Ole Kristian Hjelstuen

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.

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.

A critical study on borosilicate glassware and silica-based QMA's in nucleophilic substitution with [18F]fluoride: influence of aluminum, boron and silicon on the reactivity of [18F]fluoride.

Leachables of borosilicate glassware and silica-based anion exchange columns (QMAs) may influence nucleophilic substitution with [(18)F]fluoride ([(18)F]F(-)). Aluminum, boron and silicon, all constituents of borosilicate glass, were found as water soluble leachables in a typical PET synthesis setup. Relevant ranges of the leachable quantities were studied based on an experimental design, in which species of the three elements were added to the labeling of the precursor for anti-1-amino-3-[(18)F]fluorocyclobutyl-1-carboxylic acid ([(18)F]FACBC). Levels of 0.4-2 ppm aluminum as AlCl(3) had a strong negative influence on labeling yield while 4-20 ppm of boron as KBO(2) and 50-250 ppm of silicon as Na(2)SiO(3) did not have a significant impact. Interesting interaction effects between the elements were observed, where particularly KBO(2) reduced the negative effect of AlCl(3) on labeling yield. It can be concluded that leachables of borosilicate glassware easily can influence nucleophilic substitution with n.c.a. [(18)F]F(-) and give variable yields.