latest advances in scintigraphic imaging are introduced and followed by a report about the enzyme class of MMPs. The involvement of MMPs in cancer and atherosclerosis is exemplified and small molecule MMPIs are classified. Subsequently, the development of radiolabelled small molecule MMPIs, their synthesis and in vitro and in vivo evaluation is reviewed. Finally, an outlook on the clinical potential of labelled MMPIs in diagnostic algorithms is given.

It is assumed that the regulation of cardiac endothelin (ET) receptor density is abnormal in heart diseases. From that perspective, an ET receptor radioligand is needed to assess ET receptor density in vivo. The nonpeptidyl ETA receptor antagonist PD 169390 was labelled with radioiodine to give a putative radioligand for SPECT. Labelling with [125I]iodide and [123I]iodide was accomplished with good to excellent radiochemical yields. The affinities of the nonradioactive reference and those of selected precursor compounds for ETA receptors were determined, using [125I]iodine labelled endothelin-1 with mouse ventricular membranes. All employed substances exhibited potent in vitro pharmacological characteristics with Ki values comparable to that of the lead compound PD 156707. Biodistribution studies and scintigraphic imaging experiments in mice, however, showed no significant uptake of the [123I] derivative in the heart.

To date, some non-selective β-adrenoceptor (β-AR) positron emission tomography (PET) radioligands are in clinical use, but no PET radioligand for the selective imaging of cardiac β1-ARs is clinically available. Therefore, the aim of this study was to develop a potential high-affinity PET radioligand for the β1-subtype of ARs. Here, the synthesis and in vitro evaluation of (S)- and (R)-N-[2-[3-(2-cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N′-[4-(2-fluoro-ethoxy)-phenyl]-urea (8a–b), derivatives of the well-characterized β1-AR selective antagonist, ICI 89,406, are described. The (S)-isomer 8a shows both higher β1-AR selectivity and β1-AR affinity than the (R)-enantiomer 8b (selectivity: 40 800 vs 1580; affinity: KI1=0.049 nM vsKI1=0.297 nM). Therefore, the 18F-labelled analogue 8e of compound 8a was synthesized. While the direct nucleophilic 18F-fluorination of the tosylate precursor 8d produced 8e in low radiochemical yields (⩽2.9% decay-corrected) and specific activities (⩽3.5 GBq/µmol at the end of synthesis (EOS), n=9) the alternative two-step synthesis of 8e from ethylene glycol di-p-tosylate 9, [18F]fluoride ion and phenol precursor 8f gave satisfying results (16.4±3.2% radiochemical yield (decay-corrected), 99.7±0.5% radiochemical purity, 40±8 GBq/µmol specific activity at the EOS within 174±3 min from the end of bombardment (EOB) (n=5)). Copyright © 2006 John Wiley & Sons, Ltd.

Recently, the spectrum of molecular imaging devices such as positron emission tomography (PET) was further expanded by the now clinically available combined imaging modalities such as PET-CT and the preclinically used small animal PET scanners. These are powerful tools that can bridge the gap between preclinical and clinical evaluation studies of new radiotracers for molecular imaging of healthy and diseased states in vivo. The beta-adrenoceptor (beta-AR) radioligands discussed in this review represent a class of molecular probes for the non-invasive in vivo assessment of beta-AR density eg. in the heart with PET. The beta-AR radioligands (S)-[11C]CGP 12177 (1) or (S)-[11C]CGP 12388 (2) are currently investigated in clinical studies with PET. Additionally, subtype-selective beta1-AR radioligands are used in preclinical research which show potential for the diagnostics of the "beta1-AR organ" as such the heart can be defined. Non-invasive quantification of beta-ARs could facilitate the accurate choice and control of therapeutic interventions. Here we summarize the state-of-the-art of the radiochemistry of radioactive beta-AR radioligands.

Studies have demonstrated a positive correlation between inflammation, metastasis, or atherosclerosis and the unbalanced or culminated expression of matrix metalloproteinases (MMPs). The molecular imaging of locally upregulated MMP activity in vivo is a clinical challenge. Actually, radioligands based on nonpeptidyl MMP inhibitors (MMPIs) are currently in development as putative radiopharmaceutical agents for the noninvasive in vivo assessment of activated MMPs. Nonpeptidyl MMPIs bind to the zinc active site of the activated enzyme via mono- (e.g. carboxylate) or bidentate (e.g. hydroxamate) complexation thereby exhibiting a broad-spectrum MMP binding potency. Thus, these mentioned endopeptidase inhibitors should be useable lead compounds for the redevelopment as diagnostic MMPI radiotracers. Recently, the non-hydroxamate C-5-disubstituted pyrimidine-2,4,6-triones were disclosed as subgroup-selective MMP inhibitors. We here describe a set of fine-tuned barbiturates as a new class of MMPI radiotracers for the noninvasive in vivo visualization of activated MMPs using scintigraphic techniques such as SPECT or PET.

Molecular imaging and quantification of myocardial β1-adrenoceptor (AR) rather than total β-AR density is of great clinical interest since cardiac biopsy studies suggest that myocardial β1-AR density is reduced in patients with chronic heart failure whereas cardiac β2-AR density may vary. Positron emission tomography (PET), with appropriate radioligands, offers the possibility to assess β-AR density non-invasively in humans. However, no PET radioligand for the selective imaging of cardiac β1-ARs is clinically available. Here some derivatives of the well characterized β1-AR selective antagonist, ICI 89,406, namely the enantiomers of N-[2-[3-(2-cyano-phenoxy)-2-hydroxy-propylamino]-ethyl]-N′-(4-hydroxy-phenyl)-urea (5a and 5b) were synthesized and evaluated in vitro. The (R)-isomer 5a was more β1-selective but has lower affinity than its (S)-enantiomer 5b (β1-AR selectivity: 6100 vs 1240; β1-affinity: K1 = 0.288 nM vs K1 = 0.067 nM). Etherification of the analogous desmethyl precursors, 5e and 5f, respectively, with [11C]iodomethane gave 11C-labelled versions of 5a and 5b, namely 5g and 5h, in 44 ± 5% radiochemical yield (decay-corrected) and 97.4 ± 1.3% radiochemical purity with specific radioactivities of 26.4 ± 9.4 GBq/µmol within 41.2 ± 3.4 min from the end of bombardment (n = 14). 5g and 5h are now being evaluated as candidate radioligands for myocardial β1-ARs. Copyright © 2005 John Wiley & Sons, Ltd.

(Butadiene)zirconocene (s-trans-/s-cis-1 equilibrium mixture) reacts with the imine MeN=CHPh (7a) to form the 1:1 carbon-coupling product [κN-MeN−CHPh−CH2−η3-(CHCHCH2)]ZrCp2 (8a). The X-ray crystal structure analysis of 8a revealed a metallacyclic structure featuring a distorted π-allyl moiety and a planar-tricoordinate nitrogen atom. Only one of the two possible diastereoisomers is found in the crystal (cis-8a) as well as in solution. Treatment of 1 with the more bulky aldimine PhCH2N=CHPh (7b) selectively gave the corresponding 1:1 addition product trans-8b. Ketimines PhCH2N=C(CH2)4 (7c), HN=CPh2 (7d), or Δ1-2-methylpyrroline (7e) also reacted selectively with (butadiene)zirconocene to yield the analogous metallacyclic C−C coupling products 8c−e. Treatment of 8e with B(C6F5)3 proceeded cleanly by abstraction of a hydride anion from the carbon atom α to the nitrogen atom to form the intramolecularly coordinated imine product 10a {with the [HB(C6F5)3−] anion}. Similarly, Ph3C+ abstracts a hydride ion from 8e to yield 10b {isolated with the [B(C6H5)4−] anion}. Proton addition from HNR3+ reagents takes place selectively at the Zr−amido nitrogen atom of the complexes 8a or 8d to yield the substituted (π-allyl)zirconocene cation complexes 11 or 12, respectively, The cationic complexes 10, 11, and 12 polymerize ethene at room temperature/2 bar of ethene with moderate catalytic activities. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

To explore the feasibility of tracking thulium (Tm)-1,4,7,10-tetraazacyclododecane-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraacetic acid (DOTMA)-labeled cells in vivo by means of highly shifted proton magnetic resonance (MR) imaging as a potential alternative to established cell-tracking methods.All animal experiments were approved by the local ethics committee for animal experiments. Highly shifted proton MR imaging is based on the principle that the shifted resonances on Tm and dysprosium (Dy)-DOTMA can be detected separately from the tissue water signal at MR imaging with very short echo time and radial center-out readout (UTE, or "ultrashort echo time"). MR imaging of aqueous solutions and in mice in vivo was performed at 9.4 T. Human fibrosarcoma cells (HT-1080) and murine macrophages were labeled with different amounts of Tm-DOTMA. Labeled fibrosarcoma cells were injected subcutaneously into three mice. For cell tracking, labeled macrophages were administered intravenously into eight mice bearing local granulomatous inflammation. Three-dimensional UTE MR imaging was performed during 1 week. Macrophage viability and activity and fibrosarcoma cell viability were statistically analyzed by performing an unpaired two-tailed t test for labeled versus unlabeled cells by using data of at least six independent experiments.The strongly shifted MR lines of Tm- and Dy-DOTMA can be separated from the tissue water signal and from each other. A detection limit of about 25 µmol/L of Tm-DOTMA was calculated from in vitro MR measurements. A mean ± standard error of the mean intracellular uptake of (4.19 ± 0.88) × 10(9) (HT-1080) and (10.1 ± 3.0) × 10(10) (macrophages) of Tm-DOTMA molecules per cell was achieved. In vivo, Tm-DOTMA signal was detectable for 1 week in both tumors and macrophages, with a detection limit of approximately 10(4) HT-1080 and 600 macrophages. Histologic examination results and elemental bioimaging confirmed labeled cells as source of MR signal.Strongly shifted proton three-dimensional UTE MR imaging of Tm-DOTMA-labeled cells is a highly specific and sensitive tool for in vivo cell tracking.

tTF-NGR consists of the extracellular domain of tissue factor and the peptide GNGRAHA, a ligand of the surface protein aminopeptidase N and of integrin αvβ3. Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR shows antitumor activity in xenografts and inhibition of tumor blood flow in cancer patients. We performed random TMS(PEG)12 PEGylation of tTF-NGR to improve the antitumor profile of the molecule. PEGylation resulted in an approximately 2-log step decreased procoagulatory activity of the molecule. Pharmacokinetic studies in mice showed a more than 1-log step higher mean area under the curve. Comparison of the LD10 values for both compounds and their lowest effective antitumor dose against human tumor xenografts showed an improved therapeutic range (active/toxic dose in mg/kg body weight) of 1/5 mg/kg for tTF-NGR and 3/>160 mg/kg for TMS(PEG)12 tTF-NGR. Results demonstrate that PEGylation can significantly improve the therapeutic range of tTF-NGR.

Truncated tissue factor (tTF) retargeted to tumor vasculature by C-terminal GNGRAHA peptide (tTF-NGR) is able to induce tumor vessel infarction. Here, we describe the identification of the optimum therapeutic regimen for the use of tTF-NGR (or a random-PEGylated derivative of tTF-NGR) in combination with the chemotherapeutic agent doxorubicin in vitro and in vivo. First, we treated human HT1080 xenografts subcutaneously transplanted in athymic CD-1 mice with doxorubicin (5 mg/kg body weight (bw), i.v.) and excised the tumors at different times after injection. Fluorescence spectrometric analyses of the autofluorescence of doxorubicin revealed that intratumoral maximum of doxorubicin accumulation was reached approximately 6 hours after injection. While tTF-NGR given before doxorubicin inhibits the uptake of the latter into HT1080 human fibrosarcoma xenografts in vivo, the reverse sequence does not influence the uptake of doxorubicin in tumor tissue, but significantly inhibits the late wash-out phase entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with various concentrations of doxorubicin in vitro increases concentrations of phosphatidylserine (PS) in the outer layer of the lipid bilayer induced by pro-apoptotic stress. In Factor X activation kinetics, both HUVEC and HT1080 cells showed a significantly higher procoagulatory efficacy when used with equimolar concentrations of tTF-NGR upon doxorubicin incubation and higher PS externalization than identical numbers of control cells with low PS externalization. For proof of a causal relation between doxorubicin-induced PS externalization and procoagulant behaviour of the cells, we could show that preincubation with an phosphatidylserine-inhibiting antibody completely abolished this elevated procoagulatory state of the cells. Thus, we hypothesized that both drugs by the reciprocal mechanisms described mutually increase their therapeutic anti-tumor efficacy in vivo. Therapeutic experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in CD1 nude mice revealed a significantly increased tumor growth inhibition applying suboptimal doses of doxorubicin and tTF-NGR in sequence over single drug treatment. In conclusion, combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by a new mechanism, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in endothelial and tumor cells. Citation Format: Janine Stucke-Ring, Julian Timo Ronnacker, Caroline Brand, Verena Mantke, Carsten Höltke, Christoph Schliemann, Torsten Kessler, Rolf M. Mesters, Christian Schwöppe, Wolfgang E. Berdel. Intratumoral accumulation/entrapment of doxorubicin by tumor vascular infarction with retargeted tissue factor tTF-NGR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-223.

(19) F MRI offers high specificity but usually low sensitivity. Here, paramagnetic relaxation enhancement is assessed as a method to improve SNR efficiency in (19) F MRI. Compounds with short relaxation times are used that combine fluorine and a paramagnetic ion within the same molecule. Different molecular designs provide T1 values in the range of 1.4-15 ms and T2*/T1 ratios from 0.3 to 1. Gradient echo, as well as ultrafast radial MR sequences, is optimized to achieve highest SNR efficiency. Compared to nonparamagnetic compounds, ultrafast sequences can yield a gain of up to a factor 27 in sensitivity, whereas the gain with gradient echo is only factor 11. Comparison among the paramagnetic compounds shows that T2*/T1 close to unity is a prerequisite for highest SNR efficiency gain and that best results are obtained for compounds with T1 in the range of 1-5 ms.

The use of light-driven imaging techniques for the diagnosis of breast cancer dates back to as early as 1929. Transillumination was evaluated for potential differentiation of breast lesions, e.g. to distinguish benign cysts from solid tumours. Cutler summed the technique to be “...a simple procedure and a valuable aid in the interpretation of pathological conditions in the mammary gland. Its use is recommended in the routine examination of the breast...”(Cutler, 1929, 1931).

The expression and function of endothelin (ET) receptors are abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. A previously reported promising radioligand for positron emission tomography (PET) based on the non-peptide ET(A) receptor antagonist PD 156707 showed specific binding to target receptors in the myocardium but high accumulation in bile and intestine, probably because of its high lipophilicity. In this study we describe the synthesis of a series of fluorinated derivatives with hydrophilic building blocks. All compounds were evaluated as high affinity ET(A) receptor ligands (16, 17, 23-26, K(i) = 1.4-7.9 nM) with high subtype selectivity over the ET(B) receptor. [(18)F]3-Benzo[1,3]dioxol-5-yl-4-{3-[1-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethyl)-1H-[1,2,3]triazol-4-ylmethoxy]-4,5-dimethoxybenzyl}-5-hydroxy-5-(4-methoxyphenyl)-5H-furan-2-one ([(18)F]17) was synthesized as one of the radioligands of this series that possesses a higher hydrophilicity and an excellent stability in human serum. Improved clearance properties and specific uptake in target organs have been confirmed by biodistribution studies and small animal PET imaging.

Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent endopeptidases. Representing a subfamily of the metzincin superfamily, MMPs are involved in the proteolytic degradation of components of the extracellular matrix. Unregulated MMP expression, MMP dysregulation and locally increased MMP activity are common features of various diseases, such as cancer, atherosclerosis, stroke, arthritis, and others. Therefore, activated MMPs are suitable biological targets for the specific visualization of such pathologies, in particular by using radiolabeled MMP inhibitors (MMPIs). The aim of this work was to develop a radiofluorinated molecular probe for noninvasive in vivo imaging for the detection of up-regulated levels of activated MMPs in the living organism. Fluorinated MMPIs (26, 31 and 38) based on the pyrimidine-2,4,6-trione lead structure RO 28-2653 (1) were synthesized, and their MMP inhibition potency was evaluated in vitro. The radiosynthesis and the in vivo biodistribution of the first (18)F-labeled prototype, MMP-targeted tracer [(18)F]26, suitable for molecular imaging by means of positron emission tomography (PET) were realized.

The assessment of matrix metalloproteinase (MMP) activity in vivo is highly desirable in various human diseases such as cancers. Hydroxamic acids based on CGS27023A or CGS25966 are nonpeptidyl lead structures that specifically target activated MMPs in vivo. The aim of this study was the modification and fluorescent labeling of these lead structures to develop a highly affine, nonpeptide MMP inhibitor (MMPI)-ligand for molecular optical imaging of activated MMPs. An 11 step synthesis was developed involving a PEGylated benzyl derivative as a spacer to minimize the interactions between the activated MMP and the dye of conjugate 11 with an azide as a protected amino function. After reducing the azide (Staudinger reaction) and labeling with Cy5.5, we obtained a CGS-based MMP inhibitor 11 with a fluorescent signaling flag. To evaluate the biological properties of this photoprobe, three human cancer cell lines (A-673, HT-1080 and BT-20) were characterized with respect to their MMP-2 and -9 (gelatinases) expression levels (real-time PCR) and protein levels (Western blotting). Initially, fluorogenic inhibition assays were used to assess the MMP inhibition potential. The PEGylated CGS 10 showed complete inhibition of MMP-2 and MMP-9 activities in vitro both for purified MMP-2/-9 (active and pro-forms) and MMP-2/-9 containing cell culture supernatants. To test the imaging potential in biological tissues, gelatinase activity was measured on tumor cryostat sections of the above-mentioned tumor cells using FITC-labeled dye-quenched gelatin. Gelatinase positive tumors revealed strong binding of CGS-Cy5.5 11, while gelatinase negative tumors were not targeted. In conclusion, this new CGS-based MMP photoprobe has a high affinity for MMP-2 and -9 and is thus a promising candidate for sensitive imaging of MMP activity in various diseases in patients.

Macrophages play an important role during the development and progression of atherosclerotic plaques. alphavbeta3 integrins are highly expressed by macrophages; thus, targeting alphavbeta3 may allow targeting of culprit macrophage-loaded atherosclerotic lesions in vivo.An alphavbeta3-targeted Arg-Gly-Asp (RGD) peptide was labeled with the cyanine 5.5 (Cy 5.5) dye and applied to image atherosclerotic plaques in apolipoprotein E-deficient mice.The peptide-dye conjugate binds to alphavbeta3 integrin-positive RAW264.7 macrophages with high affinity. Competition experiments confirmed binding specificity of the probe. A significant fluorochrome accumulation in atherosclerotic plaques was demonstrated 24 h after injection by fluorescence reflectance imaging, which was blocked with high efficiency by competition with the unlabeled peptide. Conversely, the nonconjugated dye revealed only a minor fluorescence signal in the plaques. Fluorescence microscopy revealed colocalization of the probe with macrophages in the plaque of a mouse model for accelerated atherosclerosis, which was corroborated in human carotid artery specimens. In addition to macrophage-associated signals, binding of the probe to the neointima or elastica of the arteries was observed.RGD-Cy 5.5, combined with near-infrared optical imaging methods, allows the specific imaging of alphavbeta3-integrin expression on macrophages recruited to vascular lesions and may serve to estimate macrophage-bound inflammatory activity of atherosclerotic lesions.

The CGS 27023A derivative (R)-2-(N-((6-fluoropyridin-3-yl)methyl)-4-methoxyphenyl-sulphonamido)-N-hydroxy-3-methylbutanamide 1a was identified as a very potent matrix metalloproteinase inhibitor. Here, we describe a one-step radiosynthesis of the target compound [(18)F]1a. The syntheses of [(18)F]1a resulted in a radiochemical yield of 12.1+/-5.9% (decay-corrected), a radiochemical purity of 98.8+/-0.6%, and a specific activity of 39+/-27 GBq/micromol at the end of synthesis within 160+/-18 min from the end of radionuclide production (n=5). Initial small-animal PET studies in wild-type mice (C57/BL6) showed no unfavourable tissue accumulation of [(18)F]1a.