Covalent labeling of immune cells.

Inflammation is a common, fast, and innate response of the immune system to sterile or infectious tissue damage or autoimmune triggers. It aims at minimizing tissue destruction and maintaining organ function, hence is vital to life. Therefore, the immune system comprises the concerted action of a variety of different immune cells with specific tasks in the initiation, maintenance, and termination of inflammation. Visualizing their localization, trafficking, and interaction is of utmost importance to unravel the dynamics of inflammation in the living organism and requires tools for cell-specific labeling and imaging. Many concepts for covalent cell-type or protein-specific labeling have been developed, but only few have been implemented for labeling immune cells. Here, we review approaches that were already successful for fluorescent reporters and radioactive nuclides. We also provide a glimpse on emerging technologies that bear potential for immune cell labeling and imaging in vivo.

Towards Optimized Bioavailability of 99mTc-Labeled Barbiturates for Non-invasive Imaging of Matrix Metalloproteinase Activity.

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.

A novel 18F-labeled clickable substrate for targeted imaging of SNAP-tag expressing cells by PET in vivo.

Bioorthogonal covalent labeling with self-labeling enzymes like SNAP-tag bears a high potential for specific targeting of cells for imaging in vitro and also in vivo. To this end, fluorescent SNAP substrates have been established and used in microscopy and fluorescence imaging while radioactive substrates for the highly sensitive and whole-body positron emission tomography (PET) have been lacking. Here, we show for the first time successful and high-contrast PET imaging of subcutaneous SNAP-tag expressing tumor xenografts by bioorthogonal covalent targeting with a novel 18F-based radioligand in vivo.

Synthesis and biological evaluation of PET tracers designed for imaging of calcium activated potassium channel 3.1 (KCa3.1) channels in vivo.

Expression of the Ca2+ activated potassium channel 3.1 (KCa3.1) channel (also known as the Gàrdos channel) is dysregulated in many tumor entities and has predictive power with respect to patient survival. Therefore, a positron emission tomography (PET) tracer targeting this ion channel could serve as a potential diagnostic tool by imaging the KCa3.1 channel in vivo. It was envisaged to synthesize [18F]senicapoc ([18F]1) since senicapoc (1) shows high affinity and excellent selectivity towards the KCa3.1 channels. Because problems occurred during 18F-fluorination, the [18F]fluoroethoxy senicapoc derivative [18F]28 was synthesized to generate an alternative PET tracer targeting the KCa3.1 channel. Inhibition of the KCa3.1 channel by 28 was confirmed by patch clamp experiments. In vitro stability in mouse and human serum was shown for 28. Furthermore, biodistribution experiments in wild type mice were performed. Since [18F]fluoride was detected in vivo after application of [18F]28, an in vitro metabolism study was conducted. A potential degradation route of fluoroethoxy derivatives in vivo was found which in general should be taken into account when designing new PET tracers for different targets with a [18F]fluoroethoxy moiety as well as when using the popular prosthetic group [18F]fluoroethyl tosylate for the alkylation of phenols.

Synthesis of Small-Molecule Fluorescent Probes for the In†Vitro Imaging of Calcium-Activated Potassium Channel KCa 3.1.

Small-molecule probes for the in vitro imaging of KCa 3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the KCa 3.1 channels, was chosen as the targeting component. BODIPY dyes 15-20 were synthesized and connected by a CuI -catalyzed azide-alkyne [3+2]cycloaddition with propargyl ether senicapoc derivative 8, yielding fluorescently labeled ligands 21-26. The dimethylpyrrole-based imaging probes 25 and 26 allow staining of KCa 3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre-incubation with senicapoc. The density of KCa 3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells. Molecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.

New in Vivo Compatible Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitors.

Matrix metalloproteinases (MMPs) are emerging as pivotal fine-tuners of cell function in tissue homeostasis and in various pathologies, in particular inflammation. In vivo monitoring of the activity of specific MMPs, therefore, provides high potential for assessing disease progression and tissue function, and manipulation of MMP activity in tissues and whole organisms may further provide a mode of controlling pathological processes. We describe here the synthesis of novel fluorinated and nonfluorinated analogues of a secondary sulfonamide-based lead structure, compound 2, and test their efficacy as in vivo inhibitors and tracers of the gelatinases, MMP-2 and MMP-9. Using a murine neuroinflammatory model, we show that compound 2 is a highly effective in vivo inhibitor of both MMP-2 and MMP-9 activity with little or no adverse effects even after long-term daily oral administration. A fluorescein-labeled derivative compound 17 shows direct binding to activated gelatinases surrounding inflammatory cuffs in the neuroinflammation model and to pancreatic ß-cells in the islets of Langerhans, colocalizing with MMP-2 and MMP-9 activity as detected using in situ zymography techniques. These results demonstrate that compound 2 derivatives have potential as in vivo imaging tools and for future development for specific MMP-2 versus MMP-9 probes. Our chemical modifications mainly target the residues directed toward the S1' and S2' pockets and, thereby, provide new information on the structure-activity relationships of this inhibitor type.