Synthesis and evaluation of an ¹⁸F-labeled bradykinin B1 receptor-targeting small molecule for PET imaging

Objectives The bradykinin B1 receptor (B1R), a G-protein coupled receptor, is overexpressed in several pathophysiological conditions including inflammation and cancer. B1R mediates nociception in the context of inflammation, and it is believed to contribute to tumourigenesis by promoting cell proliferation, invasion and angiogenesis. Previously, our lab reported the evaluation of several promising B1R-targeting peptides for cancer imaging. These peptides have high affinity to B1R and generate high-contrasted images of B1R-expressing tumour xenografts in mice. A significant number of potent B1R small molecule antagonists have been developed by pharmaceutical companies for the treatment of chronic pain, and could potentially be exploited for the design of B1R-targeting imaging agents. In this study, we investigated the use of an ¹⁸F-labeled small molecule (4-methoxy-N,2,6-trimethyl-N-[2-[2-[4-(1-[¹⁸F]fluoroethyl-4-piperidinyl)-1-piperazinyl]-2-oxoethoxy]ethyl]benzenesulfonamide, ¹⁸F-Z02035) as an alternative to peptide-based B1R-imaging.

Methods The non-radioactive Z02035 standard and its des-fluoroethyl precursor (4-methoxy-N,2,6-trimethyl-N-[2-[2-[4-(1-H-4-piperidinyl)-1-piperazinyl]-2-oxoethoxy]ethyl]benzenesulfonamide, Z02153) were synthesized via multi-step synthesis approach. The binding affinity of Z02035 for B1R was determined by in vitro competition binding assay using B1R-expressing cell membrane. ¹⁸F-Z02035 was prepared in two steps: (1) synthesis of 2-[¹⁸F]fluoroethyl tosylate by aliphatic nucleophilic substitution using ¹⁸F-fluoride and 1,​2-​bis(tosyloxy)​ethane; and (2) coupling of 2-[¹⁸F]fluoroethyl tosylate with the precursor Z02153. PET imaging and biodistribution studies were performed at 1 h post-injection in mice bearing both B1R-positive HEK293T::hB1R and B1R-negative wild-type HEK293T tumours.

Results The overall synthesis yield for the non-radioactive standard Z02035 was 1% (seven steps), and the yield for the precursor Z02153 was 47% (seven steps). Competition assays showed that Z02035 is a potent B1R binder with a K_i value of 0.93 ± 0.44 nM. By reacting Z02153 with 2-[¹⁸F]fluoroethyl tosylate, ¹⁸F-Z02035 was obtained in 6 ± 1% non-decay corrected yield with > 99% radiochemical purity and 0.9 ± 0.8 Ci/µmol specific activity. From PET images, ¹⁸F-Z02035 enabled visualization of the B1R-positive HEK293T::hB1R tumours with better contrast than the B1R-negative HEK293T tumours. In terms of clearance, ¹⁸F-Z02035 was excreted primarily through the hepatobiliary pathway. The average uptake in HEK293T::hB1R tumours (3.77 ± 0.79 %ID/g) was higher than in HEK293T tumours (2.11 ± 0.37 %ID/g). The tumour-to-muscle ratio for HEK293T::hB1R was 3.38 ±1.05. ¹⁸F-Z02035 exhibited slight defluorination in vivo as bone uptake was 4.63 ± 0.65 %ID/g.

Conclusions We took a novel approach for targeting B1R expression by synthesizing an ¹⁸F-labeled small molecule. Although absolute uptake and overall contrast are lower than reported B1R tracers derived from peptide sequences, ¹⁸F-Z02035 was able to differentiate between B1R expressing and non-expressing tumour xenografts. Optimization to enhance tumour uptake/contrast and reduce in vivo defluorination is currently underway.

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