Fluorine-18 Labelled substrate-based radiotracers for in vivo imaging of lysyl oxidase

Objectives: The ability of solid tumours to invade surrounding tissues and, in consequence, to metastasise to distant organs is mediated by bidirectional molecular interactions between tumour cells and the extracellular matrix [1,2]. Recently, the copper-dependent amine oxidase lysyl oxidase (LOX, EC 1.4.3.13) could be identified as one of the key players in these processes [3]. Therefore, the development of molecular probes that enable the imaging
of this enzyme in vivo by positron emission tomography (PET) was in the focus of this study.
Methods: As the enzyme is catalysing the oxidative crosslinking of lysine side chains in collagen and other extracellular proteins, the design of radiotracers based on substrates seemed to be promising. Thus, the N-terminal telopeptide of the α1-chain of type I collagen containing the key sequence Asp-Glu-Lys-Ser [4] and peptides derived from this were chosen to be labelled with fluorine-18 at their N-termini (Figure 1). To achieve this, a method was developed that allows the site-selective ¹⁸F-fluorobenzoylation of peptides [5]. The metabolic stability and biodistribution of these potential radiotracers was investigated in male wistar rats. To estimate the potential of the different lysine-containing peptides for crosslinking with collagen in vivo, their interaction with bovine atelocollagen was investigated by surface plasmon resonance (SPR) experiments. A panel of tumour cell lines was screened for expression of the enzyme. The presence of LOX could be confirmed for the human breast cancer celllines MDA-MB-231, MCF-7 and the melanoma cell line A375 by RT-PCR as well as western blots. Based on the human A375 cell line, an animal model was established consisting of nude mice bearing tumours derived from these cells. Expression of LOX in the developed tumours was proven by immunohistochemical methods and western blots.
Results: The developed labelling method for site-selective radiolabelling of peptides allowed to obtain the ¹⁸Ffluorobenzoylated telopeptide in high radiochemical yields and purities. All peptides show good stability in vivo and even no metabolites could be detected for the cyclohexapeptide. The biodistribution studies indicate no organ enrichment and fast renal elimination. For the first time, the telopeptide-collagen interaction could be studied quantitatively, indicating dissociation constants in the high micromolar range.
Conclusions: Despite unfavourable pharmokinetics due to fast blood clearance, the compounds show the potential to reflect the LOX activity in vivo, as concluded from PET imaging experiments with nude mice bearing A375 tumours.
References: [1] Fidler IJ, et al. (2003) Nat Rev Cancer, 3, 453.
[2] Rowe RG, et al. (2009) Annu Rev Cell Dev Biol, 25, 567.
[3] Erler JT, et al. (2006) Nature, 440, 1222-6.
[4] Helseth DL, et al. (1979) Biopolymers, 18, 3005.
[5] Kuchar M, et al. (2012) Amino Acids, 43, 1431-43.