Ralf Schirrmacher

The Temporal Dynamics of Poststroke Neuroinflammation: A Longitudinal Diffusion Tensor Imaging–Guided PET Study with 11C-PK11195 in Acute Subcortical Stroke

Animal experiments suggest that 2 different types of activated microglia (AMG) cells occur in the brain after a stroke: local AMG in the area of the infarct and remote AMG, which occurs along affected fiber tracts. We used 11C-PK11195 PET to image AMG in vivo after stroke in humans in a prospective longitudinal study to investigate the temporal dynamics of AMG and relate local and remote AMG activity to pyramidal tract (PT) damage using diffusion tensor imaging (DTI). Methods: Eighteen patients underwent DTI–MRI, 11C-PK11195 PET, and behavioral testing within 2 wk and 6 mo of acute subcortical stroke. In 12 patients, the PT was affected by the stroke (PT group), and in 6 patients it was not (non-PT group). Standardized volumes of interest (VOIs) were placed along the PT at the level of the brain stem, semioval center, and infarct. Tracer uptake ratios (ipsilateral to contralateral) were calculated for each VOI and related to tract damage (measured as fractional anisotropy ratio) and clinical outcome. Six controls underwent the same protocol but only once. Results: In both patient groups, local AMG activity in the infarct was increased initially and significantly decreased over the follow-up period. In contrast, remote AMG was detected only in the PT group in the brain stem along the affected tract and persisted during follow-up. No AMG was observed retrograde to the lesion at any time. Remote AMG activity along the affected PT in the brain stem correlated with initial PT damage as measured by DTI in the same tract portion. Local AMG activity in the infarct correlated with anterograde PT damage only at follow-up. After controlling for PT damage, initial AMG activity in the brain stem showed a positive correlation with clinical outcome, whereas persisting AMG activity in the infarct tended to be negatively correlated. Conclusion: DTI-guided 11C-PK11195 PET in acute subcortical stroke demonstrates differential temporal dynamics of local and remote AMG. Activity of both types related to anterograde PT damage as measured by DTI and might contribute differently to clinical outcome.

Where in-vivo imaging meets cytoarchitectonics: The relationship between cortical thickness and neuronal density measured with high-resolution [18F]flumazenil-PET

MRI-based measurements of surface cortical thickness (SCT) have become a sensitive tool to quantify changes in cortical morphology. When comparing SCT to histological cortical thickness maps, a good correspondence can be found for many but not all human brain areas. Discrepancies especially arise in the sensory motor cortex, where histological cortical thickness is high, but SCT is very low. The aim of this study was to determine whether the relationship between cortical thickness and neuronal density is the same for different cytoarchitectonic areas throughout homo- and heterotypical isocortex. We assessed this relationship using high-resolution [(18)F]-labelled flumazenil (FMZ) PET and SCT-mapping. FMZ binds to the benzodiazepine GABA(A) receptor complex which is localized on axo-dendritic synapses, with a cortical distribution closely following the local density of neurons. SCT and voxelwise FMZ binding potential (BP(ND)) were assessed in ten healthy subjects. After partial volume correction, two subsets with a differential relationship between SCT and BP(ND) were identified: a fronto-parietal homotypical subset where neuronal density is relatively constant and mainly independent of SCT, and a subset comprising heterotypical and mainly temporal and occipital homotypical regions where neuronal density is negatively correlated with SCT. This is the first in-vivo study demonstrating a differential relationship between SCT, neuronal density and cytoarchitectonics in humans. These findings are of direct relevance for the correct interpretation of SCT-based morphometry studies, in that there is no simple relationship between apparent cortical thickness and neuronal density, here attributed to FMZ binding, holding for all cortical regions.

Multimerization of cRGD peptides by click chemistry: synthetic strategies, chemical limitations, and influence on biological properties.

Integrin ανβ3 is overexpressed on endothelial cells of growing vessels as well as on several tumor types, and so integrin‐binding radiolabeled cyclic RGD pentapeptides have attracted increasing interest for in vivo imaging of ανβ3 integrin expression by positron emission tomography (PET). Of the cRGD derivatives available for imaging applications, systems comprising multiple cRGD moieties have recently been shown to exhibit highly favorable properties in relation to monomers. To assess the synthetic limits of the cRGD‐multimerization approach and thus the maximum multimer size achievable by using different efficient conjugation reactions, we prepared a variety of multimers that were further investigated in vitro with regard to their avidities to integrin ανβ3. The synthesized peptide multimers containing increasing numbers of cRGD moieties on PAMAM dendrimer scaffolds were prepared by different click chemistry coupling strategies. A cRGD hexadecimer was the largest construct that could be synthesized under optimized reaction conditions, thus identifying the current synthetic limitations for cRGD multimerization. The obtained multimeric systems were conjugated to a new DOTA‐based chelator developed for the derivatization of sterically demanding structures and successfully labeled with 68Ga for a potential in vivo application. The evaluated multimers showed very high avidities—increasing with the number of cRGD moieties—in in vitro studies on immobilized ανβ3 integrin and U87MG cells, of up to 131‐ and 124‐fold, respectively, relative to the underivatized monomer.

Establishment and functional validation of a structural homology model for human DNA methyltransferase 1

Changes in DNA methylation patterns play an important role in tumorigenesis. The DNA methyltransferase 1 (DNMT1) protein represents a major DNA methyltransferase activity in human cells and is therefore a prominent target for experimental cancer therapies. However, there are only few available inhibitors and their high toxicity and low specificity have so far precluded their broad use in chemotherapy. Based on the strong conservation of catalytic DNA methyltransferase domains we have used a homology modeling approach to determine the three-dimensional structure of the DNMT1 catalytic domain. Our results suggest an overall structural conservation with other DNA methyltransferases but also indicate local conformational differences. To prove the validity of our model we used it as a template to design a novel derivative of the known DNA methyltransferase inhibitor 5-azacytidine. The resulting compound (N4-fluoroacetyl-5-azacytidine) functioned as an efficient inhibitor of DNA methylation in human tumor cell lines and also provides novel opportunities for pharmacological applications.

Kit-Like 18F-Labeling of Proteins: Synthesis of 4-(Di-tert-butyl[18F]fluorosilyl)benzenethiol (Si[18F]FA-SH) Labeled Rat Serum Albumin for Blood Pool Imaging with PET

Radiosyntheses of 18F-radiopharmaceuticals for positron emission tomography (PET) normally require an extraordinarily high effort of technical equipment and specially trained personnel. We recently reported a novel method for the introduction of fluorine-18 into peptides for PET-imaging based on silicon-18F-chemistry (SiFA technique). We herewith introduce the first SiFA-based Kit-like radio-fluorination of a protein (rat serum albumin,RSA) and demonstrate its usefulness for in vivo imaging with microPET in normal rats as well as in a rat heterotropic transplanted heart model. As a labeling agent, we prepared 4-(di-tert-butyl[18F]fluorosilyl)benzenethiol (Si[18F]FASH)by simple isotopic exchange in 40-60% radiochemical yield (RCY) and coupled it directly to a Sulfo-SMCC derivatized RSA in an overall RCY of 12% within 20-30 min. The technically simple labeling procedure does not require any elaborated purification procedures and is a straightforward example of a successful application of Si-18F chemistry for in vivo imaging with PET.

One-step 18 F-labeling of peptides for positron emission tomography imaging using the SiFA methodology

Here we present a procedure to label peptides with the positron-emitting radioisotope fluorine-18 (18F) using the silicon-fluoride acceptor (SiFA) labeling methodology. Positron emission tomography (PET) has gained high importance in noninvasive imaging of various diseases over the past decades, and thus new specific imaging probes for PET imaging, especially those labeled with 18F, because of the advantageous properties of this nuclide, are highly sought after. N-terminally SiFA–modified peptides can be labeled with 18F− in one step at room temperature (20–25 °C) or below without forming side products, thereby producing satisfactory radiochemical yields of 46 ± 1.5% (n = 10). The degree of chemoselectivity of the 18F-introduction, which is based on simple isotopic exchange, allows for a facile cartridge-based purification fully devoid of HPLC implementation, thereby yielding peptides with specific activities between 44.4 and 62.9 GBq μmol−1 (1,200–1,700 Ci mmol−1) within 25 min.

One-Step 18F-Labeling of Carbohydrate-Conjugated Octreotate-Derivatives Containing a Silicon-Fluoride-Acceptor (SiFA): In Vitro and in Vivo Evaluation as Tumor Imaging Agents for Positron Emission Tomography (PET)

The synthesis, radiolabeling, and initial evaluation of new silicon-fluoride acceptor (SiFA) derivatized octreotate derivatives is reported. So far, the main drawback of the SiFA technology for the synthesis of PET-radiotracers is the high lipophilicity of the resulting radiopharmaceutical. Consequently, we synthesized new SiFA-octreotate analogues derivatized with Fmoc-NH-PEG-COOH, Fmoc-Asn(Ac₃AcNH-β-Glc)-OH, and SiFA-aldehyde (SIFA-A). The substances could be labeled in high yields (38 ± 4%) and specific activities between 29 and 56 GBq/μmol in short synthesis times of less than 30 min (e.o.b.). The in vitro evaluation of the synthesized conjugates displayed a sst2 receptor affinity (IC₅₀ = 3.3 ± 0.3 nM) comparable to that of somatostatin-28. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be 0.96 for SiFA-Asn(AcNH-β-Glc)-PEG-Tyr³-octreotate and 1.23 for SiFA-Asn(AcNH-β-Glc)-Tyr³-octreotate, which is considerably lower than for SiFA-Tyr³-octreotate (log P(ow) = 1.59). The initial in vivo evaluation of [¹⁸F]SiFA-Asn(AcNH-β-Glc)-PEG-Tyr³-octreotate revealed a significant uptake of radiotracer in the tumor tissue of AR42J tumor-bearing nude mice of 7.7% ID/g tissue weight. These results show that the high lipophilicity of the SiFA moiety can be compensated by applying hydrophilic moieties. Using this approach, a tumor-affine SiFA-containing peptide could successfully be used for receptor imaging for the first time in this proof of concept study.

Generation of Novel Single-Chain Antibodies by Phage-Display Technology to Direct Imaging Agents Highly Selective to Pancreatic β- or α-Cells In Vivo

OBJECTIVE Noninvasive determination of pancreatic β-cell mass in vivo has been hampered by the lack of suitable β-cell–specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo. RESEARCH DESIGN AND METHODS To generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. RESULTS We report the generation of SCAs that bind highly selective to either β- or α-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to β- or α-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [125I]-labeled SCAs after intravenous administration in rats strongly predicted the β-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. CONCLUSIONS Our data provide strong evidence that the presented SCAs are highly specific for pancreatic β-cells and enable imaging and quantification in vivo.

A Universally Applicable 68Ga-Labeling Technique for Proteins

Although protein-based PET imaging agents are projected to become important tracer molecules in the future, the labeling of complex biomolecules with PET radionuclides is inexpedient and, most of the time, challenging. Methods: Here we present a straightforward labeling chemistry to attach the versatile radionuclide 68Ga to proteins. Introducing the 68Ga chelating agent NODA-GA-T (2,2′-(7-(1-carboxy-4-(2-mercaptoethylamino)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid) by reaction with proteins chemically processed with sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt) results in labeling precursors, enabling a simple and rapid kit-labeling procedure that requires no workup of the radiolabeled proteins. Various 68Ga- proteins were labeled using this method, and the radiochemical yields and specific activities of the labeled proteins were determined. To show that the radiotracers are applicable for in vivo studies, proof-of-concept small-animal PET images were acquired in healthy rats using 68Ga rat serum albumin for blood-pool imaging and 68Ga-annexin V for apoptosis imaging in mice with a left ventricular myocardial infarction. Results: The proteins could be modified, yielding 1.2–1.7 68Ga-labeling sites per protein molecule. All investigated proteins could be labeled in high radiochemical yields of 95% or more in less than 10 min in 1 step, using acetate-buffered medium (pH 3.5–4.0) at room temperature without any further purification. The labeled proteins displayed specific activities of 20–45 GBq/μmol (540–1,200 Ci/mmol). In the proof-of-concept in vivo studies, 68Ga rat serum albumin and 68Ga-annexin V were successfully used for in vivo imaging. Both radiotracers showed a favorable biodistribution in the animal models, thus demonstrating the usefulness of the developed approach for the kit 68Ga labeling of proteins. Conclusion: The preprocessing of proteins proceeds in high chemical yields and with high protein recovery rates after purification. These precursors can be stored for several months at −20°C without degradation, and 68Ga labeling can be performed in a 1-step kit-labeling reaction in high radiochemical yields. Two of the derivatized model proteins were successfully used in proof-of-concept in vivo imaging studies to prove the applicability of this kit 68Ga-labeling technique.

Oxalic Acid Supported Si–18F-Radiofluorination: One-Step Radiosynthesis of N-Succinimidyl 3-(Di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB) for Protein Labeling

N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB), a novel synthon for one-step labeling of proteins, was synthesized via a simple (18)F-(19)F isotopic exchange. A new labeling technique that circumvents the cleavage of the highly reactive active ester moiety under regular basic (18)F-labeling conditions was established. In order to synthesize high radioactivity amounts of [(18)F]SiFB, it was crucial to partially neutralize the potassium oxalate/hydroxide that was used to elute (18)F(-) from the QMA cartridge with oxalic acid to prevent decomposition of the active ester moiety. Purification of [(18)F]SiFB was performed by simple solid-phase extraction, which avoided time-consuming HPLC and yielded high specific activities of at least 525 Ci/mmol and radiochemical yields of 40-56%. In addition to conventional azeotropic drying of (18)F(-) in the presence of [K(+)⊂2.2.2.]C(2)O(4), a strong anion-exchange (SAX) cartridge was used to prepare anhydrous (18)F(-) for nucleophilic radio-fluorination omitting the vacuum assisted drying of (18)F(-). Using a lyophilized mixture of [K(+)⊂2.2.2.]OH resolubilized in acetonitrile, the (18)F(-) was eluted from the SAX cartridge and used directly for the [(18)F]SiFB synthesis. [(18)F]SiFB was applied to the labeling of various proteins in likeness to the most commonly used labeling synthon in protein labeling, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). Rat serum albumin (RSA), apo-transferrin, a β-cell-specific single chain antibody, and erythropoietin were successfully labeled with [(18)F]SiFB in good radiochemical yields between 19% and 36%. [(18)F]SiFB- and [(18)F]SFB-derivatized RSA were directly compared as blood pool imaging agents in healthy rats using small animal positron emission tomography. Both compounds demonstrated identical biodistributions in healthy rats, accurately visualizing the blood pool with PET.