Elsevier

Nuclear Medicine and Biology

Volume 39, Issue 7, October 2012, Pages 1000-1005
Nuclear Medicine and Biology

Automated GMP Synthesis of [18F]ICMT-11 for In Vivo Imaging of Caspase-3 Activity

https://doi.org/10.1016/j.nucmedbio.2012.03.004Get rights and content

Abstract

Introduction

Isatin-5-sulfonamide ([18F]ICMT-11) is a sub-nanomolar inhibitor of caspase-3 previously evaluated as an apoptosis imaging agent. Herein, an alternative radiosynthesis of [18F]ICMT-11 with increased purity and specific activity is presented. Finally, a GMP-applicable automated radiosynthesis of [18F]ICMT-11 is described.

Methods

The preparation of [18F]ICMT-11 was evaluated under a variety of reaction conditions, including reaction solvent, by employing alternative phase transfer catalysts and under different deprotection conditions. Following initial investigations, the process was transferred onto a fully automated GE FASTlab synthesis platform for further development and optimisation.

Results

The synthesis of [18F]ICMT-11 was successfully validated under GMP conditions, resulting in a yield of 4.6 ± 0.4 GBq with a radiochemical purity of > 98% at EOS and a specific activity of 685 ± 237 GBq/μmol within 90 min. Quality control was carried out in accordance with the European Pharmacopoeia and demonstrated that [18F]ICMT-11 can be consistently manufactured on the FASTlab to meet specifications.

Conclusions

A simplified methodology for the synthesis of the apoptosis imaging agent, [18F]ICMT-11, has been achieved by the SN2 displacement of a tosylate leaving group with [18F]fluoride ion. This results in an increased purity and specific activity over the original copper catalysed “Click” synthetic stratagem reaction involving 2-[18F]fluoroethylazide with an alkyne precursor and is now suitable for routine clinical application.

Introduction

The ability to detect and quantify biological processes in vivo is the primary objective of Positron Emission Tomography (PET). Apoptosis, or Programmed Cell Death, is an energy conserved process in which the contents of a cell are broken down and engulfed by macrophages prior to re-use by other cells. Excessive apoptosis is implicated in neurodegenerative diseases (e.g. Alzheimer's), ischemia and transplant rejection [1]. By contrast, insufficient apoptosis is defined as one of the hallmarks of cancer [2], [3]. It therefore follows that strategies for imaging apoptosis in vivo would be of considerable interest both in the clinic, for patient diagnosis and evaluation of response to treatment [4], and as a tool to aid evaluation of new therapeutics, both clinically and pre-clinically.

Previously, apoptosis imaging using PET has concentrated on Annexin V radiolabelled probes [5], [6], [7]. These proteins interact with phosphatidylserine residues, in the presence of calcium ions, externalised on the cell surface during apoptosis. However, this strategy suffers from several drawbacks, chiefly an inability to distinguish between apoptosis and necrosis, leading to reduced specificity in vivo. An alternative approach involves imaging caspase activity. Caspases are expressed as inactive zymogens in normal cells but cleave to their active form in response to cellular stress signals. There are two classes of caspase involved in the apoptotic process, caspases 2, 8, 9, 10 are initiator caspases that cleave the effector caspases 3, 6, 7 into their active forms. These effector caspases are then responsible for the process of cellular breakdown by cleavage of key cellular structural and repair proteins such as PARP [8].

Recently, isatin-5-sulfonamides have been proposed as suitable probes for radiolabelling and in vivo detection of caspase-3 activity [9], [10], [11]. In vivo monitoring of caspase-3 activity using a radiolabelled isatin was initially studied in a liver model of apoptosis. In this model cycloheximide treated rats showed higher liver uptake of [18F]WC-II-89 and [11C]WC-98 than control treated rats [12]. More recently, an 18F-click labelled isatin derivative ([18F]ICMT11), has been developed using 2-[18F]fluoroethyl azide [13], [14]. In a different, more clinically relevant model, 38C13 tumour bearing mice were treated with cyclophosphamide, demonstrating increased uptake of [18F]ICMT-11 relative to control treated mice. [18F]ICMT-11 has become established as the lead radiotracer for PET imaging of caspase-3 activity [15], [16]. The radiolabelling of [18F]ICMT-11 has been adapted for automation by using an acetal protected tosylate precursor and the radiosynthesis has been transferred to a cassette based automated platform (FASTlab).

Section snippets

Methods

The synthesis of protected isatin alkyne 1 ((S)-1-{[1′-[1-(2-propynyl)]-(1′2′-dihydro-2′-oxospiro(1,3-dioxane-2,3′-[3H]indol)-5′-sulfonyl}-2-(2,4-difluorophenoxymethyl)-pyrrolidine) is described elsewhere [13], [14]. All other reagents and solvents were purchased from either VWR International (Lutterworth, United Kingdom) or Sigma-Aldrich (Gillingham, United Kingdom) in the highest available purity and were used without further purification. Sodium chloride for injection 0.9% w/v and water for

Radiosynthesis of ICMT-11

The original synthetic stratagem for [18F]ICMT-11 (Scheme 1, I) utilised a copper catalysed “Click” reaction involving 2-[18F]fluoroethylazide with an alkyne precursor 5 [13]. This synthesis resulted in a modest specific activity of 1.2 GBq/μMol and a stable isatin analogue impurity at a concentration of 14 μg/mL. In an effort to improve the specific activity of [18F]ICMT-11 and reduce the concentration of stable isatin impurities we investigated a modified click radiochemistry strategy (Scheme 1

Conclusion

A simplified, more robust methodology for the synthesis of the apoptosis imaging agent, (S)-1-((1-(2-fluoroethyl)-1H-[1,2,3]-triazol-4-yl)methyl)-5-(2(2,4-difluorophenoxymethyl)-pyrrolidine-1-sulfonyl)isatin ([18F]ICMT-11) has been achieved by the SN2 displacement of a tosylate leaving group with [18F]fluoride ion resulting in an increased purity and specific activity. A fully automated GMP synthesis of [18F]ICMT-11 suitable for clinical use has been developed providing in excess of 4 GBq of [18

Acknowledgments

The authors would like to acknowledge Andrew Black (MDx Fundamental Radiochemistry Team, GE Healthcare, Amersham, U.K.) for HPLC-MS/MS data and analysis and Dave Turton (Hammersmith Imanet, GE Healthcare, London, U.K.) for Cation HPLC analysis.

This work was funded by Cancer Research UK–Engineering and Physical Sciences Research Council grant (in association with the Medical Research Council and Department of Health (England)) grant C2536/A10337. E.O.A.'s laboratory receives core funding from

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    These authors contributed equally.

    2

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