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A full understanding concerning the photophysical properties of a fluorescent label is crucial for a reliable and predictable performance in biolabelling applications. This holds true not only for the choice of a fluorophore in general, but also for the correct interpretation of data, considering the complexity of biological environments. In the frame of a case study involving inflammation imaging, we report the photophysical characterization of four fluorescent S100A9-targeting compounds in terms of UV–vis absorption and photoluminescence spectroscopy, fluorescence quantum yields (ΦF) and excited state lifetimes (τ) as well as the evaluation of the radiative and non-radiative rate constants (kr and knr, respectively). The probes were synthesized based on a 2-amino benzimidazole-based lead structure in combination with commercially available dyes, covering a broad color range from green (6-FAM) over orange (BODIPY-TMR) to red (BODIPY-TR) and near-infrared (Cy5.5) emission. The effect of conjugation with the targeting structure was addressed by comparison of the probes with their corresponding dye-azide precursors. Additionally, the 6-FAM and Cy5.5 probes were measured in the presence of murine S100A9 to determine whether protein binding influences their photophysical properties. An interesting rise in ΦF upon binding of 6-FAM-SST177 to murine S100A9 enabled the determination of its dissociation equilibrium constant, reaching up to KD = 324 nM. This result gives an outlook for potential applications of our compounds in S100A9 inflammation imaging and fluorescence assay developments. With respect to the other dyes, this study demonstrates how diverse microenvironmental factors can severely impair their performance while rendering them poor performers in biological media, showing that a preliminary photophysical screening is key to assess the suitability of a particular luminophore.

The tumor microenvironment is highly heterogeneous. For gliomas, the tumor-associated inflammatory response is pivotal to support growth and invasion. Factors of glioma growth, inflammation, and invasion, such as the translocator protein (TSPO) and matrix metalloproteinases (MMP), may serve as specific imaging biomarkers of the glioma microenvironment. In this study, noninvasive imaging by PET with [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) was used for the assessment of localization and quantification of the expression of TSPO and MMP. Imaging was performed in addition to established clinical imaging biomarker of active tumor volume ([18F]FET) in conjunction with MRI. We hypothesized that each imaging biomarker revealed distinct areas of the heterogeneous glioma tissue in a mouse model of human glioma. Tracers were found to be increased 1.4- to 1.7-fold, with [18F]FET showing the biggest volume as depicted by a thresholding-based, volumes of interest analysis. Tumor areas, which could not be detected by a single tracer and/or MRI parameter alone, were measured. Specific compartments of [18F]DPA-714 (14%) and [18F]BR-351 (11%) volumes along the tumor rim could be identified. [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) matched with histology. Glioma-associated microglia/macrophages (GAM) were identified as TSPO and MMP sources. Multitracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation (GAM, MMP). Moreover, this noninvasive technique enables characterization of the glioma microenvironment with respect to the disease-driving cellular compartments at the various disease stages. Cancer Res; 77(8); 1831–41. ©2017 AACR.

Gli36dEGFR cells were implanted. Thereafter, two imaging sequences were conducted: In sequence 1, T2w MRI and PET with [18F]DPA-714 was performed, while [18F]BR-351 PET and [18F]FET PET were conducted at day 13 and 14, respectively. In imaging sequence 2, T2w MRI and [18F]FET PET were conducted at day 12, followed by [18F]BR-351 and [18F]DPA-714 at day 13 and 14, respectively. After the last imaging experiment, mice were sacrificed and the brains immediately harvested for further analysis.

PET and CT images were co-registered using three CT-detectable, molecular sieve spheres (# 1-3) rinsed in radiotracer solution. After segmentation of spheres in the CT image (arrows), PET and CT images were superimposed by using the automated "Landmark tool" provided by the Vinci software. The "Magic tool" was applied for minor corrections of co-registrations. The resulting resliced PET image was saved for further processing. In a second step, MR and CT images were superimposed using the "Fusion- and Magic tool". For illustration MR is pseudocolored in red, while the CT was inverted in color (black). The CT contour was further superimposed to demonstrate co-registration accuracy. Selected bone structures were used as additional quality measures of image registration. The resliced CT image was saved with the new transformation matrix. In a last step, the before mentioned transformation of the CT (CTMR) was transferred to the PET, resulting in co-registration of PET/MR/CT. This workflow was repeated for every PET scan per mouse.

(A) T/B ratios, (B-D) tracer volumes, (E-J) areas of unique tracer uptake and overlapping regions. No significant effect on volumetric data was observed based on the order of imaging experiments.

Supplementary Figure Legends 1-5

(A) An elliptical VOI (yellow) was drawn in the striatum. (B) Another elliptical VOI (green) was placed over the affected hemisphere. (C) After application of a thresholding based on the control VOI. The same original VOIs (left and middle) were used for all animals for individual thresholding.

An exemplary subset of n=6 mice were randomly assessed for intra-rater reproducibility. (A-E) [18F]DPA-714 and [18F]FET images were on two consecutive days repeatedly co-registered. Volumetry was performed twice (coreg 2 and coreg 3) and compared to the data reported in the main text (coreg 1). Dotted lines represent the overall mean for all n=20 animals reported in the main text. No significant changes for all parameters, except for the "percentage of [18F]DPA-714 only" between coreg 1 and 3, was found over the three different time points of co-registration.

Introduction Dysregulated activity of matrix metalloproteinases (MMPs) drives a variety of pathophysiological conditions. Non-invasive imaging of MMP activity in vivo promises diagnostic and prognostic value. However, current targeting strategies by small molecules are typically limited with respect to the bioavailability of the labeled MMP binders in vivo. To this end, we here introduce and compare three chemical modifications of a recently developed barbiturate-based radiotracer with respect to bioavailability and potential to image MMP activity in vivo. Methods Barbiturate-based MMP inhibitors with an identical targeting unit but varying hydrophilicity were synthesized, labeled with technetium-99m, and evaluated in vitro and in vivo. Biodistribution and radiotracer elimination were determined in C57/BL6 mice by serial SPECT imaging. MMP activity was imaged in a MMP-positive subcutaneous xenograft model of human K1 papillary thyroid tumors. In vivo data were validated by scintillation counting, autoradiography, and MMP immunohistochemistry. Results We prepared three new 99mTc‐labeled MMP inhibitors, bearing either a glycine ([99mTc]MEA39), lysine ([99mTc]MEA61), or the ligand HYNIC with the ionic co-ligand TPPTS ([99mTc]MEA223) yielding gradually increasing hydrophilicity. [99mTc]MEA39 and [99mTc]MEA61 were rapidly eliminated via hepatobiliary pathways. In contrast, [99mTc]MEA223 showed delayed in vivo clearance and primary renal elimination. In a thyroid tumor xenograft model, only [99mTc]MEA223 exhibited a high tumor-to-blood ratio that could easily be delineated in SPECT images. Conclusion Introduction of HYNIC/TPPTS into the barbiturate lead structure ([99mTc]MEA223) results in delayed renal elimination and allows non-invasive MMP imaging with high signal-to-noise ratios in a papillary thyroid tumor xenograft model.

Single site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound. To explore the effect of molecular editing, selectively modified oligosaccharides were preparedviaa convergent α-selective strategy. Incubation experiments in purified α-amylase and α-glucosidase, and in human and murine blood serum, provide insight into the influence of fluorine on the hydrolytic stability of these clinically important scaffolds. Enhancements ofca. 1 order of magnitude result from these subtle single point mutations. Modification at the monosaccharide furthest from the probable enzymatic cleavage termini leads to the greatest improvement in stability. In the case of α-amylase, docking studies revealed that retentive C2-fluorination at the reducing end inverts the orientation in which the substrate is bound. A co-crystal structure of human α-amylase revealed maltose units bound at the active-site. In view of the evolving popularity of C(sp3)-F bioisosteres in medicinal chemistry, and the importance of maltodextrins in bacterial imaging, this discovery begins to reconcile the information-rich nature of carbohydrates with their intrinsic hydrolytic vulnerabilities. © The Royal Society of Chemistry 2020. Western Washington University

Tyrosine kinase inhibitors have significantly improved treatment of patients with different malignancies, but their utilization can be compromised by unintended toxicities. Ibrutinib is a first-in-class covalent inhibitor of the Bruton´s tyrosine kinase inhibitor that has been approved for the treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and Waldenstrom's macroglobulinemia and is connected with toxicities, caused by high dosage. To minimize toxicities of ibrutinib we linked ibrutinib to a cell-targeted, internalizing antibody. To this end, we synthesized a poly-anionic derivate of ibrutinib, ibrutinib-Cy3.5 that retains full functionality. This anionic inhibitor is complexed by our anti-CD20-protamine targeting conjugate and free protamine and thereby spontaneously assembles into an electrostatically stabilized vesicular nanocarrier. The complexation led to an accumulation of the drug driven by the CD20 antigen internalization to the intended cells and an amplification of its physiological effectivity. In vivo , we observed a significant enrichment of the drug in xenograft lymphoma tumors in immune-compromised mice and a significantly better response to significantly lower doses compared to the original untargeted drug . This combination of a cell-determining antibody, electrostatically connected to a molecular targeted inhibitor defines a first-in-class antibody-inhibitor conjugate.

Purpose Primary angiitis of the central nervous system (PACNS) is a heterogeneous, rare, and poorly understood inflammatory disease. We aimed at non-invasive imaging of activated microglia/macrophages in patients with PACNS by PET-MRI targeting the translocator protein (TSPO) with [18F]DPA-714 to potentially assist differential diagnosis, therapy monitoring, and biopsy planning. Methods In total, nine patients with ischemic stroke and diagnosed or suspected PACNS underwent [18F]DPA-714-PET-MRI. Dynamic PET scanning was performed for 60 min after injection of 233 ± 19 MBq [18F]DPA-714, and MRI was simultaneously acquired. Results In two PACNS patients, [18F]DPA-714 uptake patterns exceeded MRI correlates of infarction, whereas uptake was confined to the infarct in four patients where initial suspicion of PACNS could not be confirmed. About three patients with PACNS or cerebral predominant lymphocytic vasculitis showed no or only faintly increased uptake. Short-term [18F]DPA-714-PET follow-up in a patient with PACNS showed reduced lesional [18F]DPA-714 uptake after anti-inflammatory treatment. Biopsy in the same patient pinpointed the source of tracer uptake to TSPO-expressing immune cells. Conclusions [18F]DPA-714-PET imaging may facilitate the diagnosis and treatment monitoring of PACNS. Further studies are needed to fully understand the potential of TSPO-PET in deciphering the heterogeneity of the disease.

Dysregulated activity of matrix metalloproteinases (MMPs) drives a variety of pathophysiological conditions. Non-invasive imaging of MMP activity in vivo promises diagnostic and prognostic value. However, current targeting strategies by small molecules are typically limited with respect to the bioavailability of the labeled MMP binders in vivo. To this end, we here introduce and compare three chemical modifications of a recently developed barbiturate-based radiotracer with respect to bioavailability and potential to image MMP activity in vivo.Barbiturate-based MMP inhibitors with an identical targeting unit but varying hydrophilicity were synthesized, labeled with technetium-99m, and evaluated in vitro and in vivo. Biodistribution and radiotracer elimination were determined in C57/BL6 mice by serial SPECT imaging. MMP activity was imaged in a MMP-positive subcutaneous xenograft model of human K1 papillary thyroid tumors. In vivo data were validated by scintillation counting, autoradiography, and MMP immunohistochemistry.We prepared three newIntroduction of HYNIC/TPPTS into the barbiturate lead structure ([

Single site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound. To explore the effect of molecular editing, selectively modified oligosaccharides were prepared via a convergent α-selective strategy. Incubation experiments in purified α-amylase and α-glucosidase, and in human and murine blood serum, provide insight into the influence of fluorine on the hydrolytic stability of these clinically important scaffolds. Enhancements of ca. 1 order of magnitude result from these subtle single point mutations. Modification at the monosaccharide furthest from the probable enzymatic cleavage termini leads to the greatest improvement in stability. In the case of α-amylase, docking studies revealed that retentive C2-fluorination at the reducing end inverts the orientation in which the substrate is bound. A co-crystal structure of human α-amylase revealed maltose units bound at the active-site. In view of the evolving popularity of C(sp3)–F bioisosteres in medicinal chemistry, and the importance of maltodextrins in bacterial imaging, this discovery begins to reconcile the information-rich nature of carbohydrates with their intrinsic hydrolytic vulnerabilities., Single site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound.

Matrix metalloproteinases (MMPs) are involved in a spectrum of physiological processes, rendering them attractive targets for small-molecule drug discovery. Strategies to achieve selective inhibition continue to be intensively pursued, facilitated by advances in structural biology. Herein, we harness MMPs 2, 8, 9, and 13 to validate the

Zinc(II)phthalocyanines (Zn(II)Pc) have shown promising applications in photodynamic therapy due to their high quantum yield of singlet oxygen generation; however, optimization of their overall properties are required before their clinical application as photosensitizers (PSs). The photosensitization efficiency of photoprobes is strongly influenced by the nature of the conjugated moieties and often it can be efficiently tuned by variation in the substitution pattern. Through this study we examined how the structural design of amphiphilic carbohydrate-based Zn(II)Pcs affects their photophysical properties, binding affinity to human serum albumin (HSA) and photodynamic activity against human cancer melanoma cells. The replacement of oxygen with sulfur at non-peripheral positions of low-symmetry Zn(II)Pcs contributes to the bathochromic shift of maximum absorption, which is relevant for the activation of the PS in deeper tissues. Moreover, this modification also influences the overall flexibility of the macrocyclic core and results in different behaviour towards HSA. Density functional theory calculations have been carried out to substantiate the effect of the peripheral environment on the photophysical characteristics and geometry of the molecules.

Herein, we present a comparative study about the photoinactivation of Staphylococcus aureus (Gram-positive model) and Escherichia coli (Gram-negative model) employing a neutral and a dicationic axially functionalized SiIV -phthalocyanine. Depending on the charge of the siloxane moiety (neutral monosaccharide or cationic ammonium salt), different interactions with the bacteria were observed, and a differential photoinactivation was facilitated. The intensity of the fluorescence labeling correlated with the photoinactivation of the two types of bacteria: While the neutral species only significantly affected the Gram-positive cells, we observed that the positively charged photosensitizer interacted both with the Gram-positive and with the Gram-negative models. The dicationic photosensitizer labeled both models with a characteristic deep-red fluorescence and photoinactivated both classes of prokaryotes. In general, our study clearly demonstrates that axially ammoniumsiloxane-functionalized Si(IV) phthalocyaninates constitute excellent photosensitizers due to their weak aggregation in aqueous environments. In particular, we also show that charge-based targeting with axial ammonium groups leads toward broad-spectrum SiIV -phthalocyanines for photodynamic inactivation of bacteria.

In this paper we establish a methodology to predict photoacoustic imaging capabilities from the structure of absorber molecules (sonochromes). The comparative in vitro and in vivo screening of naphthalocyanines and cyanine dyes has shown a substitution pattern dependent shift in photoacoustic excitation wavelength, with distal substitution producing the preferred maximum around 800 nm. Central ion change showed variable production of photoacoustic signals, as well as singlet oxygen photoproduction and fluorescence with the optimum for photoacoustic imaging being nickel(II). Our approach paves the way for the design, evaluation and realization of optimized sonochromes as photoacoustic contrast agents. Keywords: Naphthalocyanines, Spectroscopy

The tumor microenvironment is highly heterogeneous. For gliomas, the tumor-associated inflammatory response is pivotal to support growth and invasion. Factors of glioma growth, inflammation, and invasion, such as the translocator protein (TSPO) and matrix metalloproteinases (MMP), may serve as specific imaging biomarkers of the glioma microenvironment. In this study, noninvasive imaging by PET with [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) was used for the assessment of localization and quantification of the expression of TSPO and MMP. Imaging was performed in addition to established clinical imaging biomarker of active tumor volume ([18F]FET) in conjunction with MRI. We hypothesized that each imaging biomarker revealed distinct areas of the heterogeneous glioma tissue in a mouse model of human glioma. Tracers were found to be increased 1.4- to 1.7-fold, with [18F]FET showing the biggest volume as depicted by a thresholding-based, volumes of interest analysis. Tumor areas, which could not be detected by a single tracer and/or MRI parameter alone, were measured. Specific compartments of [18F]DPA-714 (14%) and [18F]BR-351 (11%) volumes along the tumor rim could be identified. [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) matched with histology. Glioma-associated microglia/macrophages (GAM) were identified as TSPO and MMP sources. Multitracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation (GAM, MMP). Moreover, this noninvasive technique enables characterization of the glioma microenvironment with respect to the disease-driving cellular compartments at the various disease stages. Cancer Res; 77(8); 1831–41. ©2017 AACR.

Purpose Primary angiitis of the central nervous system (PACNS) is a heterogeneous, rare, and poorly understood inflammatory disease. We aimed at non-invasive imaging of activated microglia/macrophages in patients with PACNS by PET-MRI targeting the translocator protein (TSPO) with [18F]DPA-714 to potentially assist differential diagnosis, therapy monitoring, and biopsy planning. Methods In total, nine patients with ischemic stroke and diagnosed or suspected PACNS underwent [18F]DPA-714-PET-MRI. Dynamic PET scanning was performed for 60 min after injection of 233 ± 19 MBq [18F]DPA-714, and MRI was simultaneously acquired. Results In two PACNS patients, [18F]DPA-714 uptake patterns exceeded MRI correlates of infarction, whereas uptake was confined to the infarct in four patients where initial suspicion of PACNS could not be confirmed. About three patients with PACNS or cerebral predominant lymphocytic vasculitis showed no or only faintly increased uptake. Short-term [18F]DPA-714-PET follow-up in a patient with PACNS showed reduced lesional [18F]DPA-714 uptake after anti-inflammatory treatment. Biopsy in the same patient pinpointed the source of tracer uptake to TSPO-expressing immune cells. Conclusions [18F]DPA-714-PET imaging may facilitate the diagnosis and treatment monitoring of PACNS. Further studies are needed to fully understand the potential of TSPO-PET in deciphering the heterogeneity of the disease. Electronic supplementary material The online version of this article (10.1007/s00259-019-04662-4) contains supplementary material, which is available to authorized users.

Advanced stage colorectal cancer (CRC) is still associated with limited prognosis. For preclinical evaluation of novel therapeutic approaches, murine models with orthotopic tumor growth and distant metastases are required. However, these models usually require surgical procedures possibly influencing tumor immunogenicity and development. The aim of this study was to establish a minimal-invasive endoscopy-based murine orthotopic model of metastatic CRC. During colonoscopy of CD-1 nude and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, implantation of Caco-2 and HT-29 CRC cells was performed subcutaneously (s.c.) or orthotopic into the colonic submucosa. White light endoscopy (WLE) and fluorescence endoscopy (FE) were applied for tumor detection in vivo. Ex vivo, resected tumors were examined by fluorescence reflectance imaging (FRI), histology, gelatin zymography and immunohistochemistry. In CD-1 nude mice, marked tumor growth was observed within 14 days after subcutaneous implantation while submucosal implantation failed to induce CRC after 17 weeks. In contrast, in NOD/SCID mice submucosal injection of HT-29 cells resulted in pronounced tumor growth 12 days post injectionem. Subsequently, rapid tumor expansion occurred, occupying the entire colonic circumference. Importantly, post mortem histological analyses confirmed liver metastases in 28.6 % and peritoneal metastases in 14.3 % of all mice. FRI and gelatin zymography did not detect a significantly increased matrix metalloproteinases (MMPs) expression in s.c. implanted tumors while MMP-tracer uptake was significantly enhanced in orthotopic implanted tumors. Neither s.c. nor orthotopic Caco-2 cell implantation resulted in tumor development. We successfully established an endoscopy-based model of metastatic CRC in immunodeficient mice.

Carbohydrate-conjugated silicon(IV) phthalocyanines with bimodal photoactivity were developed as probes with both fluorescent labeling and photosensitizing capabilities, and the concomitant fluorescent labeling and photoinduced inactivation of Gram-positive and Gram-negative models was explored. The maltohexaose-conjugated photoprobe provides a dual readout to distinguish between both groups of pathogens, as only the Gram-positive species was inactivated, even though both appeared labeled with near-infrared luminescence. Antibiotic resistance did not hinder the phototoxic effect, as even the methicillin-resistant pathogen Staphylococcus aureus (MRSA) was completely photoinactivated. Time-resolved confocal fluorescence microscopy analysis suggests that the photoprobe sticks onto the outer rim of the microorganisms, explaining the resistance of Gram-negative species on the basis of their membrane constitution. The mannose-conjugated photoprobe yields a different readout because it is able to label and to inactivate only the Gram-positive strain.

The synthesis and photophysical properties of a tailored Pt(II) complex are presented. The phosphorescence of its monomeric species in homogeneous solutions is quenched by interaction with the solvent and therefore absent even upon deoxygenation. However, aggregation-induced shielding from the environment and suppression of rotovibrational degrees of freedom trigger a phosphorescence turn-on that is not suppressed by molecular oxygen, despite possessing an excited-state lifetime ranging in the microsecond scale. Thus, the photoinduced production of reactive oxygen species is avoided by the suppression of diffusion-controlled Dexter-type energy transfer to triplet molecular oxygen. These aggregates emit with the characteristic green luminescence profile of monomeric complexes, indicating that Pt-Pt or excimeric interactions are negligible. Herein, we show that these aggregates can be used to label a model biomolecule (bovine serum albumin) with a microsecond-range luminescence. The protein stabilizes the aggregates, acting as a carrier in aqueous environments. Despite spectral overlaps, the green phosphorescence can be separated by time-gated detection from the dominant autofluorescence of the protein arising from a covalently bound green fluorophore that emits in the nanosecond range. Interestingly, the aggregates also acted as energy donors able to sensitize the emission of a fraction of the fluorophores bound to the protein. This resulted in a microsecond-range luminescence of the fluorescent acceptors and a shortening of the excited-state lifetime of the phosphorescent aggregates. The process that can be traced by a 1000-fold increase in the acceptor's lifetime mirrors the donor's triplet character. The implications for phosphorescence lifetime imaging are discussed. Fil: Delcanale, Pietro. Università di Parma; Italia Fil: Galstyan, Anzhela. Westfalische Wilhelms Universitat; Alemania Fil: Daniliuc, Constantin G.. Westfalische Wilhelms Universitat; Alemania Fil: Grecco, Hernan Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Abbruzzetti, Stefania. Università di Parma; Italia Fil: Faust, Andreas. Westfalische Wilhelms Universitat; Alemania Fil: Viappiani, Cristiano. Università di Parma; Italia Fil: Strassert, Cristian A.. Westfalische Wilhelms Universitat; Alemania

© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Diagnosis and localization of bacterial infections remains a significant clinical challenge. Harnessing bacteria-specific metabolic pathways, such as the maltodextrin transport mechanism, may allow specific localization and imaging of small or hidden colonies. This requires that the intrabacterial tracer accumulation provided by the transporter is matched by high serum stability of the tracer molecule. Herein, radiolabeled maltodextrins of varying chain lengths and with free nonreducing/reducing ends are reported and their behavior against starch-degrading enzymes in the blood, which compromise their serum stability, is evaluated. Successful single-photon emission computed tomography (SPECT) imaging is shown in a footpad infection model in vivo by using the newly developed model tracer, [99mTc]MB1143, and the signal is compared with that of 18F-fluorodeoxyglucose positron emission tomography ([18F]FDG-PET) as a nonbacterial specific marker for inflammation. Although the [99mTc]MB1143 imaging signal is highly specific, it is low, most probably due to insufficient serum stability of the tracer. A series of stability tests with different 18F-labeled maltodextrins finally yielded clear structural guidelines regarding substitution patterns and chain lengths of maltodextrin-based tracers for nuclear imaging of bacterial infections.

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.

Stroke is the most common cause of death and disability from neurologic disease in humans. Activation of microglia and matrix metalloproteinases (MMPs) is involved in positively and negatively affecting stroke outcome. Novel, noninvasive, multimodal imaging methods visualizing microglial and MMP alterations were employed. The spatio-temporal dynamics of these parameters were studied in relation to blood flow changes. Micro positron emission tomography (μPET) using [F]BR-351 showed MMP activity within the first days after transient middle cerebral artery occlusion (tMCAo), followed by increased [F]DPA-714 uptake as a marker for microglia activation with a maximum at 14 days after tMCAo. The inflammatory response was spatially located in the infarct core and in adjacent (penumbral) tissue. For the first time, multimodal imaging based on PET, single photon emission computed tomography, and magnetic resonance imaging revealed insight into the spatio-temporal distribution of critical parameters of poststroke inflammation. This allows further evaluation of novel treatment paradigms targeting the postischemic inflammation.

CCR2 and CCR5 receptors play a key role in the development and progression of several inflammatory, cardiovascular and autoimmune diseases. Therefore, dual targeting of both receptors appeals as a promising strategy for the treatment of such complex, multifactorial disorders. Herein we report on the design, synthesis and biological evaluation of benzo[7]annulene- and [7]annulenothiophene-based selective and dual CCR2 and CCR5 receptor antagonists. Intermediates were designed in such a way that diversification could be introduced at the end of the synthesis. Starting from the lead compound TAK-779 (1), the quaternary ammonium moiety was exchanged by different non-charged moieties, the 4-methylphenyl moiety was extensively modified and the benzo[7]annulene core was replaced bioisosterically by the [7]annulenothiophene system. The naphthyl derivative 9h represents the most promising dual antagonist (Ki (CCR2) = 25 nM, IC50 (CCR5) = 17 nM), whereas the 6-isopropoxy-3-pyridyl and 4-methoxycarbonylphenyl derivatives 9k and 9r show more than 20-fold selectivity for the CCR2 (Ki = 19 nM) over the CCR5 receptor.

The establishment of novel molecular imaging tools to monitor the local activity of inflammation remains an interdisciplinary challenge. Our target, the alarmin S100A9, one subunit of the heterodimer S100A8/S100A9 (calprotectin), is locally secreted in high concentrations from immigrated and activated phagocytes at local sites of inflammation. Calprotectin is already a well established serum biomarker for many inflammatory disorders. Here we show the development and first evaluation of the novel S100A9 specific molecular imaging probe for optical imaging of local inflammatory activity in vivo.

Non-invasive assessment of inflammatory activity in the course of various diseases is a largely unmet clinical challenge. An early feature of inflammation is local secretion of the alarmin S100A8/A9 by activated phagocytes. We here evaluate a novel S100A9-targeted small molecule tracer Cy5.5-CES271 for in vivo optical imaging of inflammatory activity in exemplary disease models. Dynamics of Cy5.5-CES271 was characterized in a model of irritant contact dermatitis by sequential fluorescence reflectance imaging (FRI) up to 24 h postinjection (p.i.). Specificity of Cy5.5-CES271 binding to S100A9 in vivo was examined by blocking studies and by employing S100A9−/− mice. Finally, S100A9 secretion in acute lung inflammation was assessed by Cy5.5-CES271 and FRI of explanted lungs. In ear inflammation, we were able to non-invasively follow the time course of S100A9 expression using Cy5.5-CES271 and FRI over 24 h p.i. (peak activity at 3 h p.i.). Specificity of imaging could be shown by a significant signal reduction after predosing and using S100A9−/− mice. In acute lung injury, local and systemic S100A8/A9 levels increased over time and correlated significantly with FRI signal levels in explanted lungs. Cy5.5-CES271 shows significant accumulation in models of inflammatory diseases and specific binding to S100A9 in vivo. This study, for the first time, demonstrates the potential of a small molecule non-peptidic tracer enabling imaging of S100A9 as a marker of local phagocyte activity in inflammatory scenarios suggesting this compound class for translational attempts.

Herein, we present a comparative study about the photoinactivation of Staphylococcus aureus (Gram-positive model) and Escherichia coli (Gram-negative model) employing a neutral and a dicationic axially functionalized Si

© 2017 Elsevier GmbH A novel quantification method named isobaric dilution analysis (IBDA) is introduced for internal calibration using inductively coupled plasma mass spectrometry (ICP-MS). Unlike isotope dilution analysis (IDA), where a sample to be analysed for a target analyte element is spiked with an isotopically enriched solution of the same element, IBDA uses the fact that conventional mass analysers cannot distinguish between two isobaric isotopes of different elements. Therefore, in IBDA, the sample with the element of interest is spiked with a solution of a different element, which shares at least one isobaric isotope and shows similar chemical properties resulting in a similar response. This method offers a less expensive alternative to conventional IDA especially for long-lived radionuclides of elements for which a spiking element that fulfils the above mentioned requirements can be provided. In addition, IBDA offers the advantage of making certain metastable, sometimes monoisotopic elements accessible to internal calibration using a strategy analogous to IDA. One of the elements accessible by the new calibration strategy is technetium (Tc), which suffers from a lack of standards due to safety requirements associated with its radioactivity. In this work, the principle of IBDA is first demonstrated for a certified gadolinium standard, which was diluted with a dysprosium spike exhibiting an isobaric isotope with m/z 160. The results obtained by IBDA were compared with those obtained by a conventional IDA. The concept of IBDA was subsequently used for the determination of 99Tc in a contrast agent. Since no certified Tc standards are available, the response of 99Tc was interpolated allowing accurate determinations with an uncertainty of 1%.

The enzymes gelatinase A/matrix metalloproteinase-2 (MMP-2) and gelatinase B/MMP-9 are essential for induction of neuroinflammatory symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS); in the absence of these enzymes, the disease does not develop. We therefore investigated the cellular sources and relative contributions of MMP-2 and MMP-9 to disease at early stages of EAE induction. We demonstrated that MMP-9 from an immune cell source is required in EAE for initial infiltration of leukocytes into the central nervous system and that MMP-9 activity is a reliable marker of leukocyte penetration of the blood-brain barrier. We then developed a molecular imaging method to visualize MMP activity in the brain using fluorescent- and radioactive-labeled MMP inhibitors (MMPis) in EAE animals and used the radioactive MMP ligand for positron emission tomography (PET) imaging of MMP activity in patients with MS. In contrast to traditional T1-gadolinium contrast-enhanced MRI, MMPi-PET enabled tracking of MMP activity as a unique feature of early lesions and ongoing leukocyte infiltration. MMPi-PET therefore allows monitoring of the early steps of MS development and provides a sensitive, noninvasive means of following lesion formation and resolution in murine EAE and human MS.

Introduction The natural occurring vitamin biotin, also known as vitamin H or vitamin B 7 , plays a major role in various metabolic reactions. Caused by its high binding affinity to the protein avidin with a dissociation constant of about 10 - 15 M the biotin-avidin system was extensively examined for multiple applications. We have synthesized a fluorine-18 labeled biotin derivative [ 18 F] 4 for a potential application in positron emission tomography (PET). Methods Mesylate precursor 3 was obtained by an efficient two-step reaction via a copper catalyzed azide-alkyne cycloaddition (CuAAC) from easily accessible starting materials. [ 18 F] 4 was successfully synthesized by a nucleophilic radiofluorination of precursor 3 . A biodistribution study by means of small-animal PET imaging in wt-mice was performed and serum stability was examined. Results Compound [ 18 F] 4 was obtained from precursor compound 3 with an average specific activity of 16 GBq/μmol within 45 min and a radiochemical yield of 45 ± 5% (decay corrected). [ 18 F] 4 demonstrated only negligible decomposition in human serum. A qualitative binding study revealed the high affinity of the synthesized biotin derivative to avidin. Blocking experiments with native biotin showed that binding was site-specific. Biodistribution studies showed that [ 18 F] 4 was cleared quickly and efficiently from the body by hepatobiliary and renal elimination. Conclusion An efficient synthesis for [ 18 F] 4 was established. In vivo characteristics were determined and demonstrated the pharmacokinetic behaviour of [ 18 F] 4 .

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade structural components of the extracellular matrix and participate in pathologies such as cancer and inflammatory disorders. The development of novel contrast agents for optical imaging of MMP activity in vivo is of great interest. The commonly used near-infrared fluorescence-compatible agents are dye-quenched probes that do not emit fluorescence until they interact with MMPs. In contrast, fluorescent synthetic low-molecular-weight MMP inhibitors have not been systematically employed. The aim of this study was to evaluate the performance of our recently developed Cy5.5-labeled MMP inhibitor to image MMP activity in tumors in vivo compared with activatable fluorescent MMP-sensing probes. The dynamic uptake of Cy5.5-AF489 into four different tumor entities was analyzed in xenografted mice by intravenous injection and subsequent fluorescence reflectance imaging. Tumors were characterized in regard to their MMP-2 and −9 mRNA expressions (qRT-PCR analysis), proteins (immunohistochemistry) and gelatinase/collagenase activities (in situ zymography). Cy5.5-AF489 was compared with MMPSenseTM 680 and MMPSenseTM 750 FAST, two commercially available MMP-activatable probes. Cy5.5-AF489 showed a specific tumor uptake, which was blocked by pre-injection of the unlabeled MMPI, and discriminated between tumors with high or low MMP-2/-9 expressions. Our optical probe facilitated faster visualization of MMP-active tumors accompanied by excellent tumor-to-background ratios when compared with activatable probes. The MMP inhibitor Cy5.5-AF489 permits fast in vivo imaging of MMP expression/activity in tumors. Given its small molecular weight and non-peptidic structure, translational imaging from a preclinical application to a diagnostic tool for MMP-related diseases seems feasible. Copyright © 2012 John Wiley & Sons, Ltd.

A deeper understanding of the role of specific genes, proteins, pathways and networks in health and disease, coupled with the development of technologies to assay these molecules and pathways in patients, promises to revolutionise the practice of clinical medicine. Especially the discovery and development of novel drugs targeted to disease-specific alterations could benefit significantly from non-invasive imaging techniques assessing the dynamics of specific disease-related parameters. Here we review the application of imaging biomarkers in the management of patients with brain tumours, especially malignant glioma. In our other review we focused on imaging biomarkers of general biochemical and physiological processes related with tumour growth such as energy, protein, DNA and membrane metabolism, vascular function, hypoxia and cell death. In this part of the review, we will discuss the use of imaging biomarkers of specific disease-related molecular genetic alterations such as apoptosis, angiogenesis, cell membrane receptors and signalling pathways and their application in targeted therapies.

Radioligands, which specifically bind to a receptor or enzyme (target), enable molecular imaging of the target expression by positron emission tomography (PET). One very promising PET tracer is (S)-1-(4-(2-[(18)F]-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (isatin), a caspase-3 inhibitor, which has been developed at the University Hospital of Münster to image cell death (apoptosis). The translation of this novel tracer from preclinical evaluation to clinical examinations requires biodistribution studies, which characterize the pharmakodynamics and metabolic fate of the compound. This information is used to further optimize the radioligands and to interpret radioactive signals from tissues upon injection of the radioligand in vivo with respect to their specificity. The analysis of the metabolism of radioligands is hampered by the low amount of the compound being typically injected (nano/picomolar amount per injection). In the present study, electrochemistry (EC) is applied to elucidate the oxidative metabolism pathway of the radiotracer. Previous studies have demonstrated that EC can be utilized as a complementary tool to conventional in vitro approaches in drug metabolism studies. Thereby, potential oxidative metabolites of the isatin are determined by EC coupled to electrospray ionization mass spectrometry (EC/ESI-MS). Moreover, using EC/liquid chromatography (LC) and ESI-ion trap MS(n), structural elucidation of the oxidation products is performed. Comparatively to EC, in vitro metabolism studies with rat liver microsomes are conducted. Finally, the developed LC/ESI-MS method is applied to determine metabolites in body fluids and cell extracts from in vivo studies with the nonradioactive ((19)F) and radioactive isatin ((18)F). On the basis of the electrochemically generated oxidation products of the radioligand, the major radioactive metabolite occurring in vivo was successfully identified.