Automated synthesis of the generic peptide labelling agent N-succinimidyl 4-[18F]fluorobenzoate and application to 18F-label the vasoactive transmitter urotensin-II as a ligand for positron emission tomography

https://doi.org/10.1016/j.nucmedbio.2008.04.005Get rights and content

Abstract

Introduction

The objectives of this work were to develop an automated production of N-succinimidyl 4-[18F]fluorobenzoate ([18F]-SFB) and to test whether the vasoactive peptide urotensin-II (U-II) could be labelled by conjugation with [18F]-SFB.

Methods

A TRACERlab MXFDG synthesizer including an HPLC unit was used. The MS Excel synthesis sequence and the standard disposable FDG cassette were modified to allow the synthesis of [18F]-SFB. U-II was subsequently conjugated with [18F]-SFB, and the resulting 18F-labelled peptides were characterised using in vitro ligand binding assays.

Results

[18F]-SFB was successfully synthesised in the TRACERlab MXFDG in 44.3±2.5% (n=25) radiochemical yield in 98 min. [18F]-SFB (8–12 GBq) has been produced with specific activities in the range of 250–350 GBq/μmol and a radiochemical purity >95%. [18F]-SFB was subsequently used to label U-II. Two radiolabelled products, [18F]-(Glu1)-U-II and [18F]-(Lys8)-U-II, were formed in an isolated radiochemical yield from [18F]-SFB of 5.2±0.3% and 29.0±3.7%, respectively (n=7). Radioligand binding assays revealed that [18F]-(Glu1)-U-II had retained subnanomolar affinity. Binding to human skeletal muscle (n=3) was concentration dependent and saturable with Kd=0.84±0.51 nM, Bmax=0.69±0.14 fmol/mg protein and Hill slope (nH)=1.03±0.12.

Conclusions

[18F]-SFB has been synthesised using the TRACERlab MXFDG module, allowing production of up to 8–12 GBq of [18F]-SFB with specific activities of 250–350 GBq/μmol. [18F]-SFB was used for the labelling of U-II. In vitro characterisation demonstrated that [18F]-(Glu1)-U-II had retained desirable binding properties and may be suitable as a positron emission tomography radioligand for the imaging of the U-II receptor.

Introduction

Peptides and their interaction with receptors play a major role in normal physiology and in pathophysiology. The impact of this group of receptor systems in human biology is expected to increase further following the recent identification of novel peptide receptor systems and their cognate ligands predicted from the human genome [1]. A number of these receptor systems are present in the cardiovascular system [2], and our overall aim is to investigate the role these receptors play in the human cardiovascular system and to quantify changes associated with disease and to identify new targets for novel drugs.

Urotensin-II (U-II; cyclo5–10[H-Glu1-Thr2-Pro3-Asp4-Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10-Val11-OH]) has recently been identified as the cognate ligand for the ‘orphan’ G-protein-coupled receptor, GPR14 [3], now designated as the UT receptor [4] that is widely distributed in the human cardiovascular system [5]. In humans, U-II is a ubiquitous endothelium-derived peptide, acting as a locally released mediator on underlying vascular smooth muscle to cause potent vasoconstriction or indirect vasodilatation via endothelial cell receptors. Changes in the U-II system have been implicated in cardiovascular disease such as elevation of U-II in clinical [6] and experimental heart failure [7] and the presence of the peptide within infiltrating macrophages in atherosclerosis [8].

The use of biomolecules such as proteins, peptides and antibodies labelled with positron-emitting radionuclides as probes to image physiological and pathological processes will potentially be a significant means to rapidly translate genomic and proteomic information into man. We have established the synthesis of N-succinimidyl 4-[18F]fluorobenzoate ([18F]-SFB) (Fig. 1) originally developed by Vaidyanathan and Zalutsky [9] and modified by Wester et al. [10] to allow labelling of peptides for visualisation of peptide receptor system using positron emission tomography (PET). We have successfully used this approach to image the endothelin (ET) receptor system in PET using the endogenous transmitter [18F]-ET-1, its precursor peptide [18F]-big ET-1 and a receptor subtype specific peptide, [18F]-BQ3020 [11], [12], [13], [14], [15].

The first aim of this work was to develop an automated system for the production of [18F]-SFB. We used the TRACERlab MXFDG synthesizer from GE Healthcare (formerly Coincidence FDG synthesizer), a synthesis module originally developed for the automated production of FDG. This module has also been successfully adapted for the production of other radiopharmaceuticals such as [18F]MPPF [16], [18F]FLT [17], [18F]FMISO [18], [18F]fluoroacetate [19], 16α-[18F]fluoroestradiol [20] and [18F]fluorocholine [21], demonstrating the versatility of the system and suggesting that the TRACERlab MXFDG could potentially be used for the synthesis of [18F]-SFB. The advantage of this system is the use of a sterile, single-use cassette, allowing synthesis of radiopharmaceuticals to the GMP standard required for clinical use, and secondly, through automation, the use of higher levels of radioactivity than would be permitted by manual synthesis.

Secondly, our aim was to test whether the peptide U-II could be labelled by conjugation with [18F]-SFB generated by this automated system. Since U-II has two possible sites for conjugation (Glu1 and Lys8), labelling will potentially result in two products. We therefore used in vitro receptor binding to identify which (if any) of the two [18F]-U-II products had retained high affinity for the UT receptor.

Section snippets

Materials

Human U-II was obtained from Peptide Institute Inc. (Osaka, Japan), and leucine aminopeptidase (LAP; microsomal suspension in ammonium sulfate solution) was obtained from Sigma (Poole, UK). All other chemicals used were from Aldrich (Gillingham, UK). Solid-phase extraction (SPE) cartridges (SepPak QMA Light, SepPak C18 Plus and Oasis HLB 30 mg) were obtained from Waters (Elstree, UK). The QMA cartridge was converted to the carbonate form by treatment with an aqueous solution of 0.5 M K2CO3.

Synthesis of reference fluorobenzoyl-conjugated U-II peptides

As expected, three products were formed when U-II was reacted with an excess of unlabelled SFB. LCMS confirmed that these products corresponded to the two monoconjugated products [MS (m/z): RT 20.2 min 1510.6 [MH]+, 1532.5 [MNa]+; RT 25.0 min 1510.6 [MH]+, 1532.5 [MNa]+] and the double-conjugated product [MS (m/z): RT 27.9 min 1654.8 [MNa]+]. Fragmentation of the two monoconjugated products (MSMS) indicated that RT 20.2 corresponded to fluorobenzoyl-(Glu1)-U-II [MSMS (m/z): 1492.6 [MH-{H2O}]+,

Discussion

We have successfully adapted the TRACERlab MXFDG synthesis module to enable the synthesis of [18F]-SFB, and we have, for the first time, performed a three-step procedure in this synthesizer. [18F]-SFB was produced in reproducible radiochemical yields and to high specific activities using a modified FDG cassette. The advantage of this system is the use of a sterile, single-use cassette for radioligand production for clinical PET studies. However, for peptide labelling development and preclinical

Acknowledgments

We thank I. Abakumova, P. Burke, O. Golovko, R. Kuc, J. Maguire and R. Smith for helpful discussion and technical support. This work was supported by grants from the British Heart Foundation and the Medical Research Council.

References (33)

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