Dag Erlend Olberg

One Step Radiosynthesis of 6-[18F]Fluoronicotinic Acid 2,3,5,6-Tetrafluorophenyl Ester ([18F]F-Py-TFP): A New Prosthetic Group for Efficient Labeling of Biomolecules with Fluorine-18

The labeling of biomolecules for positron emission tomography (PET) with no-carrier-added fluorine-18 is almost exclusively accomplished using prosthetic groups in a two step procedure. The inherent complexity of the process renders full automation a challenge and leads to protracted synthesis times. Here we describe a new (18)F-labeled prosthetic group based on nicotinic acid tetrafluorophenyl ester. Reaction of [(18)F]fluoride at 40 degrees C with the trimethylammonium precursor afforded 6-[(18)F]fluoronicotinic acid tetrafluorophenyl ester ([(18)F]F-Py-TFP) directly in 60-70% yield. [(18)F]F-Py-TFP was conveniently purified by Sep-Pak cartridge prior to incubation with a peptide containing the RGD sequence. The desired conjugate was formed rapidly and in good yields. An in vitro receptor-binding assay for the integrin alpha(v)beta(3) was established to explore competition with peptide and peptidomimetic prepared from F-Py-TFP with (125)I-echistatin. The nonradioactive conjugates were found to possess high binding affinities with calculated K(i) values in the low nanomolar range.

Labeling Strategies of Peptides with 18F for Positron Emission Tomography

A variety of peptides labeled with the positron emitting radionuclide fluorine-18 have shown promise as tracers for use in positron emission tomography (PET) for the detection of malignancies. Peptides can be produced with a formidable versatility allowing them to target a vast diversity of uniquely expressed or overexpressed receptors associated with pathological conditions. The quantitative nature of PET gives the opportunity to stage and monitor the progress of the disease. The pharmacokinetics of peptides are compatible with the half-life of fluorine-18 (110 min), allowing the generation of high quality PET images within the time frame of 1-3 hours or longer. The production of high energy gamma emitting radiopharmaceuticals puts certain constraints and requirements on the production method. These are to a large extent dictated by the short half-life of the ¹⁸F and the need for appropriate shielding of the operator. For large scale productions, a fully automated production process is a requirement. Compared to low molecular weight fluorine-18 labeled tracers, the production of ¹⁸F-labeled peptides entails specific challenges. As opposed to small organic molecules where direct labeling with no-carrier added 18-fluoride is feasible, peptides do not normally allow for such a direct labeling approach. Therefore, peptides are for all practical purposes labeled by ¹⁸F-prosthetic groups, also called bifunctional labeling agents, making their synthesis relatively complicated. During the last decade, various methodologies have been developed for the introduction of ¹⁸F-fluoride into peptides. The strategies employed for the labeling of peptides with ¹⁸F all represent their own advantages and inconveniences, still some are more flexible than others. In this review, the aim is to provide an overview and discuss the strategies currently used for labeling of peptides with ¹⁸F for PET.

Prepubertal gonadotropin-releasing hormone analog leads to exaggerated behavioral and emotional sex differences in sheep.

In mammals, sex specialization is reflected by differences in brain anatomy and function. Measurable differences are documented in reproductive behavior, cognition, and emotion. We hypothesized that gonadotropin-releasing hormone (GnRH) plays a crucial role in controlling the extent of the brains sex specificity and that changes in GnRH action during critical periods of brain development, such as puberty, will result in altered sex-specific behavioral and physiological patterns. We blocked puberty in half of the 48 same-sex Scottish mule Texel cross sheep twins with GnRH analog (GnRHa) goserelin acetate every 3 weeks, beginning just before puberty. To determine the effects of GnRHa treatment on sex-specific behavior and emotion regulation in different social contexts, we employed the food acquisition task (FAT) and measurement of heart rate variability (HRV). ANOVA revealed significant sex and sex×treatment interaction effects, suggesting that treated males were more likely to leave their companions to acquire food than untreated, while the opposite effect was observed in females. Concordant results were seen in HRV; treated males displayed higher HRV than untreated, while the reverse pattern was found in females, as shown by significant sex and sex×treatment interaction effects. We conclude that long-term prepubertal GnRHa treatment significantly affected sex-specific brain development, which impacted emotion and behavior regulation in sheep. These results suggest that GnRH is a modulator of cognitive function in the developing brain and that the sexes are differentially affected by GnRH modulation.

A Novel Prosthetic Group for Site-Selective Labeling of Peptides for Positron Emission Tomography

Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.

Standardization of fluorine-18 manufacturing processes: New scientific challenges for PET

In [(18)F]fluoride chemistry, the minute amounts of radioactivity taking part in a radiolabeling reaction are easily outnumbered by other reactants. Surface areas become comparably larger and more influential than in standard fluorine chemistry, while leachables, extractables, and other components that normally are considered small impurities can have a considerable influence on the efficiency of the reaction. A number of techniques exist to give sufficient (18)F-tracer for a study in a pre-clinical or clinical system, but the chemical and pharmaceutical understanding has significant gaps when it comes to scaling up or making the reaction more efficient. Automation and standardization of [(18)F]fluoride PET tracers is a prerequisite for reproducible manufacturing across multiple PET centers. So far, large-scale, multi-site manufacture has been established only for [(18)F]FDG, but several new tracers are emerging. In general terms, this transition from small- to large-scale production has disclosed several scientific challenges that need to be addressed. There are still areas of limited knowledge in the fundamental [(18)F]fluoride chemistry. The role of pharmaceutical factors that could influence the (18)F-radiosynthesis and the gaps in precise chemistry knowledge are discussed in this review based on a normal synthesis pattern.

Radiosynthesis and Biodistribution of a Prosthetic Group (18F-FENMA) Conjugated to Cyclic RGD Peptides

We have recently reported a new N-methylaminooxy-based prosthetic group for the site-selective introduction of ¹⁸F-fluorine under mild acidic aqueous conditions into model peptides functionalized with a Michael acceptor moiety. To further investigate the utility of this methodology, the radiosynthesis of two cyclic RGD peptides was carried out, and in vivo biodistribution and microPET studies were performed in tumor-bearing mice. A cyclic RGD peptide was functionalized with the Michael acceptors trans-β-nitrostyrene carboxylic acid and 3-vinylsulfonylpropionic acid. Radiolabeling was then performed with the prosthetic group O-(2-(2-[¹⁸F]fluoroethoxy)ethyl)-N-methylhydroxylamine (¹⁸F-FENMA) yielding the ¹⁸F-conjugates in moderate yields (8.5-12%). Biodistribution, blocking, and microPET imaging studies were performed in a mouse xenograft model. The vinylsulfonyl-modified conjugate demonstrated good in vitro plasma stability. Biodistribution and microPET studies revealed excellent tumor uptake with low background in key organs and renal elimination as the predominant route of excretion. Blocking studies with coinjected nonlabeled RGD peptide confirmed the in vivo specificity for the integrin α(v)β₃. On the other hand, ¹⁸F-FENMA-nitrostyrene-RGD, although stable at conjugation pH 5, was found to rapidly degrade at physiological pH through loss of the ¹⁸F-prosthetic group.

Rapid 18F-radiolabeling of peptides from [18F]fluoride using a single microfluidics device

To date the majority of 18F-peptide radiolabeling approaches are multi-step, low yielding and time-consuming processes. Given the short half-life of 18F (109.8 min), it is critical that methods are developed to increase the efficiency of this process with simpler, higher yielding and faster reactions that can be rapidly translated into clinical use. Here, we demonstrate the first microfluidic synthesis of the [18F]F-Py-TFP prosthetic group with radiochemical yields of up to 97% and a synthesis time of 3 min. In addition, we utilized a single microfluidics device to prepare the [18F]F-Py-YGGFL peptide using [18F]F-Py-TFP, from [18F]fluoride in a two-step, fully automated approach. The model peptide NH2-YGGFL was radiolabeled with [18F]F-Py-TFP in up to 28% overall radiochemical yield within 8 minutes starting from anhydrous [18F]fluoride.

Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression

Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. LogP (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. (18)F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75 GBq/μmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.

Brain penetrant small molecule 18F-GnRH receptor (GnRH-R) antagonists: Synthesis and preliminary positron emission tomography imaging in rats

INTRODUCTION The gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood-brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and in vitro evaluation of two novel [(18)F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these (18)F-labeled compounds in brain of living rats. METHODS We synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for (18)F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability in vitro and in vivo were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration in vivo. RESULTS A palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays in vitro revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The (18)F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5-15% overall radiochemical yield. The radiolabeled compounds demonstrated good in vivo stability. PET imaging with the (18)F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding in vivo. CONCLUSIONS The novel small molecule (18)F-fluorinated GnRH-R antagonist compounds show high receptor affinity in vitro, and may prove useful for quantitative autoradiographic studies in vitro. The compounds were permeable to the blood-brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain.

Radiosynthesis of high affinity fluorine-18 labeled GnRH peptide analogues: in vitro studies and in vivo assessment of brain uptake in rats

Gonadotropin releasing hormone (GnRH) is recognized as an important neuromodulator affecting behavior and has been associated with the progression of Alzheimers disease. The peptide has been shown to have a bidirectional transport through the blood–brain-barrier (BBB), which may account for the cognitive effects of systemically administered GnRH. In this study, four novel 18F-GnRH peptide analogues were synthesized and their in vitro and in vivo characteristics studied in male rats. GnRH peptides were assembled by solid-phase peptide synthesis, either as the full length D-Lys6-GnRH (pyroGlu1-His2-Trp3-Ser4-Tyr5-D-Lys6-Leu7-Arg8-Pro9-Gly10-NH2) or as D-Lys6-desGly10-GnRH-NHEt. In all, four GnRH peptide analogues were synthesized and reacted with N-succinimidyl-4-fluorobenzoate (SFB) to yield the fluorinated versions. Binding affinities of the analogues were determined in a competitive binding assay for both human and rat GnRH receptors. Ki-values for the GnRH peptides were found to be subnanomolar, with D-Lys6(FBA)-desGly10-GnRH-NHEt (7) being most potent with a Ki-value of around 50 pM for GnRH receptor species. Radiolabeling was performed using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) in 33.3 ± 12.8% isolated decay corrected (d.c.) yield within 1.5–2 h. Rat serum stability over 2 h revealed minor degradation (≤5%). For in vivo studies, 18F-peptides (4–30 MBq) were injected intravenously via the tail vein into rats and brain uptake was evaluated by means of dynamic PET (2 h) followed by biodistribution studies. PET showed limited or no uptake in brain for the 18F-peptides which predominantly cleared rapidly by renal excretion. Specific binding in the pituitary gland was confirmed for the 18F-peptide, 7, by blocking with the GnRH agonist buserelin.