Esther Schirrmacher

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.

Opioid Receptor PET Reveals the Psychobiologic Correlates of Reward Processing

Little is known about the neurobiologic correlates of human personality. On the basis of the key role of the central opioidergic system in addiction and substance abuse, we investigated the relationship between certain personality traits that are supposed to be relevant in addiction and the opioid receptor status in healthy subjects. Methods: We investigated 23 healthy male volunteers who were extensively clinically tested to exclude substance abuse. All of the subjects underwent 1 PET scan with the subtype-nonselective opioidergic radioligand 18F-fluoroethyl-diprenorphine under resting conditions without sensory or cognitive stimulation. Subsequently, the subjects were psychologically tested for the personality traits novelty seeking, harm avoidance, reward dependence, and persistence, according to Cloningers biosocial model of personality. The binding potential (BP) as a parameter of regional cerebral opioid receptor availability was computed by means of the modified Logan plot using the occipital cortex as a reference region. Further imaging data analysis was performed using statistical parametric mapping; after stereotactic normalization, the correlations were calculated between the regional BP and the psychologic scores on a voxel-by-voxel basis. Results: The correlation analysis between personality dimensions and opioid receptor availability showed a significant (P < 0.001) positive correlation between the scores of reward dependence and the BP of the bilateral ventral striatum with nucleus accumbens (z scores, 4.52 and 4.33, respectively). The additionally performed region-of-interest–based correlation analysis yielded correlation coefficients of r = 0.84 and r = 0.81 for the left and right ventral striata, respectively. No further significant correlations were detectable between the other personality dimensions and cerebral opioid receptor binding. Conclusion: In healthy subjects, personality traits, which might be predisposing for addictive behavior, are correlated to the opioidergic neurotransmission in core structures of the human reward system.

Improved work-up procedure for the production of [18F] flumazenil and first results of its use with a high-resolution research tomograph in human stroke

INTRODUCTION The central benzodiazepine receptor (cBZR)-gamma-aminobutyric acid (GABA(A)) receptor complex in the human brain plays an important role in many neurological and psychiatric disorders. (18)F-Labeled flumazenil ([(18)F]FZ) provides a potentially useful tracer to investigate those disorders by means of positron emission tomography (PET). METHODS [(18)F]Flumazenil was synthesized from its nitro-precursor Ro 15-2344 in DMF at high temperatures between 150 degrees C and 160 degrees C. Other solvents like acetonitrile and dimethylsulfoxide were also investigated as reaction media. A new HPLC method for the final purification of [(18)F]FZ was developed to circumvent some difficulties associated with a previously published procedure sometimes led to a contamination of [(18)F]FZ with Ro 15-2344. The final purification of the radiotracer was achieved using a Waters Symmetry Prep C18 HPLC column with elution with 0.05 M sodium acetate (NaOAc) buffer (pH 5)/THF/MeOH (80:10:10). RESULTS [(18)F]FZ could be synthesized in reproducible radiochemical yields (RCYs) of 15-20% (decay corrected to EOB) after 80 min overall synthesis time. The synthesized [(18)F]FZ was applied for the first time in a human PET study in a patient with ischemic right middle cerebral artery stroke using the HRRT high-resolution research scanner (Siemens Medical Solution, Knoxville, TN, USA). CONCLUSIONS [(18)F]FZ is a potentially useful GABA receptor-binding PET ligand. A modified procedure for its preparation in reproducibly high radiochemical yields has been described and the [(18)F]FZ thus produced has been used successfully in a pilot clinical study.

Protein labeling with the labeling precursor [ 18 F]SiFA-SH for positron emission tomography

Proteins previously derivatized with the cross-coupling reagent sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxy-succinimide ester sodium salt) can be easily labeled in high radiochemical yields with the silicon-fluoride acceptor (SiFA) reagent [18F]SiFA-SH, obtained via isotopic exchange, by thiol-maleimide coupling chemistry (n = 10). The specific activity of SiFA-SH obtained in a one-step labeling reaction was >18.5 GBq μmol−1 (>500 Ci mmol−1). The number of SiFA building blocks per protein molecule is defined by the previously introduced number of maleimide groups, which can be determined by a simple and convenient Ellmans assay. Not more than two maleimide groups are introduced using sulfo-SMCC, thereby keeping the modification of the protein low and preserving its biological activity.

[18F]SiFA-isothiocyanate: a new highly effective radioactive labeling agent for lysine-containing proteins.

A highly efficient 18F‐labeling synthon for universal protein labeling is reported. Diverse 18F‐labeled proteins of 66–144 kDa were prepared with [18F]SiFA‐isothiocyanate synthesized by an isotopic 19F for 18F exchange at the silicon atom. Overall preparative radiochemical yields were 20–40 % after 40–50 min. No bone uptake of 18F radioactivity was detected until 90 min post‐injection of 18F‐SiFA‐RSA; this demonstrates the metabolic stability of the [18F]SiFA moiety.

Cholinergic Depletion in Alzheimer’s Disease Shown by [18F]FEOBV Autoradiography

Rationale. Alzheimers Disease (AD) is a neurodegenerative condition characterized in part by deficits in cholinergic basalocortical and septohippocampal pathways. [18F]Fluoroethoxybenzovesamicol ([18F]FEOBV), a Positron Emission Tomography ligand for the vesicular acetylcholine transporter (VAChT), is a potential molecular agent to investigate brain diseases associated with presynaptic cholinergic losses. Purpose. To demonstrate this potential, we carried out an [18F]FEOBV autoradiography study to compare postmortem brain tissues from AD patients to those of age-matched controls. Methods. [18F]FEOBV autoradiography binding, defined as the ratio between regional grey and white matter, was estimated in the hippocampus (13 controls, 8 AD) and prefrontal cortex (13 controls, 11 AD). Results. [18F]FEOBV binding was decreased by 33% in prefrontal cortex, 25% in CA3, and 20% in CA1. No changes were detected in the dentate gyrus of the hippocampus, possibly because of sprouting or upregulation toward the resilient glutamatergic neurons of the dentate gyrus. Conclusion. This is the first demonstration of [18F]FEOBV focal binding changes in cholinergic projections to the cortex and hippocampus in AD. Such cholinergic synaptic (and more specifically VAChT) alterations, in line with the selective basalocortical and septohippocampal cholinergic losses documented in AD, indicate that [18F]FEOBV is indeed a promising ligand to explore cholinergic abnormalities in vivo.

Imaging in Vivo Glutamate Fluctuations with [11C]ABP688: A GLT-1 Challenge with Ceftriaxone

Molecular imaging offers unprecedented opportunities for investigating dynamic changes underlying neuropsychiatric conditions. Here, we evaluated whether [11C]ABP688, a positron emission tomography (PET) ligand that binds to the allosteric site of the metabotropic glutamate receptor type 5 (mGluR5), is sensitive to glutamate fluctuations after a pharmacological challenge. For this, we used ceftriaxone (CEF) administration in rats, an activator of the GLT-1 transporter (EAAT2), which is known to decrease extracellular levels of glutamate. MicroPET [11C]ABP688 dynamic acquisitions were conducted in rats after a venous injection of either saline (baseline) or CEF 200 mg/kg (challenge). Binding potentials (BPND) were obtained using the simplified reference tissue method. Between-condition statistical parametric maps indicating brain regions showing the highest CEF effects guided placement of microdialysis probes for subsequent assessment of extracellular levels of glutamate. The CEF administration increased [11C]ABP688 BPND in the thalamic ventral anterior (VA) nucleus bilaterally. Subsequent microdialysis assessment revealed declines in extracellular glutamate concentrations in the VA. The present results support the concept that availability of mGluR5 allosteric binding sites is sensitive to extracellular concentrations of glutamate. This interesting property of mGluR5 allosteric binding sites has potential applications for assessing the role of glutamate in the pathogenesis of neuropsychiatric conditions.

PET imaging of cholinergic deficits in rats using [18F]fluoroethoxybenzovesamicol ([18F]FEOBV).

[(18)F]fluoroethoxybenzovesamicol ([(18)F]FEOBV) is one of the most promising radioligands for imaging the vesicular ACh transporter (VAChT) with positron emission tomography (PET). We report here that this method can detect subtle cholinergic terminals losses such as those associated with aging, or those following a partial lesion of the nucleus basalis magnocellularis (NBM). Twenty-one adult rats were evenly distributed in three groups including 1) aged rats (18 months); 2) young rats (3 months); and 3) rats with unilateral lesion of the NBM, following a local stereotaxic infusion of 192 IgG-saporin. In both normal and lesioned rats, our results revealed the highest [(18)F]FEOBV binding to be in the striatum, followed by similar values in both frontal cortex and thalamus, while lower values were observed in both hippocampus and temporo-parietal cortex. This binding distribution is consistent with the known anatomy of brain cholinergic systems. In the lesioned rats, [(18)F]FEOBV binding was found to be reduced mostly in the ventral frontal cortex on the side of the lesion, but some reductions were also observed in the homologous region of the contralateral hemisphere. Aging was found to be associated with a [(18)F]FEOBV binding reduction limited to the hippocampus of both hemispheres. [(18)F]FEOBV appears to be a very promising marker for the in vivo quantification of the brain VAChT; PET imaging of this agent allows in vivo detection of both physiological and pathological reductions of cholinergic terminals density.

Synthesis of 2-amino-6-(2-[18F]fluoro-pyridine-4-ylmethoxy)-9-(octyl-β-d-glucosyl)-purine: a novel radioligand for positron emission tomography studies of the O6-methylguanine-DNA methyltransferase (MGMT) status of tumour tissue

Abstract The synthesis of the novel glucose conjugated O 6 -methylguanine-DNA methyltransferase (MGMT) inhibitor 2-amino-6-(2-[ 18 F]fluoro-pyridine-4-ylmethoxy)-9-(octyl-α- d -glucosyl)-purine is reported. This compound might serve as a radiotracer for the determination of the MGMT status of tumour tissue.